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ISBN 978-952-62-2805-1 (Paperback)ISBN 978-952-62-2806-8 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1594

AC

TAR

avindra Daddali

OULU 2020

D 1594

Ravindra Daddali

MOLECULAR MECHANISMS REGULATING THE ONSETOF LABORCOMPARATIVE PROTEOMICS AND miRNAOMICS OF HUMAN SPONTANEOUS AND ELECTIVE LABORS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;OULU UNIVERSITY HOSPITAL

ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 5 9 4

RAVINDRA DADDALI

MOLECULAR MECHANISMS REGULATING THE ONSET OF LABORComparative proteomics and miRNAomics of human spontaneous and elective labors

Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defence inAuditorium F202 of the Faculty of Medicine (Aapistie 5 B),on 11 December 2020, at 12 noon

UNIVERSITY OF OULU, OULU 2020

Copyright © 2020Acta Univ. Oul. D 1594, 2020

Supervised byProfessor Mika RämetProfessor Mikko HallmanDocent Antti Haapalainen

Reviewed byProfessor Markku VarjosaloDocent Tomi Airenne

ISBN 978-952-62-2805-1 (Paperback)ISBN 978-952-62-2806-8 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

PUNAMUSTATAMPERE 2020

OpponentProfessor Jaana Rysä

Daddali, Ravindra, Molecular mechanisms regulating the onset of labor.Comparative proteomics and miRNAomics of human spontaneous and electivelaborsUniversity of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center Oulu; Oulu University HospitalActa Univ. Oul. D 1594, 2020University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Human parturition is a complicated biological process and the molecular mechanisms regulating the onset oflabor are not well understood. Several studies propose that gene variants, proteins and miRNAs play a majorrole in maintaining pregnancy and aberrations in protein and miRNA levels may lead to pregnancycomplications. The levels of proteins and miRNAs could prove to be useful biomarkers for monitoring theonset of labor.

In this study, using proteomics and a genome analysis, we identified Calcineurin-like phosphoesterasedomain-containing protein (CPPED1), as a potential biomarker of labor. CPPED1 was found to bedownregulated in spontaneous term placentas compared with placentas from elective deliveries. The siRNAknockdown of CPPED1 in trophoblasts derived cells mostly affects pathways related to inflammation andangiogenesis, and these functions may be involved in the induction of labor or in maintaining pregnancy. Inbladder cancer cells, CPPED1 has been shown to dephosphorylate AKT1 on the Ser-473 residue and topromote apoptosis via the AKT/PI3K pathway. In our studies using cells of placental origin AKT1phosphorylation levels were unaffected by CPPED1 expression, whereas its silencing led to the upregulationof negative regulators of the PI3K pathway.

In a comparative miRNAomics screen of spontaneous term vs. elective term placentas, we identified 54differentially expressed miRNAs, out of which 23 miRNAs were upregulated and 31 were downregulated byat least 1.5-fold. Among the upregulated miRNAs, miRNA-371a-5p targets CPPED1 and post-transcriptionally regulates its expression by binding to its 3’UTR region. In addition, we found that CPPED1and miR-371a-5p levels inversely correlate with each other in spontaneous and elective term placentas.Measuring the levels of CPPED1 and miR-371a-5p levels in maternal blood could be a potential diagnosticbiomarker for labor.

In order to elucidate the functions of CPPED1 we used a protein microarray to identify proteins thatinteract with CPPED1 in vitro and obtained a list of 36 proteins that potentially bind CPPED1. The interactionof CPPED1 with PAK4 and PIK3R2 was confirmed by co-immunoprecipitation and investigation of thecellular localization by BiFC studies showed that CPPED1 and PAK4 are colocalized in the cytoplasm,whereas CPPED1-PIK3R2 complex on the cell membrane. Further, by a mass spectrometry analysis, we foundthat CPPED1 dephosphorylates PAK4 on five different serine residues. The functional importance of thesephosphorylation sites is still unknown. PIK3R2 phosphorylation was unaffected by CPPED1.

Based on our studies we propose that the levels of CPPED1 and miRNA-371a-5p in the maternalcirculation could serve as biomarkers for the prediction of labor. The functional role of CPPED1 on PIK3R2and PAK4 remains to be studied.

Keywords: AKT1, CPPED1, mass spectrometry, miR-371a-5p, miRNA, PAK4,PIK3R2

Daddali, Ravindra, Synnytykseen liittyvät molekyylimekanismit. Ihmisenspontaanin ja elektiivisen synnytyksen vertaileva proteomiikka ja miRNAomiikkaOulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical ResearchCenter Oulu; Oulun yliopistollinen sairaalaActa Univ. Oul. D 1594, 2020Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Ihmisen synnytyksen käynnistyminen ja synnytys ovat monimutkaisia biologisia prosesseja, joiden kaikkiataustalla olevia molekyylimekanismeja ei vielä tunneta. Useat tutkimukset osoittavat, että tietyillä proteii-neilla ja mikroRNA-molekyyleillä on tärkeä rooli raskauden ylläpitämisessä. Näiden proteiini- ja mikroR-NA-määrien poikkeamat voivat johtaa raskauskomplikaatioihin. Proteiinien ja mikroRNA:n määrien mittaa-mista voitaisiin siten käyttää mahdollisina raskauskomplikaatioiden ja synnytyksen biomarkkereina.

Tässä tutkimuksessa löysimme proteomiikan ja genomin laajuisen assosiaatiotutkimuksen (GWAS) avul-la entsyymin nimeltä Calcineurin-like phosphoesterase domain-containing protein 1 (CPPED1), joka mah-dollisesti voi toimia spontaanin synnytyksen biomarkkerina. CPPED1-tasot laskivat spontaaneissa synnytyk-sissä, kun niistä saatuja istukoita verrattiin keisarinleikkauksen kautta saatuihin istukkoihin. Kun CPPED1-geenin ilmeneminen hiljennettiin istukan trofoblastisoluissa, tulehduksiin ja verisuonten uudismuodostuk-seen liittyvät signalointireitit muuttuivat eniten. Näillä muutoksilla saattaa olla merkitystä raskauden kes-toon. Aikaisemmassa tutkimuksessa on osoitettu, että virtsarakon syöpäsoluissa CPPED1 poistaa fosfaatti-ryhmän AKT1-entsyymin seriini-473 aminohapolta. Tämän fosfaatin poistaminen johti ohjelmoituun solu-kuolemaan AKT/PI3K-signalointireitin kautta. Meidän omissa tutkimuksissamme AKT1-entsyymin fosfory-lointi ei muuttunut istukan soluissa vaikkakin CPPED1:n määrä muuttui. Sen sijaan, CPPED1-määrän laski-essa PI3K-signalointireittiä negatiivisesti säätelevien proteiinien määrät lisääntyivät istukasta peräisin ole-vassa solulinjassa.

Vertailevassa mikroRNAomiikassa, jossa vertailtiin alatiesynnytyksistä ja keisarinleikkauksista peräisinolevien istukoiden mikroRNA-molekyylejä, havaitsimme merkittäviä muutoksia 54 mikroRNA-molekyyli-en määrissä. Näistä 54 mikroRNA:stä 23 mikroRNA:n määrät nousivat ja 31 mikroRNA:n määrät laskivatspontaaneiden täysiaikaisten synnytysten istukoissa. Eräs mikroRNA, jonka määrä nousi spontaanin synny-tyksen istukassa, oli miR-371a-5p. Osoitimme, että miR-371a-5p sitoutuu CPPED1:n lähetti-RNA:n 3’UTR-alueelle, minkä seurauksena lähetti-RNA:n määrä laski. CPPED1- ja miR-371a-5p-tasot siis korreloivatkäänteisesti toisiinsa istukoissa, jotka ovat peräisin spontaaneista synnytyksistä. Näin ollen äitien verestämitatut CPPED1 ja miR-371a-5p määrät voisivat olla potentiaalinen diagnostinen biomarkkeri spontaaninsynnytyksen ennustamisessa.

CPPED1:n toimintojen selvittämiseksi käytimme proteiinimikrosirutekniikkaa tunnistaaksemme proteii-neja, jotka voisivat mahdollisesti olla vuorovaikutuksessa CPPED1:n kanssa. Kyseinen proteiinimikrosirusisälsi noin 75% ihmisen solun kaikista proteiineista. Proteiinimikrosirumenetelmällä CPPED1:n osoitettiinsitoutuvan 36 erilaiseen proteiiniin. Näistä kahden proteiinin, PAK4:n ja PIK3R2:n, vuorovaikutuksetCPPED1:n kanssa varmistettiin kahdella muulla menetelmällä. Immuunisaostuksen avulla osoitettiin, ettäCPPED1 muodosti tiiviin kompleksin sekä PAK4:n että PIK3R2:n kanssa. Kaksoisfluoresenssikomplement-timenetelmän avulla havaitsimme, että CPPED1 sijaitsi solun sisällä samoissa paikoissa kuin PAK4 jaPIK3R2. Lisäksi massaspektrometrianalyysillä osoitimme, että CPPED1 poisti fosfaattiryhmiä PAK4:n tie-tyistä seriiniaminohapoista; näiden fosforylointikohtien toiminnallinen merkitys PAK4:lle on vielä tuntema-ton. Siten CPPED1 merkitys PAK4:n ja PIK3R2:n toiminnoille vaatii vielä lisätutkimuksia.

Asiasanat: AKT1, CPPED1, massaspektrometria, miR-371a-5p, miRNA, PAK4,PIK3R2

Dedicated to my family

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9

Acknowledgements

This work was carried out at the PEDEGO Research Unit and the Medical Research

Center Oulu, University of Oulu and Oulu University Hospital, during the years

2016-2020. I wish to acknowledge all people who have contributed to this work

and supported me during these years.

I would like to sincerely thank the trio of my supervisors including Professor

Mika Ramet, Professor Mikko Hallman and Docent Antti Haapalainen. My

principal supervisor Mika Ramet is especially acknowledged for his insightful

comments and advice in different situations as well as for his positive and

encouraging support. I am grateful to Mikko Hallman for giving me the opportunity

to work in his group and I value his curiosity and open-mindedness towards

research. I would like to thank my third supervisor Antti Haapalainen for his

support throughout this project.

I would also like to extend my sincere gratitude to the pre-examiners of this

thesis, Professor Markku Varjosalo and Docent Tomi Airenne for their constructive

comments to improve the contents of the thesis. I am grateful to Professor Jaana

Rysä for agreeing to be my opponent and I look forward to our discussion.

I warmly thank all former and present members of our research group. I wish

to thank Anu Pasanen, Maarit Haarala, Minna Karjalainen, Heli Tiensuu, Marja

Ojaniemi, Johanna Huusko and others for their assistance, peer support and

intriguing conversations in the laboratory, during coffee breaks and in many other

situations. Special thanks to Marjatta Paloheimo for the help whenever I needed it.

I also wish to thank the people of CRC for such a friendly atmosphere and indulging

in interesting conversations. I wish to thank the members of my follow-up group,

Professor Lauri Eklund and Dr. Tiila-Rikka Kiema for their time and encouraging

comments. Likewise, I also want to thank the members of the neighbouring labs of

Docent Reetta Hintala and Professor Johanna Uusimaa and Antti Nätynki, Jussi

Tuusa, Masuma Aman. I also sincerely thank the faculty members of the Clinical

Research Center.

I thank to my best friends Dr. Venkateswarlu Raavi, Dr. Gaurav Soman, Dr.

Mastan babu, Dr. Vangipurapu Rajanikanth, Dr. Madhavi Padala, Anil, Sanjeeva,

Obul reddy, Vijay, Dr. Kanuru Madhavi, Dr. Saikrishna, Dr. Kedar and all my other

friends during graduation and postgraduation.

I extend my thanks to previous workplace at the CCMB Research group to Dr.

Yogendra Sharma and the YS Group (Rajanikanth, Shashi, Amita, Rajeev Raman,

Shantiswaroop, Vijeta, Balkrishn and Raji).

10

I thank my latter workplace at the NCCS research lab of Dr. Debashis Mitra

and his group (Priyanka, Surya, Tracy, Kailash, Jay) along with my batchmates of

NCCS 2012 for their immense support for making me learn the struggles for

existence and survival of the fittest throughout my thick and thins.

I also thank Dr. Karuna Rupula, Prof. Beedu Sashidhar Rao, Dr. Raju, Suresh

and Dr. Shyam at Osmania University, Biochemistry department.

My special thanks and grateful to my friends in Oulu, Dr. Abhishek Sharma,

Dr. Dhirendra Singh, Shruthi Sridhar, Surbhi Mishra, Pooja, Dr. Bishal Singh, Dr.

Srinivas kumar, Vamsikrishna and other Indian friends in Oulu for all the good

times and educational moments we have shared during these years.

I thank my family members, my uncle Srinu Mamayya and son in laws Harsha

and Rakesh, for their love and support. I express my deep gratitude to my brothers

Hari babu and Kiran Kumar and brother in-law Akshay Rai for their enormous love

and support all throughout. I thank my sister in-law Jyothsna, Shristi Rai and my

nephews Aravind and Karthik for their charming smile and love towards me. I

thank my sister Umarani and younger brother Srikanth for their love.

My special thanks to my spouse and life partner Supriya Rai for her

unconditional love and support in every phase of my struggle and sufferings. I rely

on your support for which I feel very lucky and grateful. I hope I can bring even a

fraction of the love, happiness, and joy into your life that I have received from you.

Finally, I thank my parents for their wholehearted love and never-ending

support throughout my life till now. Nothing would have been possible without you

and your support.

The work was conducted with the support of grants from the Jane and Aatos

Erkko Foundation (MH, MR), the Sigrid Jusélius Foundation (MH), and from the

Academy of Finland, Foundation for Pediatric Research and the University of Oulu

Scholarship Foundation.

Date Ravindra.Daddali

16.11.2020

11

Abbreviations

3’UTR 3’ untranslated region

ADAM12 A disintegrin and metalloprotease 12

AF Amniotic fluid

β-hCG Beta Human Chorionic Gonadotropin

BiFC Bimolecular fluorescence complementation

CNA Calcineurin A

CPPED1 Calcineurin Like Phosphoesterase Domain Containing 1

CVF Cervico-vaginal fluid

C14MC Chromosome 14 miRNA cluster

C19MC Chromosome 19 miRNA cluster

CD Circular dichroism

co-IP co-immunoprecipitation

C1qTNF5 Complement C1q Tumor Necrosis Factor-Related Protein 5

CSTP1 Complete S transactivated protein

CRH Corticotropin-releasing hormone

CYC1 Cytochrome c1

DNA-PK DNA-dependent protein kinase

ETB Elective term birth

fFN Fetal fibronectin

GRB2 Growth Factor Receptor Bound Protein 2

HD Hydrophobic Domain

IAI Intra-amniotic infection

IGFBP-1 Insulin-like growth factor-binding protein 1

ILK Integrin-linked kinase

LC-MS/MS Liquid chromatography–tandem mass spectrometry

MPEs Metallophosphoesterases

MPPs Metalloproteinphosphatases

miRNA Micro-RNA

mTORC Mechanistic target of rapamycin complex

PAK4 p21 Activated Kinase 4

PIP3 Phosphatidyl Inositol 3, 4, 5 triphosphate

PDK1 Phosphoinositide-dependent kinase 1

PI3K Phosphoinositide-3-Kinase

PIK3R2 Phosphoinositide-3-Kinase Regulatory Subunit 2

PAMG-1 Placental alpha macroglobulin-1

12

PlGF Placental growth factor

PTEN Phosphatase and tensin homolog

PP13 Plasma protein 13

PH Pleckstrin homology

PCR Polymerase chain reaction

PE Preeclampsia

PAPP-A Pregnancy associated plasma protein A

PSG Pregnancy specific glycoproteins

PROM Premature rupture of fetal membranes

PTB Preterm Birth

PP2A Protein phosphatase 2 A

PAP Purple Acid phosphatase

q-RTPCR Quantitative Real time PCR

sEng Soluble endoglobin

sFIt-1 Soluble fms-like tyrosine kinase-1

STB Spontaneous term birth

SLS Static light scattering

SELDI Surface-enhanced laser desorption ionization

TNF Tumor necrosis factor

uNK uterine Natural killer cells

VEGF Vascular endothelial growth factor

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Original publications

This thesis is based on the following publications, which are referred to throughout

the text by their Roman numerals:

I Haapalainen AM, Karjalainen MK, Daddali R, Ohlmeier S, Anttonen J, Määttä TA, Salminen A, Mahlman M, Bergmann U, Mäkikallio K, Ojaniemi M, #Hallman M, #Rämet M. (2017). Expression of CPPED1 in human trophoblasts is associated with timing of term birth. J Cell Mol Med. 2018 Feb;22(2):968-981

II Daddali R, Ojaniemi M, Hallman M, Rämet M& Haapalainen AM. (2020). MicroRNA-371a-5p targets CPPED1, and its expression in human placenta is associated with spontaneous delivery. Plos One. 2020 PLoS One. 2020 Jun 10;15(6):e0234403

III #Haapalainen AM, #Daddali R, Hallman M, Rämet M. Human CPPED1 belongs to calcineurin-like metallophosphoesterase superfamily and dephosphorylates PI3K-AKT pathway component PAK4 (Submitted)

# Equal contribution

The contributions of the author, R. Daddali, to the work focused on molecular

biology, proteomics and miRNAomics. He did all cell culture experiments (I, II,

III), the siRNA knockdown of CPPED1 (I), confocal imaging of p-FOXO1and p-

FOXO3, miRNA isolation, miRNA transfections, luciferase assay and data analysis

(II). He carried out the experiments including cloning, protein purification, BiFC

assays, co-IP, in vitro activity assays, CD and data analysis (III).

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15

Contents

Abstract

Tiivistelmä

Acknowledgements 9 

Abbreviations 11 

Original publications 13 

Contents 15 

1  Introduction 19 

2  Review of the literature 21 

2.1  Labor/delivery and the length of pregnancy ........................................... 21 

2.1.1  Major complications during pregnancy and delivery ................... 23 

2.1.2  Biomarkers for labor and pregnancy complications ..................... 24 

2.2  Human Placenta ...................................................................................... 26 

2.2.1  Placental development and functions ........................................... 27 

2.2.2  Placental genes, proteins and miRNAs ......................................... 29 

2.2.3  Placental abnormalities and pregnancy complications ................. 30 

2.3  Proteomics and pregnancy ...................................................................... 30 

2.3.1  Proteomic changes in the placenta during pregnancy ................... 31 

2.3.2  Human placental proteomics of preterm birth .............................. 32 

2.3.3  Proteins as biomarkers of preterm birth from various body

fluids ............................................................................................. 33 

2.4  Immune cells and inflammatory biomarkers in term and preterm

labor ........................................................................................................ 38 

2.5  miRNAs in labor and pregnancy ............................................................. 40 

2.5.1  Biogenesis of miRNAs ................................................................. 41 

2.5.2  miRNA functions: ......................................................................... 43 

2.5.3  Placental specific /Pregnancy associated miRNA-clusters ........... 46 

2.5.4  miRNAs as biomarkers of pregnancy complications ................... 50 

2.5.5  Circulating placenta-derived microRNA as biomarkers ............... 52 

3  Aims of the study 55 

4  Materials and Methods 57 

4.1  Molecular Cloning (III) ........................................................................... 57 

4.1.1  Generation of plasmid constructs for a BiFC analysis (III) .......... 57 

4.2  Protein purification (III) .......................................................................... 57 

4.3  In vitro phosphatase activity assay (III) .................................................. 58 

4.4  Protein microarray (III) ........................................................................... 58 

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4.5  Immunoprecipitation of CPPED1 interacting partners and

western blotting (III) ............................................................................... 59 

4.6  Bimolecular fluorescence complementation assay (III) .......................... 59 

4.7  Phosphatase assay detection by mass spectrometry (III) ........................ 60 

4.8  Placental miRNA isolation (II) ................................................................ 60 

4.9  Quantitative analysis of CPPED1 mRNA by qPCR (II) ......................... 60 

4.10 Quantitative PCR of hsa-miR-371a-5p (II) ............................................. 61 

4.11 Luciferase assay (II) ................................................................................ 61 

4.12 siRNA knockdown of CPPED1 and transcriptomics of siRNA

samples (I) ............................................................................................... 62 

5  Results 63 

5.1  Proteomic changes associated with spontaneous term deliveries

(I) ............................................................................................................. 63 

5.1.1  Placental differential protein expression between

chorionic and basal plates ............................................................. 63 

5.1.2  CPPED1 expression levels and its polymorphism

associated with gestational age ..................................................... 64 

5.1.3  AKT1 phosphorylation levels are unaffected by variations

in CPPED1 expression levels ....................................................... 64 

5.1.4  CPPED1 silencing in HTR-8 trophoblasts and mRNA

transcriptomics (I) ........................................................................ 64 

5.2  miRNA transcriptomics of spontaneous term birth (II) ........................... 65 

5.2.1  miRNA transcriptomics identifies differential miRNA

expression in spontaneous and elective term placentas (II) .......... 65 

5.2.2  Comparison of human placental proteomics and

miRNAomics identifies a miR-371a-5 -CPPED1 pair (I,

II) .................................................................................................. 65 

5.2.3  CPPED1 mRNA levels are decreased and miR371a-5p

levels increased during spontaneous delivery (II) ........................ 66 

5.2.4  miR-371a-5p and miR-520d-5p bind to the 3′UTR of

CPPED1........................................................................................ 66 

5.3  CPPED1 classification and its functional role ........................................ 67 

5.3.1  CPPED1 sequence alignment suggests a

metallophosphotase family protein (III) ....................................... 67 

5.3.2  CPPED1 dephosphorylates AKT1 in vitro ................................... 67 

5.3.3  CPPED1 interacts with multiple proteins in an in vitro

protein microarray ........................................................................ 68 

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5.3.4  CPPED1 and binding protein interactions confirmed by

co-IP and a BiFC assay ................................................................. 68 

5.3.5  CPPED1 dephosphorylation activity on the PAK4 and

PIK3R2 proteins ........................................................................... 69 

6  Discussion 71 

6.1  Proteomic changes in spontaneous term and elective term

placentas (I) ............................................................................................. 71 

6.1.1  Placenta has a unique set of proteins at the fetal and

maternal sides ............................................................................... 71 

6.1.2  CPPED1 expression levels and polymorphisms associated

with the length of gestation and spontaneous term birth .............. 72 

6.2  The expression levels of miRNAs associate with the outcome of

labor (II) .................................................................................................. 73 

6.2.1  miR-371a-5p ................................................................................. 73 

6.2.2  CPPED1 is targeted by miR-371a-5p and their expression

levels inversely associate with each other .................................... 73 

6.3  Homo sapiens CPPED1 belongs to the calcineurin-like

metallophosphoesterase superfamily (III) ............................................... 74 

6.3.1  CPPED1 mediates the dephosphorylation of AKT1 ..................... 74 

6.3.2  Interaction between CPPED1 and selected target proteins

were confirmed using co-immunoprecipitation and BiFC

experiments .................................................................................. 76 

6.4  Prospects for future studies ..................................................................... 79 

7  Conclusions 81 

References 83 

Original publications 99 

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1 Introduction

The molecular mechanisms associated with the onset and timing of human

parturition are still poorly understood (Norwitz et al., 2015). Depending on the

length of the pregnancy, the deliveries are categorized into term, postterm and

preterm births. Births before 37 weeks of gestation are premature and those taking

place at 42 weeks of pregnancy or later are defined as post term (Muglia & Katz,

2010; Schierding, O'Sullivan, Derraik, & Cutfield, 2014). At least ten percent of all

births are premature and about fifteen million premature infants are born each year.

Prematurity is the major determinant of neonatal mortality and morbidity, as these

children have multiple, mostly acquired disorders and they are at high risk of

having long term morbidities. Nearly 70% of preterm births start spontaneously,

and the rest are elective as a result of a medical condition of the mother, fetus or

the placenta. Hereditary factors, environmental exposures, infertility treatments,

behavioral and socioeconomic factors influence the risk of premature birth.

However, spontaneous onset is the most common cause of premature birth (Esplin,

2014).

Molecular mechanisms underlying the onset and progression of labor, preterm

labor and postterm delivery, are not known in detail. The genomes of the mother

and the fetus influences the length of the pregnancy and the risk of premature birth.

Understanding the pathology and biochemistry of the maternal- fetal interface of

the placenta using a genomics, transcriptomics (including miRNA) and proteomics

approach helps to elucidate the function of candidate genes, gene variants, mRNA

and proteins (Aghaeepour et al., 2018; M. Cai, Kolluru, & Ahmed, 2017).

Comparative studies on term placentas with different gestational weeks may reveal

genes and proteins in placental tissues underlying pregnancy complications.

The aim of this research was to study the biochemical mechanisms of labor and

delivery, using hypothesis free approaches of proteomics, genomics and

transcriptomics combined with microRNA (miRNA) analyses. We discovered that

CPPED1, a protein phosphatase, was downregulated in placentas from spontaneous

births. We identified a microRNA, miRNA-371a-5p, which targets CPPED1.

Further, we found that CPPED1 interacts with the PAK4 and PIK3R2 proteins,

dephosphorylating five different serines on PAK4. In conclusion, CPPED1 or

miRNA-371a-5p could be a biomarker for labor. The biological significance of

PAK4 dephosphorylation by CPPED1 remains to be studied.

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2 Review of the literature

2.1 Labor/delivery and the length of pregnancy

Labor or delivery is a physiological process that results in the expulsion of the fetus,

placenta and fetal membranes from the uterus. It occurs in various time dependent

processes including uterine contractions and cervical dilations, which cause the

rupture of the fetal membrane leading to delivery. Labor in animals involves

changes in the hormonal levels of both the maternal and fetal circulations

(endocrine events). While, in humans labor involves complex biochemical

crosstalk between the fetoplacenta and the mother (autocrine, paracrine and

endocrine events) and immune endocrine events (Kota et al., 2013). Hormones

produced by both mother and fetus (placenta) are important in maintaining the

favorable environment and sufficient nutrition for the fetus. Modulation of the

maternal immune system is crucial for the immune survival of the semi allogeneic

fetus in the mother and this is supported by steroid (progesterone, estradiol,

corticosteroids) and peptide hormones (prolactin, oxytocin, human chorionic

gonadotropin and relaxin). All of them play a major role in maternal

immunomodulation (Nair, Verma, & Singh, 2017). The heterogeneous mechanisms

of parturition are due to the genetic conflict between maternal and paternal genes

and placental hormone synthesis regulated by genomic imprints (John, 2013). Early

studies suggested that the timing of birth depends on both the development of the

placenta, and the level of corticotropin-releasing hormone (CRH) produced by

placenta (McLean et al., 1995).

Length of pregnancy and types of birth/ labor

The mean duration of human pregnancy is between 39 to 40 weeks and varies

slightly between populations. Depending on the length of the pregnancy, births are

mainly classified into term, preterm and postterm term births (Morgan & Cooper,

2020) (Fig. 1).

Preterm birth: Babies born before 37 weeks of gestation are called premature

birth or preterm birth, which is further divided into moderately preterm (32-36 wk),

very preterm (28-31 wk) and extremely preterm (< 28 wk). Children who were born

as premature are at a higher risk of health complications such as cerebral palsy and

developmental disorders than are children delivered at term and the risk increases

22

according to the degree of prematurity and the intrauterine growth retardation

(IUGR) (Lee et al., 2017). Infections are commonly associated with premature birth.

Chorioamnionitis, a common phenotype associated with spontaneous premature

birth, may often be a manifestation of an infection.

Term birth: Babies that are born in between 39+0 to 41 weeks of gestation are

termed as full term or spontaneous term births, while babies that are born between

37-39 weeks are called as early term and between 41 to 42 weeks are late term.

Elective preterm deliveries before 37 weeks of gestation take place mostly by

cesarean section (ACOG committee opinion no 579: Definition of term

pregnancy.2013). Particularly in complications such as preeclampsia or IUGR

labor may precipitate acute asphyxia. In addition, bleeding from the placenta

around the cervix (Placenta previa) is an indication for cesarean section. In

multiple pregnancies and particularly in pregnancies with severe maternal or fetal

disease, elective deliveries may be indicated. In postterm pregnancies, the infants

are born after 42 weeks of gestation (Morgan & Cooper, 2020). In many cases labor

is induced before 42 weeks of gestation and postmature mothers often undergo

elective cesarean section. Infants born post term are prone to conditions such as

IUGR, birth asphyxia and to obstructed labor.

Labor and delivery as well as elective births by cesarean section are associated with

several complications that can cause adverse effects to the newborn or the

mother.

Hence, there is a need to understand the molecular mechanisms associated with

preterm, term and postterm births

Fig. 1. The timeline of human pregnancy: A normal human pregnancy or average

childbirth lasts for 39 to 41 weeks of gestation (Full term). Preterm birth occurs before

37 weeks, while 37 to 39 are early term, 41 to 42 are late term and post mature birth

takes place after 42 weeks of gestation. (Modified from Häggström, Mikael 2014

https://en.wikipedia.org/wiki/Gestational_age).

23

2.1.1 Major complications during pregnancy and delivery

As per reports in 2017, there has been a decline in the maternal death rate by 44

percent due to pregnancy associated complications. Conversely, 830 women die

every day due to causes related to pregnancy, childbirth, injuries and infections or

disabilities (https://www.who.int/news room/fact-sheets/detail/maternal-mortality).

The center for disease control and prevention (CDC) has initiated an effort to

improve the health of both neonates and mothers and to reduce the number of

maternal mortalities.

The regulation of the pregnancy duration is still poorly understood. In most

cases the pregnancy ends at the onset of labor and subsequent delivery. Many

pregnancy complications take place during labor. Most complications are evident

when the labor is either premature or postmature. Problems can be caused either by

the mother, fetus or the placenta. A common complication is premature rupture of

the membranes before the onset of labor. Another typical complication of

pregnancy is preeclampsia, which is seen as symptoms in the mother (high blood

pressure, proteinuria). In most severe cases the mother develops convulsions due

to eclampsia.

Detachment of the placenta (abruptio) with or without labor is a serious

although rare complications causing severe fetal asphyxia. Further complications

include the abnormal position or abnormal presentation of the fetus. Multiple

pregnancies are also associated with excessive complications; one of them is

premature birth. Complications during labor include shoulder dystocia, nuchal cord

and a prolapsed umbilical cord. Amniotic fluid embolism and placenta accreta are

complications manifesting during labor and delivery. Representative complications

are listed in Table 1.

24

Table 1. Common complications arising during labor

Complication name Features/Causes Reference

Obstructed labor

Preeclampsia

Postpartum bleeding

Postpartum infections

Gestational diabetes

Neonatal infections

Perinatal asphyxia

Too small or deformed pelvis. Prolonged labor, failure of vaginal delivery High blood pressure, proteinuria and risk of seizures in pregnant women. Restriction of fetal growth

Uterine atony, uterine constriction,

retention of parts of placenta

Consequences of heavy blood loss

Puerperal fever

Common in miscarriages

High blood sugar, preeclampsia,

polycystic ovary syndrome (PCOS)

Infection obtained intranatally: Str.

agalactiae. E. coli, Listeria infections

Insufficient O2 supply during birth

Multiple organ damage, persistent brain

damage

(Neilson, Lavender, Quenby, &

Wray, 2003)

(Lambert, Brichant, Hartstein,

Bonhomme, & Dewandre, 2014

(Weeks, 2015)

(Loudon, 1998)

(McIntyre et al., 2019)

(Chan, Lee, Baqui, Tan, & Black,

2013)

(Aslam et al., 2014)

2.1.2 Biomarkers for labor and pregnancy complications

There has been a long quest to understand the outcome of pregnancy and the

associated complications. It would be helpful to develop accurate biomarkers for

the prediction of a specific disorder and eventually to provide effective treatment

and prevention of severe consequences. Screening for fetal aneuploidy in the first

trimester is a commonly used test for the prediction of chromosomal incongruity.

The development of multiparametric tests for aneuploidy using multiple

biomarkers in early pregnancy helps in the prognosis of also other pregnancy

complications such as preeclampsia, fetal growth restriction, gestational diabetes

and preterm births.

25

In fetal growth restriction (FGR), the fetus fails to grow according to the

duration of the pregnancy. FGR is detected by fetal ultrasound and confirmed at

birth. Growth restricted fetuses have a higher risk of metabolic and cardiovascular

diseases in postnatal life than do children born at term (Malhotra et al., 2019). FGR

can arise from placental maternal or fetal factors.

For instance, FGR in severe preeclapsia is associated with the poor

development of placental villae. Many biomarker tests are available for the

prediction of FGR due to placental insufficiency, which includes fetal nuchal

translucency (NT) thickness, serum pregnancy associated plasma protein-A (PAPP-

A), free beta-human chorionic gonadotropin (beta-hCG), placental growth factor

(PlGF), placental protein 13 (PP13), and A disintegrin and metalloprotease

(ADAM12) (Kane, Costa Fda, & Brennecke, 2014) . PP13, which is majorly

expressed by the syncytiotrophoblast of the placenta and released into the

circulation and its low concentrations in the first trimester turned out to be a good

biomarker for pregnancy complications. Similarly, PIGF in the third trimester was

suggested to be potential biomarker of FGR and preeclampsia. Other studies

reported beta human beta-hCG and ADAM12 as diagnostic markers of FGR (Gullai

et al., 2013; Rausch et al., 2011; Than et al., 2004).

A wide range of maternal serum analytes have been used as biomarkers for

predicting preterm birth including PAPP-A and PP13 as markers of placental

function, and Inhibin A and Activin A as proteins of placental origin, angiogenesis

agents such as PIGF and vascular endothelial growth factor (VEGF), soluble fms-

like tyrosine kinase-1 (sFIt-1) and soluble endoglobin (sEng)(Oskovi Kaplan &

Ozgu-Erdinc, 2018). None of these is satisfactory in predictive accuracy. Fetal

fibronectin (fFN) is the most extensively investigated biomarker for the prediction

of preterm labor and delivery. Usually fFN is absent in cervicovaginal fluids from

week 24 of gestation. However, the presence of fFN implies the disruption of the

choriodecidual barrier and eventually leads to an increased risk of preterm labor.

Based on several reports on the biomarkers for the prediction of labor, the outcome

is inconclusive and extensive research is needed to identify accurate analytes from

the maternal circulation, such as is serum matrix metalloproteinase-12 for the

detection of an ectopic pregnancy (Rausch et al., 2011). Currently, there are no

reported biomarkers for the onset of term labor so far.

26

2.2 Human Placenta

The human placenta is structurally very different from most mammalian placentas,

although it resembles that of other great apes (Carter & Mess, 2007; Enders &

Carter, 2012). It is discoidal in shape and at term its mean weight is 500 g, length

22 cm and thickness 2.5 cm. It connects to the fetus by the umbilical cord, which

is 55-60 cm in length, inserted into the placenta and contains two umbilical arteries

and one vein (Fig. 2). These blood vessels emanate from the placenta and are further

divided into more branches resulting in formation of villous tree structures, grouped

into lobules called cotyledons (Turco & Moffett, 2019). The human placenta is

hemo-monochorial as the maternal blood flows from the uterine spiral arteries into

the intervillous space, where the maternal blood is in direct contact with fetal

syncytiotrophoblasts that line with the intervillous fetal capillaries.

In early human pregnancy, proliferation of invading trophoblasts from the outer

layer of the blastocyst triggers specific reactions in the uterine wall that lead to the

development of the placenta. The trophoblasts lining the extraembryonic

membranes of the placenta have essential roles throughout pregnancy. Human fetal

membranes composed of an amnion and a chorion also known as the

chorioamniotic membranes are derived from the outer trophoblast layer. The

amnion is the innermost layer in contact with the amniotic fluid, fetus and umbilical

cord, connected with the chorion. The chorion separates the amnion from the

maternal decidua and uterus (Menon, Richardson, & Lappas, 2019). The decidua

is a modified mucosal endometrium layer of the uterus, formed from the maternal

part of the placenta in a process called decidualization, which is influenced by

progesterone.

27

Fig. 2. The structure of the human placenta: The human placenta has two sides. The

fetal side is also called the chorionic side and the maternal side is known as the Basal

plate. The chorion contains the umbilical arteries, the umbilical vein and the amnion.

The maternal side contains the basal plate, decidual basalis, and myometrium (Modified

from https://mybiblioteka.su/tom2/3-98667.html).

2.2.1 Placental development and functions

After 6-7 days of fertilization, the blastocyst attaches to the uterine decidual wall

and the formation of the placenta begins. The outermost layer of blastocyst cells

consists of trophoblasts. The placenta is composed of trophoblasts, decidual cells,

endothelial cells and vascular smooth muscle cells, as well as multiple

mesenchymal, interstitial and immune cells. Morphologically the placenta has three

different trophoblasts named cytotrophoblasts, syncytiotrophoblasts and

extravillous trophoblasts. The overlying layer outer to the cytotrophoblast is the

syncytiotrophoblast, formed from the fusion of undifferentiated and highly

proliferative cytotrophoblasts. Chorionic villi are the main functional units of the

placenta within which the fetal blood and the maternal blood are separated by the

trophoblast layer and the villous capillary endothelium. Extravillous trophoblasts

formed from cytotrophoblasts detach from the placenta and migrate into the

maternal decidua (Castellucci, Scheper, Scheffen, Celona, & Kaufmann, 1990;

Huppertz, 2008).

28

The decidua is the modified mucosal lining of the endometrium, formed in

pregnancy due to decidualization. The decidua contains a large population of

endometrial granular leukocytes made up of specialized uterine natural killer cells

(uNK) and dendritic cells, whereas polynuclear leukocytes and B cells are scarce.

The decidua enabled gas and nutrient exchange by allowing the invasion of

trophoblasts into the maternal sides. It also protects the fetus from the invasion of

the maternal immune system throughout the pregnancy. Abnormalities in

decidualization associate with endometriosis, which could lead to miscarriage and

premature birth (Edmondson et al., 2009).

Placental functions:

Major functions of the placenta include nutrient and oxygen uptake and the

exchange of several constituents between the developing fetus and the maternal

circulation. In addition, the placenta is also involved in the secretion of hormones

and growth factors, thermogenesis, and the excretion of metabolic products such as

CO2 and urea. It is involved in protecting the fetus from infections, xenobiotics and

maternal diseases. Along with the maternal control of uterine functions that are vital

to the pregnancy outcome. The placenta maintains interdependent signaling

between the maternal and fetal compartments. Functional changes occur in the

placenta during pregnancy to accommodate the increasing metabolic demands of

the developing fetus (Costa, 2016; Gude, Roberts, Kalionis, & King, 2004).

Metabolic and endocrine functions:

The placenta grows throughout the pregnancy and the development of blood supply

to the placenta is completed by 14th week of pregnancy. The placenta functions as

a barrier between the chorionic (Fetal side) and the basal plate/Decidua (Maternal

side) formed from both fetal and maternal tissues. Syncytiotrophoblasts are the

major cells secreting different hormones such as human chorionic gonadotropin,

progesterone, estrogens and metabolites such as placental lactogen, placental

growth hormone, leptins, adiponectin, resistin, adipokines, PAPP-A, PP-13,

inhibins and activins. All these placental hormones and metabolites are important

and play a major role in maintaining the pregnancy, fetal development and labor

(Costa, 2016)

29

Placental exosomes:

As the gestational age progresses, the turnover of villous trophoblasts increases and

generates a variable size of micro particles or extracellular vesicles called exosomes

(typically 30-150 nm in diameter). These exosomes that are released into the

circulation and are taken up by maternal immune and endothelial cells induce

changes in maternal compartments (Redman & Sargent, 2008). Placental exosomes

can be found in maternal plasma from 6 weeks of gestation and their magnitude

increases as the gestation advances as well as in pregnancy complications like

diabetes and preeclampsia (M. D. Mitchell et al., 2015). Placental exosomes

contain a variety of miRNAs encoded by chromosome 19 (C19MC), chromosome

14 (C14MC) and other placenta specific miRNAs which attenuate various cellular

processes and confer viral resistance by inducing autophagy in engulfed cells

(Delorme-Axford et al., 2013).

2.2.2 Placental genes, proteins and miRNAs

70% of the human protein coding genes are expressed in the placenta. The human

protein atlas shows that the placenta expresses 1104 placenta specific genes which

are not detected in any other tissue (www. proteinatlas. org/humanproteome/ tissue/

placenta-function) and most of them are secreted proteins, expressed in

trophoblasts and likely have pregnancy specific functions. Examples of proteins

whose expression is elevated in the placenta compared to other organs and tissues

are Paternally Expressed 10 (PEG10) and the cancer testis antigen Prostate-

Associated Gene Protein 4 (PAGE4) (Placenta has higher expression levels than

any other tissue followed by epididymis, prostate, seminal vesicles and testis)

expressed in cytotrophoblasts, Chorionic Somatomammotropin Hormone 1 (CSH1)

and the metastasis-suppressor KiSS-1 (KISS-1) expressed in syncytiotrophoblasts,

while Pregnancy-Associated Plasma proteinA2 (PAPPA2) and Proteoglycan 2

(PRG2) are expressed in extravillous trophoblasts (Uhlen et al., 2015).

Transcriptome analyses have identified that in addition to placenta specific genes

and proteins, the placenta has its own specific miRNA clusters such as the C19

miRNA cluster (C19MC) located on chromosome 19, the chromosome 14 miRNA

cluster (C14MC) and the miRNA 371-373 clusters which are exclusively expressed

in the placenta (M. Cai et al., 2017).

30

2.2.3 Placental abnormalities and pregnancy complications

Normal structural functions of the placenta are essential for the growth of the fetus

throughout the pregnancy. Severe preeclampsia, placental abruption and abnormal

cord insertion are associated with more than 50% of stillbirths (Vergani et al., 2008).

Abnormalities in the placenta results in a decrease in the levels of PAPP-A protein

and an increase in the levels of alpha-fetoprotein (AFP) aggravates the risk of

stillbirth by 50% (G. C. Smith et al., 2006). A genetic analysis demonstrated that

genes imprinting in the placenta is altered in pregnancy loss and epigenetics studies

support the observation that stillbirths and pregnancy loss can be a consequence of

confined placental mosaicism (Goodfellow, Batra, Hall, McHale, & Heazell, 2011).

Common pathological conditions of the placenta include fetal thrombotic

vasculopathy, chronic intervillositis, chorioamnionitis, funisitis, massive

perivillous fibrin deposition, villous dysmaturity, and cord lesions. Further research

is needed to obtain a better understanding of placental pathology in stillbirths and

other pregnancy complications (Rathbun & Hildebrand, 2020; Warrander &

Heazell, 2011). Furthermore, studies have identified various placental

abnormalities such as chorioangioma, circumvallate placentation, placenta

membranacea, vasa previa, placenta accreta spectrum and abnormal

cytotrophoblastic invasions as well as gestational trophoblastic disease or neoplasia.

2.3 Proteomics and pregnancy

Proteomics broadly refers to the identification and quantification of overall proteins

derived from the genome or the entire set of proteins produced by an organism or

system. The term proteomics was coined by Marc Wilkins as an analogy to the term

genomics (Wilkins et al., 1996). After genomics and transcriptomics, proteomics

has gained a lot of importance in understanding and addressing biological questions.

Cells or tissues make different sets of proteins under different cellular processes

during the cell cycle, differentiation, proliferation, development and further

complexity of the proteome arises due to a wide range of posttranslational

modifications. Proteomic strategies mainly based on protein expression, structure

and functional aspects can link dynamic changes to various physiological stimuli

or diseased conditions.

Identifying the biomarkers for the diagnosis of the onset of parturition

contributes to understanding the mechanisms of labor and other complications such

as preterm birth, preeclampsia and fetal growth restriction. These complications

31

can have adverse effects on the health of the woman and child (Law, Han, Tong, &

Baker, 2015). Currently there are no accurate predictive tests available for the

diagnosis of the onset of term labor nor for pregnancy related complications.

Advancements in mass spectrometry (MS) based proteomics such as electrospray

ionization (ESI), matrix-assisted laser desorption/ionization (MALDI)-based MS,

top-down proteomic and peptidomic profiling by laser mass spectrometry, liquid

chromatography or capillary electrophoresis coupled to MS, bottom-up

quantitative proteomics and targeted proteomics by liquid chromatography MS

have been explored to elucidate protein biomarkers and biological mechanism

underlying pregnancy-related complications and various cancers. These

methods have emerged as a powerful tools to provide breakthroughs in

understanding the pathophysiology of complex pregnancy diseases (Kolialexi,

Mavrou, Spyrou, & Tsangaris, 2008; Law et al., 2015; Shankar et al., 2005).

2.3.1 Proteomic changes in the placenta during pregnancy

The biological process of human parturition is not yet completely understood. The

human placenta secretes several hormones and peptides to maintain the intricate

balance throughout pregnancy and this balance is disturbed in gestational diseases.

Identification of these changes may eventually be used to predict the onset of labor

and placental disorders leading to FGR. For example, measuring the levels of

human chorionic gonadotropin (hCG) levels allows the detection of early

pregnancy problems and gestational trophoblastic disease (GTD) (Kharfi, Giguere,

De Grandpre, Moutquin, & Forest, 2005). Similarly, other protein biomarkers

related to PE including, VEGF, sFlt1, PlGF, sENG, PAPP-A, PP13, HSP70 and

other proteins have been shown to be used in the prediction or diagnosis of PE,

which helps in understanding the pathogenesis of PE (He, Zhou, Wei, & Li, 2020).

Lowered oxygen tension is known to affect trophoblast differentiation through

transcription factors such as hypoxia-inducible factor-1(HIF-1) and transforming

growth factor β3 (TGFβ) (Caniggia et al., 2000; Genbacev, Zhou, Ludlow, & Fisher,

1997). A reduction in oxygen tension, like in preeclampsia, leads to changes in less

than 3% of expressed proteins, mainly resulting in the downregulation of

antioxidants and the upregulation of glycolytic enzymes. A significant increase in

the level of annexin II, a protein associated with proliferation and fibrinolysis under

hypoxic conditions, was reported (Menaa et al., 1999; Rao, Denslow, & Block,

1994). Functional proteomics studies have identified annexin II, which binds to

32

placental protein 13 (PP13)/galectin 13, whose dimerization affects oxygen

changes in the placenta (Than et al., 2004).

Despite the importance of the placenta in pregnancy, very little is known about

the proteomic changes in the human placenta. Mushahary et al., investigated the

protein expression profile of the full term human placenta using a two-dimensional

gel electrophoresis Mass spectroscopy (2-D gel-MS/MS) approach and identified

117 novel proteins from 2D-gel based analysis of the placenta, which belongs to

different functional classes such as cell stress, metabolism, cytoskeletal proteins,

transport proteins and signal transduction pathways (Mushahary, Gautam,

Sundaram, & Sirdeshmukh, 2013)

There has been only a limited number of proteomic studies comparing different

gestational ages during pregnancy. Fard et al., performed comparative studies of

normal human placental proteomes between the first and third trimester, identified

the variations in the expression of 11 proteins in the first trimester when compared

with the third trimester. Among the identified proteins, four proteins including

protein disulfide isomerase, tropomyosin 4 isoform 2, enolase 1, and 78-kDa

glucose-regulated protein were upregulated, while seven proteins including actin

g1 pro-peptide, heat shock protein gp96, 1-antitrypsin, EF-hand domain family

member D1, tubulin a1, glutathione S-transferase, and vitamin D binding protein

were downregulated in the first trimester vs the third trimester (Gharesi-Fard,

Zolghadri, & Kamali-Sarvestani, 2015).

2.3.2 Human placental proteomics of preterm birth

Characterization of the proteome of ex-vivo perfused placental effluents can

provide insights into the repertoire of proteins secreted by the placenta in in vivo.

Advancement in applications of these high throughput methodologies enable the

characterization of placental and cellular proteomes, unravel functional networks

along with the identification of disease markers, and can therefore significantly

improve maternal health during pregnancy (Hanash, 2003; Marko-Varga &

Fehniger, 2004; Robinson, Ackerman, Kniss, Takizawa, & Vandre, 2008).

Protein expression levels of the fetal side are different from those of the

maternal sides of the placenta and this can be a preliminary source for the discovery

of biomarkers associated with preterm births. Niu et al., collected the samples from

placentas of both the fetal and maternal sides of spontaneous preterm labors with

intact membranes (sPTL-IM) and analyzed with two-dimensional gel-

electrophoresis (2D-GE) coupled with matrix-assisted laser desorption/ionization-

33

time of flight/time of flight (MALDI TOF/TOF) mass spectrometry. Twelve

proteins with significant differential expression levels were identified in sPTL-IM

and these changes were more prominent on the fetal sides as compared to the

maternal sides. The identified proteins are involved in the regulation of the

cytoskeleton, immune response, fetal as well as placental development and the

anticoagulation cascade. Among the identified proteins S100-A9 and Ig chain C

region were upregulated, whereas Vimentin, Calpain small subunit-1, protein PP4-

X, Ferritin light chain subunit, Septin2, Peroxire-doxin 3, Cytokeratin 8,

Cytokeratin 1, Tropomo-dulin 3, Annexin A1 were downregulated in sPTL-IM

placentas (Niu et al., 2018).

In another study by Butt et al., proteomic analysis, of human preterm labor

placental membranes vs term placental membranes identified that 11 proteins,

which varied between the two phenotypes. Among the identified proteins Keratin

type I cytoskeletal 19, Annexin A4, Endoplasmin precursor, beta and Gamma actins

ERp29 are more expressed in term placentas, whereas Vimentin, 78kDa glucose

regulated protein grp78, Lactoylglutathione lyase, Transgelin were over-expressed

in preterm cases. These differentially expressed proteins belong to

structural/cytoskeletal components, ER lumenal proteins with enzymatic or

chaperone functions, and proteins with anticoagulant properties (Butt et al., 2006).

In a recent study the placenta was found to secrete thirty-four proteins into the

maternal circulation, including placental growth factor, growth/differentiation

factor 15, and matrix metalloproteinase 12, while 341 proteins were secreted into

the fetal circulation. Only 7 proteins were found to be common for both the fetal

and maternal circulations. This study signifies a distinct directionality in placental

proteins released into the fetal and maternal compartments. Among the 34 proteins

secreted into the maternal circulation, 8 were changed significantly throughout

gestational ages. Protein profiles of these placental proteins help to identify novel

minimally invasive biomarkers for human placental function across gestation and

shed light on maternal physiology and fetal development (Michelsen, Henriksen,

Reinhold, Powell, & Jansson, 2019).

2.3.3 Proteins as biomarkers of preterm birth from various body

fluids

Proteomics hase made a great impact on modern obstetrics and aided in the

identification of biomarkers for various obstetric diseases and fetal conditions such

as oocyte maturation, spermatogenesis, fertilization, endometriosis, down's

34

syndrome, preterm birth, amniotic infections, preeclampsia, intrauterine growth

restriction and obstructive uropathies (C. S. Buhimschi, Weiner, & Buhimschi,

2006; Lekhwani, Shankar, & Vaswani, 2011). The clinical proteomics provides

multiple sources for studies including maternal blood, maternal urine, placental

tissue, amniotic fluid, cervical mucus, vaginal secretions, and saliva. Proteomics

may help in the prediction, diagnosis, management, and treatment of several

obstetric conditions, which are associated with an increased risk of

maternal/perinatal mortality and morbidity (Klein et al., 2014).

The development of new technologies and methodological approaches during the

past decade has provided a motive to search for predictive and diagnostic PTB

biomarkers and many of them have been identified in several biological fluids.

Saliva biomarkers

Saliva contains progesterone and its low concentrations during 24 – 34 weeks of

gestation in women have been linked to early preterm labor, in a study following

women with a singleton pregnancy with at least one risk factor for PTB (Lachelin

et al., 2009). Fetal fibronectin (fFN) was measured at 24 and 27 weeks of gestation

in the same cohort and no correlation was observed between fFN and salivary

progesterone. In another study Priya et al., showed the utility of salivary

progesterone in the prediction of preterm birth singletons. In this study, salivary

progesterone was measured at 24 and 28 weeks of gestation and the measurement

was repeated after 3-4 weeks. Authors found that a progesterone level below

2575 pg/mL was indicative of preterm birth with a sensitivity of 83% and 86%

specificity. Thus, estimating the salivary progesterone in high risk pregnant women

may identify those who might benefit from progesterone therapy (Priya et al., 2013).

Cervical fluid

A systematic review form the early 2000’s reported that the accuracy of fetal

fibronectin in predicting spontaneous PTB varied in different studies and it is most

accurate in predicting preterm birth in women without advanced cervical dilatation

within 7-10 days after testing (Honest, Bachmann, Gupta, Kleijnen, & Khan, 2002).

In a more recent meta-analysis, it was reported that fFN levels in singleton

gestations were not predictive for PTB (Berghella & Saccone, 2016).

IL-6 and IL-8 are chemokine family members of C-X-C chemokines acting as

chemoattractants for inflammatory cells whose levels in CVF were associated with

35

PTB within 7 days. The test is more reliable when combined with a cervical length

measurement test showing 92.8% specificity in predicting PTB. However, its low

sensitivity limits its clinical utility (Jung et al., 2016). Placental alpha

microglobulin-1 (PAMG-1), reported to be a potential biomarker, assessed by

PartoSure, was compared with fetal fibronectin, cervical length measurement and

it was reported that measuring PAMG-1 protein in vaginal secretions by the

PartoSure® test have good positive prediction values (PPV) for spontaneous

preterm delivery within 7 days and it is more accurate in predicting PTB with 80%

sensitivity and 95% specificiy. PartoSure has the greatest utility in patients with a

cervical length of 15-35 mm (Nikolova, Bayev, Nikolova, & Di Renzo, 2015).

Insulin-like growth factor binding protein-1 (IGFBP-1) was suggested to be a

potential biomarker for predicting PTB in CVF of women. Premaquick developed

a triple biomarker for native and total IGFBP-1 and IL-6 was reported as a

successful test with 87.1% sensitivity, 92.4 specificity, and 95% accuracy in

predicting PTB within 7 days. IGFBP1 when combined with cervical length is an

alternative for fFN in the PTB test (Eleje et al., 2017; Tripathi et al., 2016).

Amniotic fluid

An intra-amniotic infection is one of the major complications associated with PTB.

Gravett et al., studied the protein peptide profiles of women who delivered

prematurely with or without an intra-amniotic infection and woman who delivered

at term. Calgranulin B and an insulin-like growth factor binding protein 1 (IGFBP-

1) peptide fragment were identified as useful biomarkers for the early detection of

an intra-amniotic infection associated with preterm birth (Gravett et al., 2004).

Two heparan sulphate proteoglycans, agrin and perlecan, identified by 2D-GE

were suggested as potential biomarkers for the premature rupture of fetal

membranes (PROM), a condition that often precedes spontaneous preterm labor

and is responsible for a quarter of preterm deliveries (Fortunato & Menon, 2001;

Vuadens et al., 2003). These two proteins have been identified solely in the

amniotic fluid of women with PROM.

Investigation of the amniotic fluid biomarkers during second trimester showed

that low amniotic fluid glucose was associated with subsequent preterm delivery,

while the levels of IL-6, amniotic fluid ferritin tend to be higher in the preterm

mothers (Ozgu-Erdinc et al., 2014). In contrast to this, Kesrouani et al., did not find

any significant differences in terms of IL-6, matrix metalloproteinase-9 (MMP-9),

36

glucose, and C-reactive protein (CRP) in mid-trimester amniotic fluid (Kesrouani

et al., 2016). This discrepancy remains unexplained.

According to the studies conducted by Buhimschi et al., there are four

biomarkers, defensins 1 and 2 and calgranulins A and C, in the amniotic fluid which

can predict perinatal infections (I. A. Buhimschi, Christner, & Buhimschi, 2005).

The proteomic biomarkers of intra amniotic inflammation appear sequentially,

starting with human neutrophil defensin 2, followed by human neutrophil defensin

1, calgranulin C, and ending with calgranulin A. The presence of these four

biomarkers confirm fetal and neonatal infections and may be reliable in the

prediction of infections and preterm birth (C. S. Buhimschi et al., 2009).

Maternal serum markers

There are few reported potential biomarkers for identifying PTB and other

pregnancy related complications from maternal serum: maternal serum calponin

1 for PTB and the ratio of maternal serum alpha fetoprotein (AFP) and amniotic

fluid AFP, which was suggested as a potential predictor for intrauterine growth

restriction and preterm delivery. Maternal serum progesterone-induced blocking

factor (PIBF) and maternal salivary estriol are other reported biomarkers of PTB

(Hudic et al., 2015).

Gunko et al., performed the mass spectrometric profiling of serum in women

at 16-17 weeks of gestation to detect protein biomarkers of preterm delivery and

identified 25 proteins (13 proteins downregulated and 12 upregulated) in the sera

of mothers who delivered prematurely. The listed proteins were Transgelin-2,

SOD1, β2-Glycoprotein-1, Peroxiredoxin-2, Peroxiredoxin-3, Gelsolin, VEGF-A,

Prolactin-inducible protein, JNK2 (fragment), Placental folate transporter, E-

cadherin, Heat shock 70 kDa protein 8, HspA8, Endoplasmin, Apolipoprotein A-

IV, Insulin-like growth factor-binding protein 1, Bikunin, MMP-8, IL-6, IL-7,

Fibrinopeptide B, Transcription elongation factor S-II, Retinol-binding protein-2,

Ribosomal protein S6 kinase alpha-3, Pigment epithelium-derived factor and

Lipocalin-1. These proteins are linked to antioxidant enzymes, chaperons, the

cytoskeleton, cell adhesion, angiogenesis, proteolysis, transcription, inflammation

processes, binding and/or transportation of various ligands, and such a proteomic

imbalance as early as during the 2nd trimester eventually leads to premature delivery

(Gunko, Pogorelova, & Linde, 2016).

A number of studies have identified multiple putative biomarkers for predicting

the risk of preterm birth. However, these studies have been unable to identify

37

repeatedly a common biomarker so far due to differences in the analytical

techniques for proteins as well as in the results. This warrants for further studies

using carefully selected cohorts. The Preterm Birth International Collaborative

(PREBIC; www.prebic.org) performed a systematic review of all of the literature

published over the past four decades, on single maternal biomarker for PTB and

reported 116 different biomarkers in 217 studies (Table 2) (Menon et al., 2011).

Table 2. List of proteins identified as PTB biomarkers. Listed proteins identified from

various biological fluids were reported in two different studies (Bastek & Elovitz, 2013;

Menon et al., 2011)

Immune function and

inflammation

Activin A, CRP, Defensin, ENA-78, Ferritin, G-CSF, GM-CSF, GRO-a, I-309/

CCL1, IFN-γ, IGF-1, IGFBP-1, IL-1A, IL-1B, IL-1 RA, IL-2, IL-2R, IL4-6, IL-8, IL-

10, IL-13 IL-15-18, InhA, ILGF-BP-1, ICAM-1, IP1-/CXCL10, ITAC/CXCL11,

Leukemia inhibitory factor, Lysozyme, Macrophage inflammatory protein-1,

MCP-1, MDC/CCL22, MIP-1a/CCL3, MIP-1b/CCL4, MIP-3α/CCL20, MIP-

3β/CCL19, Neopterin, nplGFBP-1, phlGFBP-1, Placental isoferritin, Prolidase,

sCD163, Selectin, Sialidase, sICAM-1, sIL-6R-α, Soluble CD8, Soluble IL-2R,

Soluble TNFR2, sTNFR-1, sVcam-1, TARC/CCL17, TGF-β, TNF-R1, TNF-R2,

TNF-α, TREM-1

Stress ACTH, Activan, Cortisol, CRH, CRHBP, DHEA, Dopamine, Noradrenaline,

Urocortin

Extracellular matrix

degradation

SLPI, TIMP I, Elastase, Active MMP2, Collagenase, fFN, HA, MMP-1, MMP-2,

MMP-7-9, MSAFP, SHAP-HA

Fetal anomalies AFP, beta-HCG, PAPP-A

Estrogen metabolism Androstenedione, estradiol, estriol, estrogen

Uterotonin Oxytocinase, PGE2, PGF2, relaxin

Cellular metabolism Lactoferrin, retinol-binding protein, transferrin, transferrin receptor

Progesterone

metabolism

Progesterone

Apoptosis Nucleosome, soluble Fas

Growth/immune

function

Prolactin

Placental dysfunction Placental lactogen, P-LAP

Hematologic disorders Complement C3

Hemostasis/uterotonin Angiogenin

Hemostasis Procalcitonin

Some other reviews on PTB biomarkers presented similar findings, endorsing the

notion that there is no single biomarker that is able to reliably predict PTB (Conde-

Agudelo, Papageorghiou, Kennedy, & Villar, 2011; Honest, Hyde, & Khan, 2012).

38

PTB involves multiple and intricate pathways and a single biomarker may not

accurately define the risk of PTB. These findings led PREBIC to perform another

systematic review to examine the biomarkers identified by proteomic technologies

specifically through 2D electrophoresis and MALDI or surface-enhanced laser

desorption/ionization (SELDI) protein profiling. They identified 64 dysregulated

proteins and none of them was reproducible or accurately able to predict PTB either

(Kacerovsky et al., 2014).

2.4 Immune cells and inflammatory biomarkers in term and

preterm labor

The maintenance of the structural integrity of the decidual–fetal membrane–

placental unit and its gradual decline towards the end of pregnancy is crucial for

parturition. Various cytokines, growth factors and proteases are involved in the

decidual–placental interactions and decidual lymphocytes are majorly involved in

separation during parturition by rupturing the fetal membranes (Abadia-Molina,

Ruiz, King, Loke, & Olivares, 1997; Lash, 2015; Menon, 2016).

The fetus resembles a semi allogeneic graft that grows and develops within the

mother without being rejected by the maternal immune system, which depends on

the establishment of a feto-maternal tolerance (Finn, St Hill, Davis, Hipkin, &

Harvey, 1977; Guleria & Sayegh, 2007). Immune tolerance is initiated by the

presentation of paternal-fetal antigens from the seminal fluid facilitated by seminal

plasma proteins (Bromfield, 2018; Robertson & Sharkey, 2001). Fetal antigens are

processed by maternal dendritic cells and present to T cells in uterine draining

lymph-nodes. This results in the proliferation of regulatory T cells (Tregs), which

maintain peripheral tolerance to the fetus throughout the pregnancy creating

tolerance, an anti-inflammatory state or hyporesponsiveness towards paternal

antigens until late gestation (Ander, Diamond, & Coyne, 2019; Rowe, Ertelt, Xin,

& Way, 2012). Parturition is linked to feto-maternal endocrine and immune changes

in the intrauterine cavity and initiation is triggered from senescent fetal tissues via

extracellular vesicles functions in paracrine mechanism (Menon, 2019; R. Smith,

1998). Dysregulation of the homeostatic balance derived from both endocrine and

paracrine actions produces an inflammatory burden which results in the transition

of the quiescent endometrium into an active status, eventually leading to the

disruption of pregnancy. During late pregnancy, circulating maternal leukocytes

(innate and adaptive) are recruited to reproductive tissues (the cervix and

myometrium) and to the feto-maternal interface (decidual tissues) by a chemotactic

39

process, which leads to the development of a proinflammatory state, resulting in

the delivery of the fetus (Fig. 3)(Gomez-Lopez, StLouis, Lehr, Sanchez-Rodriguez,

& Arenas-Hernandez, 2014).

Fig. 3. Involvement of maternal immune cells in human term/preterm labor. 1) A stimulus

from maternal/fetal origin activates innate and adaptive immune cells. 2) At the fetal

maternal interface CXCL10, CXCL8, CCL2 and CCL5 molecules attract and recruit

immune cells. 3) Infiltrating leukocytes amplify the proinflammatory microenvironment

in labor. Infection or inflammation activates the anti-inflammatory microenvironment in

preterm birth (Modified from Gomez-Lopez et al., 2014).

Tomblom et al., have quantified the concentrations of IL-8, IL-6, monocyte

chemotactic protein-1 (MCP-1), regulated upon activation normal T cells expressed

and secreted (RANTES) and tumor necrosis factor (TNF) in four groups of

40

pregnant women including preterm labor, term labor, preterm not in labor and term

not in labor. It was found that IL-8, IL-6 and MCP-1 levels were higher in the labor

compared to the nonlabor groups, whereas there were no changes in RANTES and

TNF levels between the groups. IL-8, IL-6, MCP-1 levels in turn were even more

increased in non-infected preterm parturition from the human cervix (Tornblom et

al., 2005). Complement activation plays a role in cervical remodeling and PTB and

it was found that increased C3a and C5a deposition leads to an increase in collagen

degradation and the MMP-9 activity of macrophages, which in turn leads to PTB.

A systematic review was conducted on the pro and anti-inflammatory

biomarkers reported in intrauterine tissues (amnion, chorion, decidua, placenta and

myometrium) at term labor. Each of these tissues expresses a unique set of

biomarkers at the time of term labor, but there is a significant overlap between these

tissues. All tissues express IL-6, IL-8, IL-1β, COX-2, PGE-2, TNF, and hCAP18 at

term labor and the secretion of anti-inflammatory markers has hardly been reported

in term labor (Hadley, Richardson, Torloni, & Menon, 2018).

There is no single or combinational screening method for preterm birth with

high sensitivity and specificity. So far, measuring the length of the cervix is the

most effective method for clinical practice. In addition to this, tests for detecting

biomarkers like fFN, IGFBP-1, IL-6, and placental alpha-microglobulin-1 are in

clinical use. Ultrasound markers, in addition to the common cervical length

measurements, such as the uterocervical angle and the placental strain ratio have

been proposed. Studies on metabolomics, proteomics and microRNA profiling

have brought a new facet to this subject that will likely help in identifying women

who possess a high risk for preterm birth and developing more effective and

preventive strategies in the future.

2.5 miRNAs in labor and pregnancy

miRNAs are small noncoding RNA molecules that play an important role in the

regulation of gene expression. miRNAs are transcribed from DNA sequences into

pri-miRNA which is further processed to form mature miRNAs. The placenta has

its own specific miRNAs, which play a role in the maintenance of pregnancy and

parturition, and changes in their expression are associated with pregnancy

complications.

41

2.5.1 Biogenesis of miRNAs

The biogenesis of miRNAs occurs in canonical and noncanonical pathways.

The canonical pathway is the most predominant pathway by which the majority of

miRNAs are processed. The pri-miRNA has a cap at its 5’ end and is

polyadenylated at its 3’ end. The pri-miRNA binds to and is processed by the

microprocessor complex, which consists of a ribonuclease III called Drosha and a

RNA binding protein DiGeorge syndrome critical region 8/Pasha (DGCR-8)(Denli,

Tops, Plasterk, Ketting, & Hannon, 2004). DGCR 8 recognizes an N-6 methylated

adenine residue in the GGAC sequence and other motifs in the pri-miRNA, whereas

Drosha cleaves the RNA duplex at the base of a hairpin structure which leads to

the generation of a pre-miRNA with 2 nucleotide 3’ overhangs (Alarcon, Lee,

Goodarzi, Halberg, & Tavazoie, 2015; J. Han et al., 2004).

In non-canonical pathway, miRNA processing and maturation take place in

Drosha/DGCR8 as well as in Dicer independent pathways. Pre-miRNAs produced

in this pathway resemble dicer substrates. For example, miRNAs are produced

from the introns of mRNA during splicing and such pre-miRNAs are called

mirtrons. Another example is 7-methylguanosine (m7G) capped pre-miRNAs that

are directly transported to the cytoplasm without Drosha cleavage via exportin-1

(Fig. 4). In this process, the 3p strand is loaded into Argonaute instead of the 5p

strand, because the m7G cap prevents the 5p from loading into the RISC complex

(Xie et al., 2013).

42

Fig. 4. MicroRNA biogenesis and mechanism of action. Canonical miRNA biogenesis

generates the pri-miRNA transcript, cleaved by the microprocessor complex (Drosha

and DiGeorge Syndrome Critical Region 8 (DGCR8)) to produce the precursor-miRNA

(pre-miRNA). The pre-miRNA is exported to the cytoplasm via Exportin5/RanGTP and

processed to produce the mature miRNA duplex. Finally, either the 5p or 3p strand of

the mature miRNA duplex is loaded into the Argonaute (AGO) complex to form a miRNA-

induced silencing complex (miRISC). In the non-canonical pathways, small hairpin RNA

(shRNA) are processed and transported like in the canonical pathway and further

processed via AGO2-dependent, but Dicer-independent, cleavage. Mirtrons and 7-

methylguanine capped (m7G)-pre-miRNA are dependent on Dicer to complete their

cytoplasmic maturation. Mirtrons are exported via Exportin5/RanGTP while m7G-pre-

miRNA are exported via Exportin1. All pathways ultimately lead to a functional miRISC

complex, which acts via degrading the target mRNA or inhibiting translation initiation

(Modified from O'Brien, Hayder, Zayed, & Peng, 2018).

The directionality of the miRNA determines the name of the mature miRNA. The

5p arises from the 5’ of the pre-miRNA, whereas the 3p arises from the 3’ of the

43

pre-miRNA. Both strands of the mature miRNA are loaded into the Argonaute

(AGO) family proteins in an ATP dependent manner (Yoda et al., 2010). Generally,

the strand with 5’lower stability or with uracil preferentially loaded onto AGO is

considered the guide strand. The unloaded strand, called the passenger strand, is

unwound from the guide strand through various mechanisms and degraded (M. Ha

& Kim, 2014).

2.5.2 miRNA functions:

Significant conservation of individual miRNAs across different species suggests

their functional importance. It has been shown in several animal models that

miRNAs participate in determining cell fate, in pattern formation during embryonic

development, and in controlling cell proliferation, cell differentiation, and cell

death. Several groups of miRNAs have been found to regulate the expression of

tumor-associated genes, while others seem to hold prognostic value in predicting

the survival of cancer patients. The major functions of miRNAs are the regulation

of gene expression by binding to specific sequences at the 3’ UTR of target mRNAs

to induce translational repression by mRNA deadenylation and decapping

(Huntzinger & Izaurralde, 2011; Ipsaro & Joshua-Tor, 2015).

MicroRNA-mediated gene silencing via miRISC

The miRNA induced RISC complex (miRISC) contains a guide strand and AGO

(Kawamata & Tomari, 2010). The target specificity of miRISC depends on the

interaction of miRNA with complementary sequences on its target mRNA called

miRNA response elements (MRE). The magnitude of the complementary

sequences determines whether the silencing is achieved through AGO2 dependent

silencing of target mRNA or miRISC based mRNA decay or translational inhibition

(Jo et al., 2015). Several studies showed that the fully complementary sequences

between the miRNA and the target miRNA induces AGO2 endonuclease activity

that leads to mRNA decay, whereas the partial complementary sequences lead to

weak binding that further results in the repression of translation initiation

(Krutzfeldt et al., 2005). In animal cells, miRNA-MRE interactions are partially

complementary and central mismatches prevent AGO2 endonuclease activity. In a

majority of cases the miRNA interaction occurs at the 5’ seed region (nucleotide 2-

8) and additional pairing at the 3’ end promotes the stability and specificity of

44

miRNA and target interactions (Broughton, Lovci, Huang, Yeo, & Pasquinelli,

2016).

The formation of miRISC is a multistep process that starts with the recruitment

of GW182 proteins which provide the platform for recruitment of other proteins

such as poly(A)-deadenylase complexes PAN2-PAN3 and chemokine receptor 4-

negative on TATA (CCR4-NOT) (Jonas & Izaurralde, 2015). PAN2/3 proteins

initiate the poly-(A)-deadenylation of mRNA and CCR4-NOT completes

continuation of the process.

Translational repression

The mechanism by which miRNA represses the translation of its target mRNA is

still unclear. Petersen et al., proposed that miRNA exerts its action by inhibiting the

elongation process by promoting ribosome dissociation from mRNA. Three

different theories have been proposed to explain the mechanism of miRNA

mediated translation repression (Fig. 5) (Petersen, Bordeleau, Pelletier, & Sharp,

2006).

In the first mechanism, miRNA competes with eucaryotic initiation factor 4E

(eIF4E) to bind to the 5’ cap structure of the mRNA that leads to failure of

translation initiation. However, other studies deny this model and suggest that

GW182 or any downstream factor could compete with eIF4E (Eulalio, Huntzinger,

& Izaurralde, 2008; Mathonnet et al., 2007; Thermann & Hentze, 2007). A second

model proposed that miRISC prevents mRNA circularization that prevents

initiation. The CCR4-NOT is a multiprotein complex containing chromatin

assembly factor 1 subunit (CAF-1), CCR4 and NOT 1-5, that are involved in

translation inhibition (Parker & Song, 2004). A third mechanism is that miRNA

prevents the association of the 60S ribosomal subunit with the 40S preinitiation

complex and prevents translation (Jackson, Hellen, & Pestova, 2010). Another

proposed mechanism is that miRISC inhibits translation by promoting the

accumulation of target mRNAs in processing bodies (P bodies). As P bodies do not

contain a translation machinery, it suggests that these mRNAs are not translated

into proteins (Parker & Sheth, 2007).

45

Fig. 5. Mechanisms of miRNA-Mediated Gene Silencing. (A) MicroRNA-mediated mRNA

decay. Proteins required for this process are shown including components of the major

deadenylase complex (CAF1, CCR4, and the NOT complex). Post initiation mechanisms:

(B) Argonaute proteins recruit eIF6, which prevents the large ribosomal subunit from

assembling with the small subunit. (C) MicroRNAs trigger deadenylation and

subsequent decapping of the mRNA target. (D) Argonaute proteins prevent the

formation of the closed loop mRNA that includes deadenylation. (E) Cotranslational

protein degradation. This involves the degradation of the nascent polypeptide chain

cotranslationally. The putative protease is unknown. (F) MicroRNAs repress the

translation of target mRNAs by blocking translation elongation or by promoting the

premature dissociation of ribosomes (ribosome drop-off) (Modified from Eulalio et al.,

2008).

Methylation regulates miRNA expression:

The expression of miRNA genes is regulated by epigenetic inactivation due to

aberrant hypermethylation and characterized in many cancers (Brueckner et al.,

2007; Lujambio et al., 2007). Expression of many of the miRNA genes located at

CpG islands, are affected by methylation as shown in the case of miR-34b and miR-

34c, which are the two components of the p53 pathway that are silenced due to

46

hypermethylation of nearby CpG islands in colorectal cancer (Lodygin et al., 2008;

Toyota et al., 2008). Another possible indirect mechanism of miRNA regulation is

that methylation can affect the expression of transcription factors that control

miRNA expression (Fig. 6) (L. Han, Witmer, Casey, Valle, & Sukumar, 2007). Still,

the methylation dependent regulation of miRNA expression is unclear and a further

understanding of these mechanisms at the molecular level is needed to determine

its significance in biological processes.

Fig. 6. miRNA gene expression affected by methylation. The degree of methylation in

the up-stream sequence of miRNAs regulates miRNA gene expression;

hypermethylation represses and hypomethylation activates miRNA genes (Modified

from Y. Cai, Yu, Hu, & Yu, 2009).

2.5.3 Placental specific /Pregnancy associated miRNA-clusters

The quest of identifying the placental miRNAs started in 2005 by Bentwich et.al.,

who determined the expression of computationally predicted miRNAs by high-

throughput miRNA microarray which in turn defined placental specific clusters.

There are three distinct miRNA clusters reported in the placenta, which are the

chromosome 19 miRNA cluster (C19MC), the chromosome 14 miRNA cluster

(C14MC) and the miR371-3 miRNA cluster. It is not clear whether the miRNAs of

all three clusters are coregulated by the same cis elements (Tsai, Kao, Chen, Chen,

& Lin, 2009).

47

The chromosome 14 microRNA cluster (C14MC)

Recent studies on miRNA expression analyses have shown that many miRNAs are

specifically expressed in a group of organisms or in some single species only.

Glazov et al., stated that the origin of the C14MC precursor sequences appeared

around 100 million years ago in early mammalian ancestors, post the evolution of

Metatheria (marsupial) or Prototheria (monotreme). A detailed analysis of this

miRNA cluster described that the miRNA cluster is (also known as mir-379/mir-

656 cluster) located within the imprinted DLK-DIO3 (delta-like homolog 1 gene

and the type III iodothyronine deiodinase) region on human chromosome 14q32

(Fig.7), but on distal chromosome 12 in the mouse (Glazov, McWilliam, Barris, &

Dalrymple, 2008).

Fig. 7. Schematic representation and genomic organization of the chromosome 14

miRNA cluster (C14MC). B) C14MC in the imprinted DLK-DIO3 domain on the human

14q32 chromosomal interval. C) Highlighted miRNAs are relevant in pregnancy (Red)

reported by Morales Prieto et al. (Blue) reported by Fallen et al. (Green) Reported in both

studies. D) The expression of C14MC gradually decreases from 1st to 3rd trimester

(Modified from Morales-Prieto, Ospina-Prieto, Chaiwangyen, Schoenleben, & Markert,

2013).

48

This gene cluster domain contains three protein coding genes, Dlk1, Rtl 1 and Dio3,

inherited from the paternal chromosome and several large and small noncoding

RNA genes, inherited from the maternal chromosome (da Rocha, Edwards, Ito,

Ogata, & Ferguson-Smith, 2008).

Evaluation of gene targets for this miRNA cluster has revealed that they associate

with biological processes such as neurogenesis, embryonic development,

transcriptional regulation and RNA metabolism. C14MC is also referred to as the

Mirg cluster, the miR-379/miR-410 cluster or the miR-379/miR-656 cluster

(Bortolin-Cavaille, Dance, Weber, & Cavaille, 2009; Noguer-Dance et al., 2010).

It is one of the largest miRNA clusters and comprises 52 miRNA genes, of which

40 are organized as large clusters located within two close neighboring segments

spanning about 40 kb (Gardiner et al., 2012; Morales-Prieto et al., 2012; Seitz et

al., 2004). This cluster is mainly regulated by methylation of a distal Intergenic

Germline-derived Differentially Methylated Region (IG-DMR). Expression

analysis of healthy human tissues revealed that some members of C14MC are

predominantly expressed in the placenta and to some extent in epithelial tissues.

The expression of C14MC decreases from the first trimester to the third trimester

and their altered expression levels have been shown to be associated with various

pregnancy complications (Liang, Ridzon, Wong, & Chen, 2007).

The chromosome 19 microRNA cluster (C19MC)

C19MC is one of the largest miRNA gene clusters in humans, identified so far. It

is located in the 19q13.41 region (Fig. 8) and spans around 100 kb in length.

C19MC miRNAs are primate-specific and comprised of 46 miRNA genes

(Bentwich et al., 2005; Bortolin-Cavaille et al., 2009; Lin et al., 2010). The miRNA

members of this cluster share common seed sequences and originate from a

common ancestor of the miR-371-3 cluster which is juxtaposed with C19MC on

chromosome 19 (Zhang, Wang, & Su, 2008). Similar to C14MC, C19MC is also

localized within imprinted genes except that it is imprinted from the paternally

inherited chromosome and regulated by methylation of the promoter CpG rich

region, which is 17.6 kb upstream of C19MC (Noguer-Dance et al., 2010).

Remarkably, C19MC miRNA genes are enriched in dispersed Alu elements,

approximately 10 times more than in any other regions, denoting the co-evolution

of Alu sequences and miRNAs (Lehnert et al., 2009; Zhang et al., 2008). However,

the functions of these Alu repeat elements and their connection with miRNAs

remain unclear.

49

Fig. 8. Genomic organization of the chromosome 19 miRNA cluster. A) Chromosome 19

containing miRNA clusters C19MC (Hg19: 54 169 933–54 265 683). B) Location of C19MC

miRNAs and miR-371-3 cluster mapping at chromosome 19q13.42 located in between

the protein coding regions of ZNF761 and MYDAM and their directionality. C) List of all

C19MC miRNAs. Altered expression of different C19MC miRNAs in pregnancy

complications are highlighted in red reported by (Fallen et al., 2018). D) The expression

of C19MC increases exponentially from the 1st trimester to the 3rd trimester (Modified

from Morales-Prieto et al., 2013).

Moreover, the expression of miRNAs seems to be partly regulated by Alu elements.

The expression of C19MC is found to be mainly restricted to the reproductive

system and placenta. Its homologues are absent in mouse, rat and dogs which

strongly emphasizes that it is specific to primates only (Bentwich et al., 2005; Liang

et al., 2007; Zhang et al., 2008). Comparative genomic studies showed that 99%

conservation of chromosome 19 miRNA genes between chimpanzees and humans

and this cluster may be related to human-primate evolution and maybe important

for embryo development as well (Cao, Yang, & Rana, 2008; Tsai et al., 2009).

The expression of the paternal imprinted placenta specific C19MC is regulated by

the epigenetic modification of DNA methylation. Surprisingly, this methylated

region originates from maternal imprinting in oocytes and a large noncoding RNA

population compartmentalizes near to the C19MC transcription site but not in the

Alu-rich domain region. Interestingly, C19MC is located adjacent to another

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maternally imprinted protein coding region, the ZNF331 gene that defines a novel

large primate specific paternal imprinted chromosomal domain (Noguer-Dance et

al., 2010). The C19MC accounts for about ∼8% of all known human miRNAs (59

out of 695) and their primate specificity emphasizes its role in phenotype plasticity.

C19MC miRNAs are intron encoded and processed by DGCR8-Drosha and the

non-protein coding RNA Pol II (Bortolin-Cavaille et al., 2009).

The miR-371-3 cluster

The miR-371-3 cluster mainly comprises 3 miRNAs hsa-miR-371a-3p, hsa-miR-

372 and hsa-miR-373-3p which share the same seeding sequence “AAG UGC”. In

humans, this cluster is a homologue of miR-290-295 sharing the same seed

sequence with the mouse. Two miRNAs are synthesized from the opposite site of

the pre-microRNA (hsa-miR-371-5p and hsa-miR-373-5p), as well as hsa-miR-

371b-3p of the cluster (Houbaviy, Murray, & Sharp, 2003; Persson et al., 2011).

Surprisingly, several members of C19MC miRNAs (miR-520s), CM2C (miR-467s)

and miR-17/92 cluster(oncomiR1) have the same seed sequence AAGUGC, which

is found in the ES cell specific miR-371-3 cluster.

The miR-371-3 cluster is located on chromosome 19 within a 1050 bp region

adjacent to the C19MC cluster and is conserved in mammals like C14MC and

C19MC, and is predominantly found to be expressed in the placenta (Fig.9)

(Bentwich et al., 2005; Houbaviy et al., 2003). Interestingly, members of the miR-

371-3 cluster are highly expressed in human ESCs and their levels decrease during

development, possibly to enhance the reprogramming of fibroblasts to induced

pluripotent stem cells (Laurent et al., 2008; Subramanyam et al., 2011; Wilson et

al., 2009). This murine miR-290 cluster or the mammalian miR-371-3 cluster, also

known as embryonic stem cell-specific cell cycle regulating (ESCC-) miRNAs,

plays an essential role in cell cycle maintenance and is also involved in the

regulation of cell proliferation and apoptosis (Voorhoeve et al., 2006; Y. Wang et

al., 2008).

2.5.4 miRNAs as biomarkers of pregnancy complications

Several studies have suggested that miRNA expression changes with physiological

conditions during pregnancy and facilitates a successful pregnancy outcome,

whereas dysregulation of miRNAs contribute to disorders of pregnancy such as

preterm birth, preeclampsia and intrauterine growth restriction etc (Fig. 10).

51

Measuring alterations in placenta and uterus specific miRNAs can be used as

potential biomarkers of pregnancy complications.

miRNAs in preterm birth

Placenta-associated miRNAs have been reported to be implicated in preterm birth

and abortion. Mayor-Lynn et al., analyzed the miRNA expression profiles between

preterm and normal term placentas and identified twenty differentially expressed

miRNAs, including some well-known preeclamptic or small for gestational ages

(SGA)-associated miRNAs. The expression of miR-15b, miR-181a, miR-200C,

miR-210, miR-296-3p, miR-377, miR-483-5p, and miR-493 varied significantly

and these miRNAs target matrix metalloproteinases (MMP-1, MMP-9), a

disintegrin and metalloproteinase domains (ADAM-17, ADAM-30), tissue

inhibitor of metalloproteinase 3 (TIMP-3), suppressor of cytokine signaling 1

(SOCS1), Stanniocalcin 2 (STC2), corticotropin-releasing hormone (CRH), CRH-

binding protein (CRHBP) and endothelin-2 (EDN2). Some of these proteins have

been reported as potential protein biomarkers in pregnancy complications (Mayor-

Lynn, Toloubeydokhti, Cruz, & Chegini, 2011). The expression levels of miR-17

and 19b were downregulated in placentas of early pregnancy loss compared to

normal term placentas and these miRNAs are known to target PTEN, CREB-1,

TGFβ-1 and TGFβ-RII. Surprisingly, the PTEN mRNA was significantly up-

regulated in early pregnancy loss, while TGF-β1, CREB-1 and TGFβ-RII were not

significantly different (Ventura et al., 2013). In case of women with recurrent

spontaneous abortion (RSA) miR-133a was found to be overexpressed in placental

villi which led to the down regulation of Human leukocyte antigen G (HLA-G) at

protein levels that might be involved in fetal rejection by maternal immune cells

leading to RSA (X. Wang et al., 2012). In placenta accreta patients, miR-34a

expression was found to be reduced in trophoblast cells and this led to an increase

in the trophoblast invasion potential (Umemura et al., 2013). Recently in an miRNA

omics screen of plasma from pregnant women with preterm birth and/or cervical

shortening, nine miRNAs including hsa-let-7a-5p, hsa-miR-374a-5p, hsa-miR-

15b-5p, hsa-miR-19b-3p, hsa-miR-23a-3p, hsa-miR-93-5p, hsa-miR-150-5p, hsa-

miR-185-5p and hsa-miR-191-5p were found to be differentially expressed in PTB

(Cook et al., 2019). In another miRNA comparative study between the placentas of

term in labor, PTB and PROM pregnancies, the expression profile of microRNAs

was found to be different between PPROM and PTB, term in labor pregnancies,

and between gestational age-matched PPROM and PTB groups. When compared

52

with term in labor pregnancies, C19MC microRNAs were downregulated in

PPROM pregnancies (miR-525-5p), whereas in PTB pregnancies C19MC

microRNAs were upregulated (miR-515-5p, miR-516-5p, miR-518b, miR-518f-5p,

miR-519a, miR-519e-5p, miR-520a-5p, miR-520h, and miR-526b-5p) or showed

a trend towards upregulation (miR-519d and miR-526a). In comparison with PTB,

the PROM group showed a significant downregulation in C19MC microRNAs

(miR-516-5p, miR-517-5p, miR-518b, miR-518f-5p, miR-519a, miR-519d,

miR-519e-5p, miR-520a-5p, miR-520h, miR-525-5p, miR-526a and miR-526b-5p).

The PROM group showed a negative correlation between gestational ages while

the PTB group showed a positive correlation between gestational age and the

miRNA of placental tissues (Hromadnikova, Kotlabova, Ivankova, & Krofta, 2017).

2.5.5 Circulating placenta-derived microRNA as biomarkers

MicroRNA profiling of the human placenta has led to the identification of certain

placenta specific miRNA clusters such as C19MC, C14MC and miR-371-3 that are

highly expressed in placentas. miRNA omics data demonstrated that the expression

of C14MC is higher in the first trimester and gradually decreases towards the end

of the third trimester. Conversely, C19MC expression levels are low in the first

trimester and exponentially increase upto 100-fold towards the end of the

pregnancy based on data both from continuous trophoblast cell lines as well as from

corresponding placental tissues (Liang et al., 2007; Luo et al., 2009; Morales Prieto

& Markert, 2011). Recent studies have shown that trophoblast and stromal cells of

the villi release some of the C19MC miRNA members into the maternal circulation

in the form of exosomes. These circulating miRNAs have interesting features like

high stability, resistance towards enzymatic cleavage, pH alterations and freeze

thaw cycles (Luo et al., 2009; P. S. Mitchell et al., 2008; Z. Zhao et al., 2012). Due

to these characteristics, miRNAs have been proposed as potential blood-based

biomarkers for the detection of tumors and pregnancy-associated diseases (Cortez

& Calin, 2009; Donker et al., 2012; Fallen et al., 2018; Hromadnikova, Kotlabova,

Doucha, Dlouha, & Krofta, 2012).

Although no individual miRNA has yet been confirmed as a biomarker, as no

significant changes in miRNA expressions have been detected in the maternal

circulation between the 12th and 16th week in pregnant women who later developed

preeclampsia and pregnancies complicated with FGR (Hromadnikova et al., 2012;

Mouillet et al., 2010). Next generation sequencing technologies for miRNA

expression profiling have revealed that the expression profiles of 29 miRNAs were

53

dysregulated in the serum of PE women (Yang et al., 2011). Despite these

conflicting results, miRNAs are implicated in the pathogenesis of PE and PTB and

can serve as potential candidate biomarkers in the future.

Fig. 9. Role of placental and uterus miRNAs in pregnancy. The pregnancy process is

regulated by genetic, environmental, and physiological factors. miRNAs in the placenta

and uterus respond to changes in these factors during pregnancy. Altered expression

of miRNAs leads to pregnancy disorders (Modified from M. Cai et al., 2017) .

54

55

3 Aims of the study

The aim of this study was to determine the differential protein and miRNA

expression levels in the human placenta associated with spontaneous and elective

term labors. The specific aims were as follows:

1. To carry out a comparative proteomics study of term and elective term

pregnancies (article #1)

2. To identify miRNAs associated with the onset of labor followed by the

functional characterization of selected miRNAs (article #2)

3. To study the functional role of CPPED1 in the cellular milieu of the placenta

(article#3)

The goal of this study was to detect variations in the expression levels of proteins

and miRNAs in spontaneous and elective delivery phenotypes. A further study on

the functional roles of proteins and miRNAs and measurements of protein and

miRNA levels in pregnancy may provide biomarkers for predicting the onset of

labor.

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57

4 Materials and Methods

4.1 Molecular Cloning (III)

The protein coding region of CPPED1 was amplified by PCR using placental

cDNA as a template. The amplified product was gel excised using Gel Extraction

Kit (Invitrogen) and subjected to restriction enzyme double digestion of both vector

and insert. The ligation product was transformed into E. coli DH5α competent cells.

Positive clones were confirmed by restriction enzyme double digestion and further

by DNA sequencing. For mammalian cell expression, CPPED1 was subcloned into

BamH1, HindIII sites in the pCMV Flag 2B plasmid. Similarly, the PAK4 and

PIK3R2 genes were cloned into the pCDNA 3-Myc expression plasmid using gene

specific primers (listed in Table-3) and these constructs were used in protein-

protein interaction studies.

4.1.1 Generation of plasmid constructs for a BiFC analysis (III)

The coding regions of the CPPED1, C1qTNF5, GRB2, PAK4 and PIK3R2 genes

were subcloned and amplified as explained previously. To generate the BiFC

constructs for a fluorescence complementation analysis, we cloned CPPED1 into

the VN plasmid and its putative binding partners such as C1qTNF5, GRB2, PAK4

and PIK3R2 were cloned into VC plasmids. The cloning primers with restriction

sites for each gene are given below (Table 3).

4.2 Protein purification (III)

CPPED1 cloned into pSFOXB20, which is a constitutive expression plasmid, was

expressed in the E. coli Bl21 (DE3) strain. The culture was grown at 37oC for 7-8

hours, cells were harvested and lysed by sonication on ice for 20 mins followed by

centrifugation at 18,000 g for 15 mins and most of the protein was found to be in

soluble form after expression checking on SDS-PAGE. Initially, the protein was

purified by Ni-NTA affinity chromatography followed by ammonium sulphate

buffer exchange for hydrophobic interaction chromatography and further by gel

flltration (Detailed description in study III). The purity of the protein was analyzed

on SDS PAGE and further confirmed by western blotting using both CPPED1 and

V5 tag antibodies.

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Table 3. List of primers used in this study for cloning

Gene Name and Plasmid Sequence

CPPED1- PSF-OXB20 sense 5'- GCAT GAATTC GATG TCG GCT GCA GAG GCG GG -3'

antisense 5'- GC GGATCCTCATTT TTT CTT GAT CAA ATC CAT GAG -3'

CPPED1-pCMV-Flag 2B sense 5'- GCAT GAATTCGATG TCG GCT GCA GAG GCG GG -3'

antisense 5'- GC GGATCCTCATTTTTT CTT GAT CAA ATC CAT GAG ATC -3'

CPPED1-pBiFC-VN173 sense 5'- GAGC AAGCTTATG TCG GCT GCA GAG GCG GGG -3'

antisense 5'- GACC GTCGACTTT TTT CTT GAT CAA ATC CAT GAG ATC-3'

PAK4-pcDNA3-myc sense 5'- GGCC AAGCTTATGTTTGGGAAGAGGAAGAAGCGG -3'

antisense 5'- GGCC CTCGAGTCTGGTGCGGTTCTGGCGCATG -3'

PAK4-pBIFC-VC155

sense 5'- GAGCGAATTCTTATGTTTGGGAAGAGGAAGAAGCGG -3'

antisense 5'- GACG GGTACCTCTGGTGCGGTTCTGGCGCATG -3'

PIK3R2- pcDNA3-myc

sense 5'- GGCC GAATTCATGGCGGGCCCTGAGGGCTTCC -3'

antisense 5'- GGCC CTCGAGGCGGGCGGCAGGCGGCGGGCC-3'

PIK3R2-pBIFC-VC155

5'- GAGC GAATTCTTATGGCGGGCCCTGAGGGCTTCC -3'

5'- GGACCTCGAGCGCGGGCGGCAGGCGGCGGGCC -3'

4.3 In vitro phosphatase activity assay (III)

To check the activity of purified CPPED1 we performed a phosphatase or

dephosphorylation assay using AKT1 as a substrate. Previously, it was reported that

CPPED1 dephosphorylates AKT1 on the Ser 473 residue (D. X. Zhuo et al., 2013).

The dephosphorylation reactions were carried out as previously reported (T. Gao,

Furnari, & Newton, 2005). Briefly, 25 ng of purified CPPED1 and 30 ng of

commercially purchased His tagged AKT1 (009-001-P21; Rockland) were

incubated in reaction buffer and the phosphorylation levels of Ser-473 were

measured using a phosphoserine specific antibody.

4.4 Protein microarray (III)

The HuProtTM v3.1- Human Proteome Microarray contains a vast number of human

proteins, encoding more than 20,000 recombinant proteins that covers ~ 75% of the

annotated protein coding genome. Prior to incubation with the bait protein

59

(CPPED1), HuprotTM arrays were blocked with a buffer containing 2% BSA in

HEPES and incubated at 4o C for overnight. The array slides were then washed with

HEPES buffer and 40 µg of CPPED1 protein prepared in blocking buffer was added

to the array and incubated at room temperature for 2 h with gentle shaking, followed

by incubation with a fluorophore conjugated Anti V5 antibody (1 µg/ml in blocking

buffer). Finally, the arrays were scanned on a Tecan LS400 microarray scanner at

532 nm excitation for the detection of GST staining of all proteins and at 633 nm

for the detection of interactions of the sample with a resolution of 10 µm, and the

fluorescence data was collected.

4.5 Immunoprecipitation of CPPED1 interacting partners and

western blotting (III)

HEK 293T cells were transfected with CPPED1-pCMV Flag 2B and PAK4,

PIK3R2 in pc-DNA 3Myc plasmids and 24 hours of post transfection cells were

lysed in IP lysis buffer supplemented with protease inhibitors (Roche diagnostics)

and 10 mM PMSF on ice for 1 hour with intermittent mixing using a vortex, and

the supernatant was separated after centrifugation at 18,000 g at 4oC. Prior to co-

IP, both anti-c-Myc agarose and control agarose resins were blocked with 1% BSA

at 4°C for 1 h to prevent nonspecific binding. 500 µg of protein samples subjected

to immunoprecipitation using1 µg of an anti c-Myc Ab (sc-40, Santa Cruz) to

immunoprecipitate Myc tagged (PAK4, and PIK3R2) proteins.

After immunoprecipitation, the samples were separated by SDS-PAGE.

Proteins were transferred onto a nitrocellulose membrane (Thermo Scientific). The

membrane was washed with 1x PBS and incubated with blocking buffer (1x PBS,

3% BSA and 0.2% Tween 20) for 1 hour at room temperature followed by

incubation with primary antibodies against CPPED1, PAK4 and PIK3R2 (Detailed

description about dilutions in study III) followed by incubations with secondary

antibodies (Described in study III). Finally, the blots were washed with 1x PBS and

scanned in the visible region to detect the protein bands with an Odyssey Infrared

Imaging System (LI-COR Biosciences).

4.6 Bimolecular fluorescence complementation assay (III)

HEK293T cells (50,000 cells) were seeded per semicircle in a four semicircle 10

mm petri dish coated with a round bottom glass slip (CELL view Cell Culture Dish

(35mm; Greiner Bio-One) and transfected when the cell confluency was 50% with

60

corresponding plasmids in combination or single plasmids and controls (Described

in study III). CPPED1 cloned into pEGFPN1 was used as a control to check the

transfection efficiency, which was found to be 90% in HEK 293T cells. After 18

hours of transfection, cells were fixed with a 4% formaldehyde solution in 1X PBS

for 20 mins at room temperature and washed three times with 1X PBS. After

fixation, cells were observed under an Olympus FluoView FV1000 confocal

microscope and images were acquired using a 20x objective and higher resolution

images for cellular localization studies were acquired using a 60x objective with

oil immersion.

4.7 Phosphatase assay detection by mass spectrometry (III)

The in vitro dephosphorylation reactions of CPPED1 performed with PAK4 and

PIK3R2 were done as described earlier with AKT1 and the detection of changes in

phosphorylation in PAK4 and PIK3R2 was done by mass spectrometry. In the

reaction mixture (50 µl), the final concentration of CPPED1 and PAK4 (TP302302;

Origene) and PIK3R2 (009-001-S95S; Rockland) were 0.13 µM, 0.16µM and 0.15

µM, respectively. The sample processing and preparation are described further in

study III.

4.8 Placental miRNA isolation (II)

For miRNAomics we included the placental tissues from the basal plate collected

after either spontaneous term or elective caesarean term birth (n=6 per each group).

miRNAs were isolated with the NucleoSpin miRNA kit (Macherey-Nagel) and

their quality was analyzed with the Advanced Analytical Fragment Analyzer.

miRNA library preparation and HiSeq 2500 sequencing were done at the Finnish

Functional Genomics Centre, Turku, Finland. While filtering for the differential

expression of miRNAs, a fold change of 1.5 and a p-value of 0.05 were set as

threshold values.

4.9 Quantitative analysis of CPPED1 mRNA by qPCR (II)

For a qRT-PCR analysis, we included samples from both the chorionic plate and

the basal plate of placentas from full term, spontaneous preterm, elective term and

elective preterm pregnancies (further details described in study II). mRNA was

isolated using the RNeasy Micro Kit (Qiagen) and cDNA synthesis was done using

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the Transcriptor First Strand cDNA Synthesis Kit (Roche). qPCR was done as an

intron-spanning assay with a LightCycler®96 instrument (Roche). Cytochrome C1

(CYC1) mRNA was used as a reference gene. All samples were measured in

triplicate and normalized in accordance with the ΔΔCt method. Statistical analyses

were conducted with SPSS Statistics 20.0 (IBM Corporation). Significant

differences in expression levels were identified by the nonparametric Mann–

Whitney U test.

4.10 Quantitative PCR of hsa-miR-371a-5p (II)

After miRNA isolation and quality assessment, the first strand cDNA synthesis was

done with the miRCURY LNA RT Kit (Qiagen). The qPCR was done using the

miRCURY LNA miRNA PCR Assay kit (Qiagen) with a LightCycler®96

instrument (Roche). The hsa-miR-103a-3p mRNA was used as a reference gene.

All samples were measured in triplicate and normalized in accordance with the

ΔΔCt method. The statistical analysis was done with SPSS Statistics 20.0 (IBM

Corporation). Significant differences in expression levels were identified by the

nonparametric Mann–Whitney U test (Detailed description in study II).

4.11 Luciferase assay (II)

HEK-293T cells were plated into a 96-well plate and allowed to reach

approximately 90% confluence. Cells were co-transfected with 100 ng of plasmid

DNA (WT-pmirGLO or Mut-pmirGLO) with Lipofectamine3000 (Invitrogen Life

Technologies) and 10 nM miRNA (mimic or negative control) was used per well.

Cells were lysed 24 hours post-transfection, and luciferase reporter assays were

performed with the Promega Dual Luciferase Reporter Assay System (Promega)

according to the manufacturer’s instructions. The Firefly and Renilla luciferase

activities were measured, and the background signal was subtracted for each

transfection. Firefly (experimental reporter) and Renilla (control reporter)

luciferase ratios were calculated and compared to negative controls (Described in

study III).

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4.12 siRNA knockdown of CPPED1 and transcriptomics of siRNA

samples (I)

For the silencing of CPPED1 we used HTR8/SVneo cells (ATCC, CRL‐3271),

which were transfected with 30 nM of a negative control siRNA or 100 nM of

pooled CPPED1 siRNAs (siRNA pairs 1–3) (Ambion) in suspensions using the

Lipofectamine 3000 transfection protocol and incubated. After 24 hours of

incubation, adherent cells were re‐transfected with siRNA pairs 1–3 or siRNA

negative controls with the same concentration as the transfection performed in

suspension. Cells were harvested 48 hours after the second transfection and RNA

was isolated using the Qiagen RNA isolation kit. RNA quality was assessed by an

Agilent 2100 Bioanalyzer system at the Biocentre Oulu Sequencing facility,

Finland. The transcriptomes of the CPPED1 silenced cells and negative control

cells were sequenced using the Illumina HiSeq high‐throughput sequencing system

(Described in study I).

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5 Results

5.1 Proteomic changes associated with spontaneous term

deliveries (I)

Proteomic studies of placentas from full term and elective term placentas were used

to identify differential protein expression patterns. After a mass spectrometry

analysis, we found 10 proteins, whose expression levels varied in spontaneous term

placentas compared to elective placentas (Figure 1, Table 1 in original article I).

Among the ten proteins, four proteins, actin cytoplasmic 1 (ACTB), β‐2‐

microglobulin, keratin type II cytoskeletal 8, keratin type II cytoskeletal 19 were

upregulated, whereas six proteins, α‐2‐macroglobulin, CPPED1, cytochrome b5,

haemoglobin subunit γ‐2, peroxiredoxin‐2 and plasminogen activator inhibitor 2

were downregulated in spontaneous term placentas compared with elective term

placentas. We confirmed the proteomics results for CPPED1 using western blotting

and observed a corresponding decrease in the protein level (Figure 1 in original

article I).

5.1.1 Placental differential protein expression between chorionic and

basal plates

A proteomics analysis of placentas from either spontaneous term or elective term

deliveries revealed that totally 19 proteins were differentially expressed in the

chorionic plate and basal plate regions of the same placenta. Out of 19 proteins,

eleven proteins, ACTB, annexin A3 (ANXA3), annexin A5 (ANXA5), serum

amyloid P‐component (APCS), clusterin (CLU), fibrinogen β (FGB), fibrinogen γ

(FGG), gelsolin (GSN), lumican (LUM), tropomyosin α‐1 chain (TPM1) and

tropomyosin β chain (TPM2) had higher expression levels in the chorionic plate,

whereas eight proteins, elongation factor 2 (EEF2), ferritin light chain (FTL), 3‐

hydroxyisobutyrate dehydrogenase (HIBADH), heterogeneous nuclear

ribonucleoprotein K (HNRNPK), estradiol 17‐β‐dehydrogenase 1 (HSD17B1),

lamin‐B1 (LMNB1), vacuolar protein sorting‐associated protein 35 (VPS35) and

tryptophan‐tRNA ligase (WARS) were present at lower levels in the chorionic plate

compared to the basal plate (Figure 2, Table 1 in original article I).

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5.1.2 CPPED1 expression levels and its polymorphism associated

with gestational age

We investigated whether SNPs within 20 kb of the genes encoding for

ACTB, A2M, B2M, CPPED1, CYB5A, HBG2, KRT8, KRT19, PRDX2 and SERPI

NB2 associated with the length of gestation. Of all the analyzed SNPs only two

alleles rs11643593 (A) and major allele of rs8048866 (A) (Table S4 and S5 in

article I) located near the CPPED1 gene associated with the length of gestation,

indicating that CPPED1 may be involved in the initiation of labor.

Next, we analyzed whether changes in CPPED1 protein levels are due to

altered mRNA levels. To this end, mRNAs were isolated from the placentas of

spontaneous term (N=6) and elective term pregnancies (N=6) at both basal plate

and chorionic plate regions and revealed that CPPED1 mRNA levels were

significantly lower in spontaneous term placentas compared with elective term

placentas ((P < 0.05)) (Figure 4 in article I).

5.1.3 AKT1 phosphorylation levels are unaffected by variations in

CPPED1 expression levels

Previously it has been shown that CPPED1 dephosphorylates Ser473 of AKT1 in

bladder cancer and supresses tumor growth (D. X. Zhuo et al., 2013). AKT1 has

been shown to phosphorylate its downstream targets, such as forkhead box protein

1, (FOXO1) also known as forkhead homologue in rhabdomyosarcoma (FKHR),

and together with progesterone receptor B acts as a transcriptional complex that

regulates the transcription of certain decidual genes (Takano et al., 2007; Vasquez

et al., 2015). We investigated whether lower CPPED1 levels lead to an increase in

p-AKT and FOXO1 phosphorylation levels in spontaneous term placentas and

found that the phosphorylation levels of both AKT1 and FOXO1 (Figure 5 in article

I) were unaffected despite the variations in CPPED1 expression levels.

5.1.4 CPPED1 silencing in HTR-8 trophoblasts and mRNA

transcriptomics (I)

To investigate the functional aspect of CPPED1 in the placenta, we silenced

CPPED1 in HTR-8 trophoblasts, a continuous cell line (ATCC) derived from first

trimester placentas with small interfering RNAs (siRNA) (Figure S2 in article I).

When gene expression levels were analyzed using RNA sequencing, 147 genes

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were either up (n = 77) or down (n = 70) regulated by CPPED1 silencing compared

to control cells (Table S6 & S7 article I). When the differentially expressed genes

were grouped according to their Gene ontology (GO) biological processes and

molecular functions, the inflammation, blood vessel and vasculature development

related pathways were among the most affected ones (Table S8 in article I).

5.2 miRNA transcriptomics of spontaneous term birth (II)

miRNAs are among the most important regulators of gene expression and their

deviant expression levels associate with various diseases and pathological

conditions. Several studies proposed that altered expression of the miRNAome in

maternal circulation or in placental tissue has a role in pregnancy maintenance and

gestational diseases such as preterm birth. To identify the differential miRNA

expression profiles between spontaneous term and elective term placentas, we

performed comparative studies using miRNAomics.

5.2.1 miRNA transcriptomics identifies differential miRNA

expression in spontaneous and elective term placentas (II)

To determine the variations in the levels of miRNA expression between

spontaneous term labor and elective term labor placentas, we used miRNAomics

of human placentas from spontaneous deliveries (n = 6) and elective births (n = 6).

miRNAomics revealed that 23 miRNAs were upregulated, whereas 31 miRNAs

were downregulated in spontaneous term labor compared to elective term placentas

(Table 1 in original article II). Among the identified differentially expressed

miRNAs, some belonged to placental specific clusters, in which miR-323b-3p is a

member of the C14MC cluster, while miR-371a-5p, miR-371b-3p, miR-372-3p,

miR-372-5p and miR-373-3p are the members of the miR-371-3 cluster, located

adjacent to the chromosome 19 miRNA cluster. The miRNA genes of the rest of the

differentially regulated miRNAs are in different chromosomes.

5.2.2 Comparison of human placental proteomics and miRNAomics

identifies a miR-371a-5 -CPPED1 pair (I, II)

In miRNAomics, we found 54 miRNAs which were differentially expressed

between placentas from elective and spontaneous deliveries. Further, we

investigated whether there was any overlap between the proteomics and predicted

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targets of miRNAs and identified three miRNA–protein target matches in the basal

plate of the placenta: miR-371a-5p–CPPED1, miR-3614-3p–ACTB and miR-

6872-3p–ACTB. We found that in spontaneous term deliveries, miR-371a-5p was

upregulated, whereas its target CPPED1 was downregulated indicating a potential

association between CPPED1 and miR-371a-5p.

5.2.3 CPPED1 mRNA levels are decreased and miR371a-5p levels

increased during spontaneous delivery (II)

In our comparative proteomic analysis and mRNA quantification analysis, we

found a decrease in the expression of the CPPED1 protein and mRNA levels in

both the basal plate and chorionic plates of the placenta from spontaneous term

labor. Further, we continued our study to measure CPPED1 mRNA levels by a

quantitative PCR (qPCR) analysis in spontaneous (n = 20) and elective caesarean

(n = 33) and preterm delivered placentas from both the chorionic and basal plate

regions. We found that CPPED1 mRNA levels decreased (1.5-fold, p< 0.001) in

the chorionic plate of the placenta at spontaneous preterm birth, but we did not

observe the same in the basal plate (Figure 1 in article II). Using miRNAomics we

observed that miR-371a-5p levels increased by 1.5-fold (p =0.04) in the basal plate

of spontaneous term placentas (Figure 2 in article II) compared to those of preterm

deliveries. The inversely correlated expression levels of miR-371a-5p and CPPED1

suggests that miR-371a-5p might regulate the levels of CPPED1 post-

transcriptionally.

5.2.4 miR-371a-5p and miR-520d-5p bind to the 3′UTR of CPPED1

In order to validate the binding of miR-371a-5p to the 3′UTR of the CPPED1

mRNA, we used a dual luciferase reporter gene assay to study the post-

transcriptional regulation of target mRNAs in intact cells. In addition to the miR-

371a-5p of miR-371-3 cluster, we also analyzed two other miRNAs of the C19MC

cluster, miR-520d-5p and miR-524-5p for their binding to the UTR and

posttranslational regulation of CPPED1 (Figure 3 in article II).

Both whole (WT) and truncated (lacking the terminal 33 bps, Mut) 3′UTRs of

CPPED1 mRNAs were cloned into the pmiRGLO vector and transfected into HEK-

293T continuous cell line. The mutant construct lacked the seed regions of both

miR-520d-5p and miR-524-5p, while both constructs contained the seed regions

for miR-371a-5p binding. The luciferase reporter assay revealed a significant

67

decrease (p = 0.004) in the relative luciferase activity of cells co-transfected with

the WT construct and the miR-371a-5p mimic (WT/miR-371a-5p) (n = 6)

compared to those transfected with the WT construct and the miRNA mimic

negative control (WT/miR-neg) (n = 6). We observed a similar decrease when the

cells were co-transfected with the WT construct and the miR-520d-5p mimic

(WT/miR-520d-5p) (n = 6, p = 0.004) when compared to WT/miR-neg (n = 6).

Altogether, these results indicated that miR-371a-5p of the miR-371-3 cluster and

miR-520d-5p of the C19MC cluster negatively regulates CPPED1 expression

(Figure 3 in article II).

5.3 CPPED1 classification and its functional role

5.3.1 CPPED1 sequence alignment suggests a metallophosphotase

family protein (III)

Zhuo et al., reported that based on a sequence analysis, CPPED1 contains a PP2A

catalytic domain from amino acids 50 to 250. According to the classification of the

protein phosphatase superfamily, members of the PP2A protein family do not

require any metal ions for their catalytic functions. CPPED1 phosphatase activity

was abrogated by trifluoroperazine, which is a PP2B specific inhibitor that does not

affect PP2A members (D. X. Zhuo et al., 2013), indicating a possible error in

classification, and prompting us to investigate CPPED1 further. Based on previous

reports, we searched for homology sequences using NPS Blast, and showed that

purple acid phosphatases from plants and phosphoesterases from Mycobacterium

and enterobacterial members are the closest homologues of CPPED1 (Figure 1 and

2 in article III).

5.3.2 CPPED1 dephosphorylates AKT1 in vitro

Previously, Zhang et al. reported that CPPED1 dephosphorylates AKT1 at the Ser-

473 residue and prevents bladder cancer progression in a mouse model (D. X. Zhuo

et al., 2013). In contrast to this finding, in our studies p-AKT of Ser-473 levels

remained unchanged, irrespective of CPPED1 expression levels in different term

placentas (Haapalainen et al., 2018). Using recombinant CPPED1, we performed

an in vitro phosphatase assay essentially as described by Gao et al., to confirm

whether CPPED1 dephosphorylates AKT1 (T. Gao et al., 2005). In this assay, we

68

incubated 30 ng of AKT1 and 25 ng of CPPED1 in Tris-HCl pH 7.5 buffer

containing 5 mM Mn2+ according to the protocol and another set without metal ions.

We observed that CPPED1 dephosphorylates AKT1 in the presence of both Mn2+

(maximum activity) and Ca2+ (moderately active) but no effect was observed in the

presence of Mg2+ (Figure 3 in article III).

5.3.3 CPPED1 interacts with multiple proteins in an in vitro protein

microarray

HuProt protein microarray has vast number of applications which includes

autoantibody-screening, protein-protein interactions, protein posttranslational

modifications and analyzing the modulation of protein expression levels in health

and diseased conditions (Huang et al., 2012). To identify proteins, which might

interact with CPPED1, recombinant purified CPPED1 with both V5 and His tags

were sent to a protein microarray core facility at Cambridge, the UK. After analysis

of protein-protein interaction studies, we obtained a list of 36 proteins (Table 1 in

article III), which interact with CPPED1 in vitro. A further pathway analysis was

done using DAVID functional annotation studies of all proteins to identify the

biological pathways affected by these proteins. Positive signal transduction

pathways and insulin pathways (PI3K/AKT pathways) were enriched in the list

(Table 2 in Article III).

5.3.4 CPPED1 and binding protein interactions confirmed by co-IP

and a BiFC assay

After the pathway analysis, we chose PAK4 and PIK3R2 for further studies. In

order to confirm the interaction between CPPED1 and binding partners, we

performed a co-IP of PAK4 and PIK3R2 with CPPED1. HEK 293T cells were co-

transfected with pCMVFlag Tag-CPPED1 and c-Myc tagged interacting partners.

CPPED1 and PAK4, PIK3R2 co-transfected cell lysates were pulled down with the

c-Myc antibody and probed with the anti-CPPED1 antibody (Figure 4 in article III).

We further used a BiFC assay to confirm the interaction between CPPED1 and

its putative binding partners the PAK4 and PIK3R2 proteins. We observed that cells

transfected with CPPED1 and PIK3R2 showed a fluorescence signal in the cytosol

with high intensity spots at the cell membrane, whereas PAK4 and CPPED1 over-

expressing cells gave the strongest signal in the cytoplasm (Figure 5 in article III).

Previously, it has been reported that phosphorylation of PAK4 is essential for its

69

nuclear translocation and functions (Li et al., 2012). Our studies confirmed the

interaction of CPPED1 with PAK4, however the functional importance of this

interaction remains to be elucidated.

5.3.5 CPPED1 dephosphorylation activity on the PAK4 and PIK3R2

proteins

After corroborating the interactions of CPPED1 with PAK and PIK3R2, we further

investigated the effect of CPPED1 on the phosphorylation status of PAK4 and

PIK3R2 proteins by an in vitro phosphatase activity assay. After the activity assay,

we analyzed the total phosphorylation levels on both the protein peptides identified

after a mass spectrometry analysis (Figure S7 in article III). It was found that

CPPED1 dephosphorylates five different serine residues in PAK4 including Ser 104,

Ser 167, Ser 173, Ser 174, Ser 181 and Ser 195 amino acids (Figure 6 in article III).

Out of the five, three have not been reported so far and the importance of these

phosphorylation sites in the functional regulation of PAK4 is not known. In contrast,

we did not observe any changes in the phosphorylation levels of the PIK3R2 protein,

which is mainly regulated by tyrosine-based phosphorylation/dephosphorylations

mediated by phosphotyrosine kinases/phosphatases.

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71

6 Discussion

The molecular mechanisms associated with the onset of human parturition are

incompletely understood. We carried out proteomic and miRNAomic analyses of

human placentas, comparing samples from spontaneous term and elective term

labors to identify the proteins and miRNAs involved in the onset of labor.

6.1 Proteomic changes in spontaneous term and elective term

placentas (I)

We compared the human placental proteomes after spontaneous and elective term

delivery and detected statistically significant differences in the levels of ten proteins

between spontaneous and elective term deliveries. The quantitative changes were

significant in either the basal or the chorionic plates or in both. When we performed

a pathway analysis, we found that the majority of the GO terms were linked to

either the immune system or to structural/cytoskeletal organization. Structural

molecule activity (ACTB, KRT8 and KRT19) represented the major molecular

function. In addition, A2M and SERPINB2 were in the same blood coagulation

pathway. CPPED1 was not associated with any of the enriched GO terms. As a

phosphatase, CPPED1 is supposed to be involved in blocking cell cycle progression,

promoting apoptosis and affecting glucose metabolism (Vaittinen et al., 2013; D.

X. Zhuo et al., 2013). In addition, among the genes encoding the ten proteins whose

expression differed in spontaneous and elective births, genetic variants of the

CPPED1 gene associated with the duration of term pregnancies.

6.1.1 Placenta has a unique set of proteins at the fetal and maternal

sides

There is only a limited number of studies on the differential mRNA and protein

expression in fetal (chorionic) and maternal tissues (Decidua basalis). Two

independent studies found oxytocin as well as oxytocin receptor (OTR) levels to

be higher in the decidua and the fetal regions, in proximity of the decidua compared

to distal regions of the fetus (Chibbar, Miller, & Mitchell, 1993; Takemura et al.,

1994). Another study, which involved in identifying the low molecular weight

proteins and peptides between the chorionic and basal plates of the same placenta

reported 16 species with significant differences between the two regions (Kedia,

Nichols, Thulin, & Graves, 2015). When we compared the proteomes of the

72

chorionic and basal plates, we identified 19 proteins differentially expressed

between the foetal (chorionic plate) and the maternal (basal plate) sides of placenta.

When we compared the list of proteins from our studies with the proteins which

were reported in different studies and listed in a systematic review by Kacerovsky

et al., we found several proteins in common such as ACTB, keratin type I,

cytoskeletal 19, SERPINB3, annexin A3, annexin A5, and isoform 4 of

tropomyosin a-1 chain.

6.1.2 CPPED1 expression levels and polymorphisms associated with

the length of gestation and spontaneous term birth

CPPED1 is a phosphoprotein phosphatase encoded on chromosome 16p13.12. This

protein has 3 isoforms. Isoform 1 is a full-length protein comprising 314 amino

acids, while variant 2 is shorter form lacking amino acid residues 97-238 and

variant 3 varies in its sequence from isoform 1 at residues 97-122 and lacks amino

acids 123-314. The importance of these variants is not known yet. A qPCR analysis

of CPPED1 showed lower expression in spontaneous term labor compared to

elective term placentas and two of its SNPs were shown to strongly correlate with

the length of gestation. These notions emphasize its role in the onset of labor.

Previous studies have shown that CPPED1 dephosphorylates AKT1 on Ser473 and

inhibits AKT1 activation. However, in our studies p-AKT levels remain unchanged

despite varying CPPED1 expression levels. It is known that AKT1 phosphorylates

FOXO1 and FOXO3 and inhibits FOXO transcription factor mediated gene

expression by recruiting FOXO from the nucleus to the cytoplasm. FOXO interacts

with the progesterone receptor and this complex binds to DNA and activates

decidual genes, which play a major role in implantation. In addition to p-AKT

levels we did not observe any changes in FOXO1 and FOXO3 and their

phosphorylation levels. Interestingly, siRNA knockdown of CPPED1 in HTR-8

cells leads to the upregulation of the PI3K pathway negative regulatory gene

expression levels such as phosphoinositide-3-kinase interacting protein 1 (PIK3IP1) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG) indicating that CPPED1 might be involved in the

regulation of the PI3K/AKT pathway via other proteins rather than AKT1. Hence,

further studies are needed to understand the cellular functions and mechanism of

CPPED1 action in regulating the PI3K pathways in detail.

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6.2 The expression levels of miRNAs associate with the outcome

of labor (II)

In this study we compared the miRNA expression levels between term placentas

from spontaneous births and elective births without labor. We were able to identify

a total of 53 different miRNA expression levels when comparing spontaneous and

elective births. Further, we compared our miRNAomics data with proteomics data

to find out whether these miRNAs target any of the reported protein candidates.

Indeed, using qpCR we found that miRNA371a-5p targets CPPED1 and its levels

were upregulated in spontaneous term placentas vs electively delivered placentas.

6.2.1 miR-371a-5p

The regulation of miR-371-5p is an epigenetic process which is dependent on

methylation and histone deacetylation mechanisms. The role of miR371-3 in the

placenta and during pregnancy was not known and further studies were needed to

understand its role in the placenta. Zhao et al., found that miR-371a-5p levels are

upregulated in gestational trophoblastic neoplasia (GTN) as compared to complete

hydatidiform moles and the elevated levels are associated with enhanced

proliferation, differentiation and invasion in choriocarcinoma cells. This dentotes

that miR371-3 may serve as a diagnostic marker for GTN (J. R. Zhao et al., 2018).

Other pregnancy disorders, in which miR-371-5p is involved, are intrahepatic

cholestasis, where patients have elevated levels of miR-371-5p in their serum (Zou

et al., 2018). Recurrent pregnancy loss is a condition in which apoptosis dominates

trophoblast growth which results in an adverse pregnancy outcome. The expression

of miR-371-5p and its target gene X-linked inhibitor of apoptosis protein (XIAP)

are greatly reduced, which is associated with increased apoptosis which further

leads to pregnancy loss (Du et al., 2019). In our studies we discovered that the

levels of miR-371a-5p are higher in term placentas from spontaneous deliveries

compared to elective term placentas.

6.2.2 CPPED1 is targeted by miR-371a-5p and their expression levels

inversely associate with each other

In our previous studies we have shown that the expression level of CPPED1

associates with the timing of birth. In addition, spontaneous term placentas have

decreased levels of CPPED1 compared to elective term placentas. Our

74

miRNAomics data showed that miRNA-371a-5p levels are upregulated in term

placentas. Furthermore, we showed that miRNA-371a-5p targets CPPED1 in vitro

assay. Interestingly, we did not observe any changes in the levels of CPPED1 in

preterm delivered placentas compared to spontaneous term placentas. Quantifying

CPPED1, along with miR-371a-5p may reveal an association, which indicates the

length of pregnancy or the onset of labor.

6.3 Homo sapiens CPPED1 belongs to the calcineurin-like

metallophosphoesterase superfamily (III)

Previously, CPPED1 was categorized as a protein phosphatase 2B family member

(D. X. Zhuo et al., 2013). Our homology search using the NPS Blast tool suggested

that CPPED1 belongs to the metallophosphatase (PPM) superfamily. However,

CPPED1 lacks MPPs signature sequence motifs. Metalloproteinphoshatases

contain two important signature sequence motifs, GDCDN-RTWGD and

DXH(X)nGDXX D(X)nGNHD/E, which are found in several PPM family

members. CPPED1 does not have these motifs except for the presence of GNHD

residues, which may aid in metal ion binding and catalysis. The lack of signature

motifs indicates that CPPED1 does not belong to MPPs despite the metal binding.

In another study, Guasch et al., performed a comparative analysis of amino

acid residues of the catalytic domain containing FSAPNYxxxxxNx from

calcineurin A with other PPP family members, identified that this motif is highly

conserved in all protein phosphatase family members including

metalloproteinphosphatases, while CPPED1 does not have this sequence,

indicating its divergence from other PPPs members (Guasch et al., 2015).

Further we have noticed that CPPED1 has many similar features to that of class

III cyclic nucleotide phosphodiesterases (also called Calcineurin-like

metallophosphoesterases, MPEs) and we conclude that CPPED1 may belong to

metallophosphoesterases (Matange, 2015) .

6.3.1 CPPED1 mediates the dephosphorylation of AKT1

In our studies we found that AKT1 levels were not affected by variations in

CPPED1 expression levels in spontaneous and elective term placentas and AKT1

was the only protein shown to be dephosphorylated by CPPED1. In order to

confirm whether CPPED1 can dephosphorylate and to assure the purified

recombinant CPPED1 was functionally active, we performed an in vitro

75

phosphatase assay with AKT1 as substrate. AKT is a member of the AGC family

of protein kinases and was discovered in 1977 as the oncogene in the transforming

retrovirus AKT8. AKT1 is also known as protein kinase B (PKB), a serine

threonine specific protein kinase which plays a major role in many cellular

processes such as cell proliferation, cell migration, glucose metabolism,

transcription and apoptosis. AKT1 is a pivotal downstream component of the

phosphatidylinositol 3-kinase (PI3K) pathway and it regulates the balance between

cell survival and apoptosis (Scheid & Woodgett, 2001).

AKT Activation

AKT resides in the cytoplasm in its inactive form until the cell is activated by an

extracellular stimulus at which point AKT translocates to the plasma membrane.

The pleckstrin homology (PH) domain of AKT has high affinity for the secondary

messenger PIP3 (PI 3,4,5 triphosphate) and PI3K pulls AKT to the membrane and

this interaction causes conformational changes resulting in the phosphorylation of

Thr-308 in the kinase domain and of Ser-473 in the C-terminal domain. AKT is

partially activated by phosphorylation of Thr-308 by PDK1 and becomes fully

active when phosphorylated on Ser-473, which is catalyzed by many proteins

including phosphoinositide-dependent kinase 2 (PDK2), integrin-linked

kinase (ILK), mechanistic target of rapamycin complex (mTORC) and DNA-

dependent protein kinase (DNA-PK)(Hemmings & Restuccia, 2012;

Vanhaesebroeck & Alessi, 2000).

Regulation of AKT

The PI3K/AKT pathway is a key player in many cellular signaling pathways, it

must be regulated carefully, and its dysregulation leads to various disease

conditions including cancer. One such mechanism of regulation is reducing the

levels of PIP3. PTEN is a tumor suppressor, which antagonizes PI3K by converting

PIP3 to PIP2 and therefore a loss of PTEN leads to hyperactivation of AKT.

Another way of regulation is by Protein Phosphatase 2A (PP2A) which

dephosphorylates AKT on Thr-308, and by the phosphatase PHLPP which

dephosphorylates AKT on Ser-473 (Manning & Toker, 2017).

CPPED1 is a recently identified protein phosphatase, found to dephosphorylate

AKT1 on Ser-473 in vivo, and blocks the cell cycle and promotes apoptosis via its

phosphatase domain. A gene expression analysis on the mRNA level has shown

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that CPPED1 levels are downregulated in bladder cancer and its overexpression

suppressed the size of tumors in nude mice, indicating a role in cancer regulation

by inhibiting AKT1 activation through a dephosphorylating mechanism (D. X.

Zhuo et al., 2013). In vitro, CPPED1 mediated AKT1 dephosphorylation is metal

ion dependent. We analyzed the effect of three different metal ions Mn2+, Ca2+ and

Mg2+ on AKT dephosphorylation. It was observed that in presence of Mn2+, AKT1

dephosphorylation was most prominent, whereas the activity was moderate in the

presence of Ca2+, and no phosphate activity towards AKT1 was observed with Mg2+

ions.

6.3.2 Interaction between CPPED1 and selected target proteins were

confirmed using co-immunoprecipitation and BiFC

experiments

After carrying out a pathway analysis for proteins found in the protein microarray

interactome study, we found that the PI3K/AKT pathway was enriched in the list.

From the pathway analysis we chose the PAK4 and PIK3R2 proteins for further

studies. It is well known that GRB2, PAK4 and PIK3R2 are involved in the same

pathway as well as in many other cellular signaling pathways (Thillai, Lam, Sarker,

& Wells, 2017; Wagner, Stacey, Liu, & Pawson, 2013)

p21 Activated Kinase 4 (PAK4)

PAK4 is a member of the p21-activated kinase (PAK) family of serine/threonine

kinases that contains six protein members named PAK 1-6, which are further

subdivided into two groups based on domain structure, sequence homology and

regulation. Group I PAKs are PAK 1-3 and group II are PAK 4-6. Structurally PAK4

contains an N-terminal PBD (p21-GTPase -binding domain) and a highly

conserved C-terminal catalytic Ser/Thr kinase domain and a newly identified auto-

inhibitory domain (AID) or AID like pseudosubstrate sequence (B. H. Ha et al.,

2012).

Pak4 is highly expressed throughout the developmental stages ubiquitously and

in adults at low levels. PAK4 activity is mainly regulated by phosphorylation of Ser

474 which is mediated by autophosphorylation as well as protein kinase D 1

(PKD1) and by an autoinhibitory domain that is released upon RhoGTPase binding.

Another important regulation mechanism for PAK4 is cellular translocation. It has

been shown that phosphorylation of Ser 99 by PKD1 and binding of the 14-3-3

77

protein promotes the formation of a PAK4/LIMK/PKD1 complex, which regulates

directed cell migration to the leading edge (Bastea et al., 2013). PRP4 is a kinase,

identified to phosphorylate PAK4 on Ser 104 and the functional importance of this

phosphorylation in PAK4 is not known yet (Q. Gao et al., 2013). Based on previous

studies it is evident that phosphorylation events play an important role in the

cellular translocation of PAK4 and its functional regulation. A mutation in the Ser

99 residue leads to the cytoplasmic retention of PAK4, whereas a mutation in Ser

474 impairs its nuclear translocation. In our studies we identified that CPPED1

interacts with PAK4 and dephosphorylates it on different serine residues.

Elucidating these dephosphorylation events helps to better understand the cellular

translocation of PAK4 and its functional regulation in the cellular milieu.

Phosphoinositide-3-Kinase Regulatory Subunit 2 (PIK3R2)

The most important function of the p85 subunit is the regulation of PI3K activation

by modulating the stability, conformation, and localization of the catalytic subunit.

Structurally p85 contains an N-terminal SH3 domain followed by a RhoGap

homology region located between the two proline rich domains. Upon binding to

activated receptor tyrosine kinases (RTK), the SH2 domain of p85 mediates

translocation of PI3K to the plasma membrane and initiates conformational

changes which lead to the activation of the PI3K pathway (Cariaga-Martinez et al.,

2014). P85β is mainly localized to the cytosol and concentrates at focal adhesions.

In the nucleus, it interacts with PI3Kβ which is required for the nuclear functions

of PI3Kβ (Kumar et al., 2011). An expression level analysis of p85β in colon and

breast cancer samples showed that the elevated expression PI3Kβ is correlated with

elevated AKT activation and a high-grade tumor stage and similar observations

were found in metastatic melanoma (Cortes et al., 2012). Recently it was shown

that the epigenetic regulation of the PIK3R2 promoter contains CpG islands and its

methylation is reduced by IL-6 that affects genes involved in angiogenesis and

insulin signaling (Balakrishnan, Guruprasad, Satyamoorthy, & Joshi, 2018).

Vallejo-Díaz et al., proposed that the oncogenic role of PIK3R2 in different cancers

by the elevated activation of the PI3K pathway indicates its involvement in tumor

progression (Vallejo-Diaz, Chagoyen, Olazabal-Moran, Gonzalez-Garcia, &

Carrera, 2019) . Our HuProt microarray interactome studies showed that CPPED1

interacts with PIK3R2 and the interaction was confirmed with co-

immunoprecipitation and BiFC studies. Further, mass spectrometry analysis of

78

PIK3R2 revealed that its phosphorylation levels are unaffected by CPPED1, but

the effect of CPPED1 on the expression levels of p85β is not known.

Fig. 10. Compex interplay of CPPED1 with different proteins of the PI3K/AKT and PAK4

signaling pathways. Binding of growth factors to tyrosine receptors leads to activation

of GRB2, which further activates PI3K via Ras GDP/GTP proteins. CPPED1 interacts

with GRB2. Activated PI3K converts PIP2 to PIP3 which recruits PDK1 and AKT1 to the

cell membrane. CPPED1 interacts with p85β which is a regulatory subunit of PI3K, but

the significance of this interaction is yet to be elucidated. PDK1, PDK2 and ILK

phosphorylate AKT1, which leads to the full activation of AKT1. Similarly, PP1, PP2A,

PHLPP dephosphorylate AKT1 which leads to AKT1 inactivation. CPPED1 is shown to

dephosphorylate AKT1 on Ser-473 and promote apoptosis. Activated AKT1

phosphorylates FOXO1/3 transcription factors leading to the nuclear exclusion of FOXO

proteins. On the other hand, the PAK4 protein is activated by the KRAS or PI3K protein,

which further activates AKT1 by phosphorylation. Both AKT1 and PAK4 reciprocally

regulate each other. PAK4 functions are regulated by phosphorylation/

dephosphorylation events. Autophosphorylation of PAK4 on Ser-474 leads to nuclear

translocation and activation of the expression of certain genes. CPPED1 interacts and

dephosphorylates several serine residues which might prevent nuclear translocation

and regulation of PAK4 functions are yet to be elucidated. In our studies we have shown

the interaction of CPPED1 with PAK4 and PIK3R2 which might regulate the PI3K

pathway at various stages either by p85β or PAK4 in addition to previously known AKT1

dephosphorylation (Modified based on Crivellaro et al., 2016; Thillai et al., 2017).

In a recent study, Zhuo et al., showed that the mechanism of CPPED1 tumor

suppressor activity in bladder cancer via decreasing IL-6 secretion which

79

inactivates the STAT3 signaling pathway. A gene expression analysis of CPPED1

overexpressing bladder cancer cells by microarray identified that IL-6 was

downregulated (D. Zhuo, Wu, Luo, Deng, & Niu, 2019) , whereas in contrast to

this, in our previous findings the knockdown of CPPED1 in HTR-8 trophoblast

cells led to a three-fold decrease in IL- 6 levels, implying that the function of

CPPED1 might be dependent on cell type and origin. It is interesting to note that

IL-6 reduces PIK3R2 levels by epigenetic modifications, whereas CPPED1

silencing leads to upregulation of IL-6 in bladder cancer. This suggests that

CPPED1 might influence p85β expression levels either directly or via upregulating

the levels of IL-6 in the cellular milieu. Further investigation is needed to

understand the connection between CPPED1, IL-6 and PIK3R2. This information

may help in the elucidation of the cancer progression.

6.4 Prospects for future studies

Using proteomics and miRNA-omic studies, we have identified different proteins

and variations in miRNA expression levels between term and elective term

placentas.

Our comparative proteomics studies have identified 10 proteins which were

differentially expressed in term placentas compared with elective term placentas.

Among the ten proteins, the following four were upregulated: ACTB, b-2-

microglobulin (B2M), keratin type II cytoskeletal 8 (KRT8) and 19 (KRT19),

whereas six proteins, including a-2-macroglobulin (A2M), CPPED1, cytochrome

b5 (CYB5A), haemoglobin subunit c-2 (HBG2), peroxiredoxin- 2 (PRDX2) and

plasminogen activator inhibitor 2 (SERPINB2) were downregulated and all

proteins showed differences in their expression at the chorionic (fetal side) and the

basal plate (maternal sides) of the placenta, suggesting a fetal - maternal gradient.

Further, we confirmed the associations of CPPED1 SNPs in term deliveries and

that its silencing in trophoblasts affects inflammation and angiogenesis. More

studies are needed with larger placental samples to confirm these changes and to

further strengthen the association of CPPED1 with the onset of labor.

Our miRNAomics demonstrated the differential expression levels of 54

miRNAs and we validated the significant differences in miR-371a-5p levels

between term and elective term placentas. Further, we characterized the connection

between miR-371a-5p and CPPED1 protein levels and they were found to be

inversely correlated in term placentas. Measuring the levels of both miR-371a-5p

80

and the CPPED1 protein during different gestational ages using more placentas

would reveal the kinetics in their variation and importance for the onset of labor.

CPPED1 is an unexplored protein of the protein phosphatase family and known

to play an important role in inhibiting bladder cancer progression via affecting the

AKT/PI3K pathway by dephosphorylating the Ser-473 residue on AKT1. We have

studied in vitro protein-protein interactions of CPPED1 and its binding partners

using the HuProt protein array and confirmed the interaction of CPPED1 with

PAK4 and PIK3R2. An in vitro phosphatase activity assay and mass spectroscopy

revealed that CPPED1 dephosphorylates PAK4 on several different serine residues.

Characterizing the importance of these phosphorylation events will help to

understand the functional regulation of PAK4. Furthermore, studying the effect of

CPPED1 on PIK3R2 expression levels will likely elucidate the functional

mechanism of CPPED1 in the cellular milieu.

81

7 Conclusions

Our comparative proteomics study of spontaneous and elective term labors

identified different protein profiles for the maternal and fetal sides of the placenta.

The levels of these proteins are altered in spontaneous labor. Previous studies

showed that the levels of CPPED1 are reduced with the progression of bladder

cancer and lower levels are associated with low survival, indicating its potential as

a biomarker for cancer progression. Similarly, we found lower levels of CPPED1

to be associated with spontaneous term birth.

miRNAomics were used to determine the differential miRNA expression

patterns between term and elective term phenotypes of labor. Measuring these

identified miRNAs between these two groups and identifying their functional and

biological roles in the placenta would contribute to understanding the roles of these

miRNAs in the onset of labor. In this study we have demonstrated that the

expression levels of a protein and miRNA pair, CPPED1-miR-371a-5p, showed a

significant difference between spontaneous term and elective term placentas and

measuring their levels could be used as a biomarker for labor.

We showed that CPPED1, a novel protein phosphatase dephosphorylates

AKT1 on the Ser-473 residue. However, AKT1 phosphorylation levels remain

unaffected despite of the variations in CPPED1 levels in different term placentas.

The siRNA knockdown of CPPED1 in HTR8 trophoblasts led to an increase in the

upregulation of negative regulators of the PI3K pathway, suggesting that CPPED1

might play a central orchestrating role in fine tuning of the AKT pathway via other

mechanisms if not by AKT1.

Finally, we characterized the protein interactome of CPPED1 using a protein

microarray and corroborated the interaction of CPPED1 with PAK4 and PIK3R2,

both involved in the regulation of the AKT/PI3K pathway. Based on proteomics,

genetics and epigenetics CPPED1 associates with spontaneous birth and may turn

out to be a specific biomarker for the onset of labor. CPPED1 might be a

pleotrophic protein which is involved in the regulation of different cellular

processes by altering the functions of the target protein by either posttranslational

modifications such as dephosphorylation, as seen in case of AKT1 and PAK4, or

by inhibiting or decreasing the levels of certain genes, as in the case of IL-6. Further

studies, identifying the significance of CPPED1 interactions with its binding

partners, might reveal the functional role of CPPED1 in the cellular milieu.

82

83

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Original publications

I Haapalainen AM, Karjalainen MK, Daddali R, Ohlmeier S, Anttonen J, Määttä TA, Salminen A, Mahlman M, Bergmann U, Mäkikallio K, Ojaniemi M, #Hallman M, #Rämet M. (2017). Expression of CPPED1 in human trophoblasts is associated with timing of term birth. J Cell Mol Med. 2018 Feb;22(2):968-981

II Daddali R, Ojaniemi M, Hallman M, Rämet M& Haapalainen AM. (2020). MicroRNA-371a-5p targets CPPED1, and its expression in human placenta is associated with spontaneous delivery. Plos One. 2020 PLoS One. 2020 Jun 10;15(6):e0234403

III #Haapalainen AM, #Daddali R, Hallman M, Rämet M. Human CPPED1 belongs to calcineurin-like metallophosphoesterase superfamily and dephosphorylates PI3K-AKT pathway component PAK4 (Submitted)

Reprinted (I and II) articles with the permission from corresponding journals.

Original publications are not included in the electronic version of the dissertation.

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D 1594

Ravindra Daddali

MOLECULAR MECHANISMS REGULATING THE ONSETOF LABORCOMPARATIVE PROTEOMICS AND miRNAOMICS OF HUMAN SPONTANEOUS AND ELECTIVE LABORS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;OULU UNIVERSITY HOSPITAL