diu internship report2 jk reviewed

8/8/2019 Diu Internship Report2 JK Reviewed

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[TYPETHECOMPANYNAME]

VALIDATIONOFMIR-125BPUTATIVETARGETSINVOLVEDINLEUKEMIA

InternshipReport

Student:DiuT.T.Nguyen

Duration:JanuaryJune2010

Supervisors:

Prof.HarveyF.LodishDr.JanM.Kooter

Dr.MarinaBousquetDepartmentofGenetics

WhiteheadInstituteforBiomedicalResearchVrijeUniversityAmsterdam

9CambridgeCenter,Cambridge,DeBoelelaan1085,1081HV

MA02142Amsterdam

June2010


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D.T.T.Nguyen MiR-125b in Leukemia

2

Contents

Abstract 3

Introduction 4

MaterialsandMethods 7

ResultsandDiscussion

Differentiationblockageandapoptosisinhibitionof32DCl3andHL60celllinesby

miR-125boverexpression

15

ApoptosissuppressionbymiR-125binHL60and32DCl3 16

Bindingsitescloningintopsicheck2vector 18

Generationofbindingsitemutants 19

Validationofputativetargetsinvolvedincellproliferation,apoptosisandcell

signaling

DUSP6andETS1 20

MAP4K2 23

PPP2R1B 27

Validationofputativetargetsinvolvedinmyeloiddifferentiation

SP1 29

CBFB 32

Generaldiscussionandconclusion 35

Acknowledgement 37

References 38

Appendix 39


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3

ABSTRACT

MiR-125b was shown to be overexpressed in leukemic patients carrying the chromosomal

translocationt(2;11)(p21;q23),whichresultedinblockeddifferentiationandapoptosisofmyeloid

progenitors invitro.Thiswashypothesizedtobethecauseofleukemiainthesepatients.The

mechanismunderlying thisphenomenon remains tobeelucidated.The aimofthis studyis to

determinethedirecttargetsofmiR-125bwhichleadtodifferentiationandapoptosisblockage.In

myfirstinternship,sixputativetargetswereselectedbasedonmRNA-sequencing,RQ-PCRand

bioinformaticapproaches,includingDUSP6,ETS1,MAP4K2,PPP2R1B,SP1 andCBFB.Tovalidate

thesegenecandidatesasrealtargetsofmiR-125b,thebindingsiteswereinsertedinto3UTRof

luciferase reportergeneandthenco-transfectedwithmiR-125binto293Tcells. Mutations of

these binding sites were created and used as controls. In a further step, miR-125b was

overexpressed in three different cells lines, namely NB4, HL60 (human cell lines) and 32DCl3

(mousecellline)whichcouldbeinducedtodifferentiateortodieinprogram.Proteinlevelsof

the putative targets were examined before and after induction according to physiological

functionsofthegenes.Theresultsrevealedthat CBFBismostlikelyadirecttargetofmiR-125b.

Since CBFB forms complexes with Runx protein family, which is essential for maturation of

hematopoietic cells, the down-regulation of it by miR-125b could explain the differentiation

blockageobservedinvitro.


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INTRODUCTION

MicroRNAsmodulateavarietyofcellularpathways,includingdevelopment,differentiation,cell

proliferation and apoptosis (Bartel 2004;Bartel 2009) and dysregulation ofmiRNAexpression

underliesspecificoncogeniceventsinhumancancer(CalinandCroce2006;Esquela-Kerscherand

Slack2006;Garzon,Fabbrietal.2006;Calin,Liuetal.2007)

In a previous study, Bousquet M. et al. showed that a chromosomal translocation

t(2;11)(p21;q23)in19patientssufferingfromacutemyeloidleukemia(AML)andmyelodysplastic

syndrome (MDS) caused a six to ninety fold up-regulation ofmiR-125b compared to healthy

individualsandleukemicpatientslackingthetranslocationt(2;11)(p21;q23)(Bousquet,Quelenet

al. 2008). The gene formiR-125b is located on chromosome 11 (miR-125b-1 locus), but it is

unclearhowthe t(2;11)(p21;q23) translocation leads tomiR-125boverexpression.WhenmiR-

125bwastransientlytransfectedintothehumanpromyelocyticleukemiacelllinesNB4andHL60

itblockedthedifferentiation ofthese cell linesuponchemical treatment. Itis notedthatNB4cellsbecomematuregranulocytesandHL60 undergoesmonocyticmaturationafter treatment

withATRAandDMSO,respectively.TheywerealsoobservedtohaveendogenousmiR-125bup-

regulated at the very end of cell maturation. Transient transfection of miR-125b resulted in

maturationarrest,shownbyareductioninthemarkersofterminaldifferentiatedmyeloidcells

(CD11b, CD14 and CD15) and by morphological observation(Bousquet, Quelen et al. 2008).

Recently,miR-125bwasshowntoinhibitapoptosisinNB4cellsuponinductionofapoptosiswith

camptothecin (unpublished data, Bousquet M. et al.). These properties may account for the

differentiation and apoptosis blockage in leukemic cells in vivo bymiR-125b(Bousquet et al.

2008).

Moreover, miR-125bwas found tohave oncogenic functions inprostate cancer where itwas

foundtosuppressbak1andinduceandrogen-independentgrowthofthecancercells(Shi,Xueet

al.2007).Itwasalsodescribedasanegativeregulatorofpro-apoptoticgenep53andBMPR1B

associatedwithbreastcancerpathogenesis(Le,Tehetal.2009;Saetrom,Biesingeretal.2009).

TosearchfortargetsofmiR-125binvolvedinleukemia(AMLandMDS),inthefirstinternshipI

used mRNA sequencing (next generation sequencing) and RQ-PCR, together with integrative

bioinformatics approach to screen for putative target candidates. To this end, I used the

promyelocyticcellline,NB4,transfectedwithmiR-125bmimic.Sixgenes,namelyDUSP6,ETS1,MAP4K2,PPP2R1B,SP1andCBFBwerefoundtobedown-regulatedbymiR-125b.Amongthem,

DUSP6,ETS1,PPP2R1Bhavebeendemonstratedtoplayrolesincellgrowthcontrolandapoptosis

andwerereportedtobedown-regulatedinseveraltypesofcancer(Furukawa,Sunamuraetal.

2003;Tamaki,Goietal.2004;Esplin,Ramosetal.2006;Okudela,Yazawaetal.2009). SP1and

CBFB are both key players in hematopoiesis, particularly in myeloid maturation (Clarke and

Gordon 1998;Hauses, Tonjes etal. 1998;Kunduand Liu 2003). SP1, a universal transcription


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factor, regulates many genes, such as HCK and CD11b which are essential for myeloid

differentiation, through its binding to their promoter regions (Hauses, Tonjes et al. 1998).

Interestingly,our data indicated significantdecreases inmRNA levels of thesetwoSP1 down-

streamtargets.CBFBdoesnotfunctionbydirectinteractionwithDNAbutformscomplexeswith

otherDNA-binding transcription factors,Runx proteins. Itwas reported that adefect inCBFB

could largely prevent definitive hematopoiesis and lead to lethality in embryogenesis. The

expectation was that if these candidates are direct targets, they could explain the myeloid

differentiationinhibitionandapoptosissuppressionobservedinNB4cellline.

ItisimportanttovalidatearealtargetofamiRNAattheproteinlevelfortworeasons:first,itis

theprotein,notmRNA,thatperformstheultimatefunctionofageneandsecond,thedetected

changes inmRNAleveldonot always andnecessarily leadtochanges inproteinlevel. Infact,

thereisapossibilitythatadecreaseinmRNAlevelasaconsequenceofdestabilizationbymiR-

125bwouldnotcauseanychangeattheproteinlevel;however,thattargetinghasnobiological

significance and hence is not worth being taken into account in attempt of deciphering the

phenotypescausedbymiR-125boverexpression.

This internshipwas aimedat validatingthe six putativetargetgenesofmiR-125b,whichwere

foundintheprimarystudy.Toachievethisgoal,thetargetbindingsitesofmiR-125inthemRNAs

ofthesegenesweretestedinanartificialsystem,inwhichthetargetsequenceswereinserted

into the 3UTRof luciferase reportergenewhichwas then co-transfectedwithmiR-125b. The

verificationofthefunctionofmiR-125bythisassayindicatesthepotentialofbeingadirecttarget

ofthegenebutdoesnotguaranteeitsfunctioninvivo.Therefore,westernblotwasfinallyused

toquantitateandcomparetheeffectofmiR-125bontheproteinleveloftheputativetargets.Forthispurpose, besides theNB4 cell line, inwhichoverexpressionofmiR-125bwasobtainedby

transienttransfectionwithmimics,twootherpromyelocyticcelllines,HL60(humancellline)and

32DCl3(mousecell line),wereinfectedwithretroviralvectorsexpressingmiR-125b.Theresults

revealedthatCBFBwasthemostlikelytobedirecttargets.

MATERIALSANDMETHODS

Cell lines andpre-microRNAs.NB4 cell linewas purchased fromDSMZ.32DCl3ERand HL60ER

weregenerousgiftsfromDr.WeiTongandDr.BradFletcher,respectively.32DCl3andHL60ER

stablyinfectedwithvectorXZorXZmiR-125bweregiftsfromMarinaBousquet.293Tcelllinewas

purchased from ATCC. MiR negative control #1 and miR-125b mimics were purchased from

Dharmacon.

Cellcultureandtransfection.NB4cellswereculturedinRPMI1640(Invitrogen) supplemented

with10%fetalbovineserum,L-glutamine,penicillinandstreptomycin.Transienttransfectionof


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miRnegativecontrol#1andmiR-125b(22.5uLofeachmimicattheconcentrationof50uM)into

NB4cells(3x106)wereperformedbyelectroporationat200Vand950uF,usingPulser(BioRad).

After24h, the transfectedcellswere induced todifferentiate into granulocytes byaddition of

ATRAatthefinalconcentrationof10uMfor5days.

HL60ERand32DCl3ERarecelllinesexpressingmurineecotropicreceptor,whichallowsastable

genetransferintothecellswithmurineretroviralvectors.Inthisstudy,thesetwocelllinescarry

vectors(XZ)containingmiR-125bgeneandGFPasareporterforselection.HL60ERwerecultured

in IMDM (GIBCO) supplemented with 1.5g/L sodium bicarbonate, 10% fetal bovine serum.

32DCl3ERweregrownin10%fetalbovineserum,10%IL3(WEHImedia)and1%penicillinand

streptomycinRPMI(GIBCO).

InductionofapoptosisinHL60ERand32DCl3ER

ToinduceapoptosisinHL60ER,CamptothecinwasaddedintothegrowingcultureofHL60ERXZ

andXZmiR-125batthedensityof3x10 5cell/mLwiththefinalconcentrationof10M.Thecells

wereharvestedatfourtimepointsafter2hours,4hours,8hoursand24hoursofinduction.For

32DER,apoptosiswastriggeredbyremovingIL3fromthemedium.Thecellswerewashedthree

timeswiththemediumwithoutIL3andthenresuspendedinthesamemediumat5x105cell/mL.

Thecellswereharvestedatday1,day2andday4afterIL3removal.

ApoptosisassaywasperformedusingtheAnnexinV-PEapoptosisdetectionkit(BDBiosciences)

according to the manufacturers instructions. Briefly, cells were washed twice with PBS and

resuspendedat1x106cell/mLin1XBindingBuffer.5lofAnnexinVand5lof7-AADwereadded

to100lofcellsandincubatedfor15mininthedark.Afteradding400lof1Xbindingbuffer,thecellswereanalysedbyFACS.

Inductionofdifferentiationin32DCl3ER

Todifferentiate 32DCl3ER intomyeloid cells, the cellswerewashed threetimes in IL3 lacking

medium and then resuspendedat3x105 cell/mL in the samemedium plus G-CSF at the final

concentrationof100ng/ml.Thecellswereharvestedforproteinquantificationafter1,2and4

days of induction. The rest of the induced culture was kept until day 5 for FACS and

morphologicalanalysis.

Constructing3UTRgenefragmentsflankingbindingsitesofmiR-125b


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a

Using primers listed in the table below, fragments surrounding the binding sites of the

correspondinggeneswereamplified byPCR.The PCR productswere cloned into the reportervector psicheck2 (Promega) usingXhoI andNotI (at its 5end and3end respectively).E. coli

DH5 was transformed with the constructs and plated onto semi-solid medium containing

Ampicillinforselectionoftheplasmidscontainingthewantedinserts.Toconfirmthepresenceof

theinserts,selectedplasmidsweredigestedwithXhoIandNotI,followedbyDNAsequencing.

Table1.Primersforcloningbindingsites.TherecognitionsitesofXhoIandNotIareinred

Primername Oligonucleotidesequence(5->3)DUSP6 F taaCTCGAGATTTGCAGCATGCTTGACTTTACCA

DUSP6R ataGCGGCCGCAGCTCTAAACTTTACACCACGAACA

ETS1 F atgcCTCGAGgaagcaaagatggacttcagtg

ETS1 R gcatGCGGCCGCGCCCTGGTTCTACTCTTACCCA

CBFB F taaCTCGAGACTCTGTACTCTGCCCTAGATTGT

CBFBR ataGCGGCCGCGACTTAGCACTGTTTTCCACTCTG

SP1.2 F atgcCTCGAGTTCAGGTTTTCAGGTTGCCCAT

SP1.2 R gcatGCGGCCGCCTGCAGCTTCCAGTATTCACAC

M4K2.1 F taaCTCGAGAATGACCGCTTGTGGATCTGCA

M4K2.1 R ataGCGGCCGCCGTCACATAGCTCATTGTAGCC

M4K2.2 F taaCTCGAGAACTGCATGAGGATACGCTGGA

M4K2.2 R ataGCGGCCGCTCAGCAGCAGAAACTTCTGCAG

PPP2R1B F atgcCTCGAGACTCTTCGGGAGACATGTTGAG

PPP2R1BR gcatGCGGCCGCTGGCCAAGTTATGAGCCAAAGC

Figure1.Psicheck-2map

HRluc encodes the Renilla luciferase.

The multicloning sites, including

several restriction sites, are within3UTRofhRluc.Hluc+isfirelyluciferase

gene,whichplaysaroleasaninternal

controloftheassay.


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TP53INP1 f taaCTCGAGGTGTATCACATAATGCCTTGCCT

TP53INP1 R ataGTTTAAACGCACTAATGGGTTAGTTACAGAC

ZNF148 F atgcCTCGAGTGGGTCTACATGCAAATGTGGT

ZNF148R gcatGCGGCCGCTAGCACATTGGTACTCACTTCA

APAF1 F atgcCTCGAGCATACCTTGTTGTACTGTTGGTA

APAF1 R gcatGCGGCCGCTCATGAGGTCAAGAGATCGAGA

APCF atgcCTCGAGAGGCACTCTTGATGGTTAGGAA

APCR gcatGTTTAAACCCATCTCACCTCAAATACCAGA

STAT3 F atgcCTCGAGTGGCCTTTTAGTGAGTAAGGCT

STAT3R gcatGCGGCCGCTTCCACAGAAACTCTGATCAGC

PPP1CA F taaCTCGAGCCAGATGATGGATTGATTGTACAG

PPP1CA R ataGCGGCCGCTGCGAGAATCCAGCTTTGACCT

PPP2CA F taaCTCGAGCAGTTTCTGGCATAGCGCTA

PPP2CA R ataGCGGCCGCAACACTGCAGTGCTCTTGTGCA

Site-directedmutagenesis

Constructscarryingbindingsitesweremutatedatthe6-nucleotidesequencecomplementaryto

the seed sequenceofmiR-125busingprimerswith the desiredmutation , designedwith the

supportfromhttp://www.stratagen.com/qcprimerdesign(Table2.Theprincipleofsite-directed

mutagenesisis illustratedinFigure2.FirstmutagenesisPCRreactionwasperformedwith50ng

ofplasmid,1.0Lofprimers(10M,forwardandreverse),1.0LdNTPsmixture(10mMeach)

andPfuTurboDNApolymeraseattheconcentrationof2.5U/L.Themixturewereruninthermalcycler(BioRad)at95

oCfor30s,then22cyclesof95

oCfor30s,53

oCfor1minand66

oCfor8min.

After,itwaschilleddownto4 oCbeforeaddingDpnIwhichdigestedthenon-mutatedparental

DNAtemplates.Thedigestionwasincubatedovernightat37oCbeforebeingtransformedintoE.

coliDH5.Selectedplasmidswerethensequencedtoverifythemutations.


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Figure2.Overviewofsite-directedmutagenesis(source:Stratagene)


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Table2.Primersforgenerationofmutatedbindingsites.DEL:Deletion,M:Substitution

Primername Oligonucleotidesequence(5->3)

DUSP6 F Del gctaaacagtatattacctctgtataaaattctagtgtcacctcaaatgc

DUSP6R del gcatttgaggtgacactagaattttatacagaggtaatatactgtttagc

CBFB FM cacaggctggcttctgttttattcatcgatttttttaaaaagtcaatcagaaa

CBFBRM tttctgattgactttttaaaaaaatcgatgaataaaacagaagccagcctgtg

SP1.2 FM Tggctcacagcctgtcacccacgtgactaccatcagttctgcc

SP1.2 RM ggcagaactgatggtagtcacgtgggtgacaggctgtgagcca

SP1.2 F dEL Cccacaccgtcttccttttccaactggcactc

SP1.2 R dEL Gagtgccagttggaaaaggaagacggtgtggg

M4K2.1 Fm Gggctccaccacctgcattccacgtggaagatccacagagacatc

M4K2.1 Rm Gatgtctctgtggatcttccacgtggaatgcaggtggtggagccc

M4K2.2 Fm Acaacgtgctgctgtcactccacgtgaaatccacgcacatctggg

M4K2.2 Rm Cccagatgtgcgtggatttcacgtggagtgacagcagcacgttgt

PPP2R1B FM1 ctcatgtgaaaggggaatgtggaagccacgtgttaatgtagtggttgttttggtttt

PPP2R1BRM1 aaaaccaaaacaaccactacattaacacgtggcttccacattcccctttcacatgag

Luciferaseassay

One day before transfection, 293T cells were seeded into 96 well white plates at 1-2x104

cell/well. After 24h, when the cells reached a confluence of 60-70%, they were used for co-

transfection.Theassaywasperformedinatriplicateforeachconstructandrepeatedatleasttwo

timesindependently.Non-insertedpsicheck2vectorandmiR-125bperfectmatchinsertedvector

wereincludedintoalloftheexperimentsasthecontroloftransfectionandproperworkingofthe

mimics.

Twomixtureswerepreparedforthetransfection.MixtureAcontained25LofOptiMEM(GiBCO)

and1LofLipofectamin2000andwasincubatedatroomtemperaturefor10minutes.MixtureB

included 25L ofOptiMEM, 10ng of constructs, 1L of 1M mature microRNA (miR-125b or

miRcontrol,Dharmacon).After10min,mixtureAwasaddedtomixtureBandincubatedatroom

temperaturefor20minbeforetheyweretransferredontotheplatecontainingcells.Priortothe

transferring,theoldmediuminthewellswasaspiratedbytapingtheplateontoapapertowelorby vacumm and 50L of new medium (10% FBS, 1% L-Glutamin DMEM without Peni-

Streptomycin)wereadded.


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Figure 3. Principle of Luciferase assay. Source: Promega. Note that in this case, shRNA is

replacedbymiRNAbutthemechanismofactionissimilar.

After48hofco-transfection,themediumwasdiscardedbyinvertingtheplate.Luminescencewas

measured usingDual-Glo Luciferase kit (Promega) and TECAN luminescence reader. The cells

werelysedbylysisbufferinthefirstreagentofthekitwhichalsocontainedsubstrateforFirely

luciferase reaction (internal control). The mixture was shaken at RT for 10 min before

measurement. Reagent 2 (Renilla luciferase substrate: buffer as 1:100) was added after to

measureRenillaluminescence.Renillaluciferasesignal,whichaccountsfortheeffectofmiR-125b

onthe3UTRoftheRenillagene,wasnormalizedwithFireflyluciferasesignal,whichisaninternal

controloftransfectionefficiency.

Real time quantitativePCR.TotalRNAwasisolatedfrommiR-125bandmiRcontroltransfected

cells treated and untreated with ATRA. The first strand cDNA synthesis was performed in a

mixtureof20uLincluding4ugRNA,0.2ugrandomprimers,0.5mMdNTPsand1LSuperscriptII

reversetranscriptase(Invitrogen).Themixturewasincubatedat25oCfor10minutes,42

oCfor50


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minutesand70oCfor15minutes.Afterthat,cDNAproductsolutionwasdiluted20timeswith

waterand5Lofthedilutedsolutionwasusedforreal-timePCRreactionwithSYBRMasterMix

(AppliedBiosystems)andthefollowingprimersforgenesofinterest(designedbyDiuNguyen,

purchasedfromInvitrogen).

Table3.RQ-PCRprimers

Primername Oligonucleotidesequence(5->3)

Apaf1 hF TTAGCTGCAGAAGCTGTTAGAG

Apaf1 hR CAGTTTCATCAGAAGCCCAGAT

Stat3hF AACATCTGCCTAGATCGGCTAG

Stat3hR TTGCTGCAACTCCTCCAGTTTC

Znf148hF TCCTCAAGGACTACAGTATGCA

Znf148hR TGTAAGTCTACTGGCTCTCTGA

Etv6hF

TGAACCACATCATGGTCTCTGTEtv6hR GTCAGAAAGCAACTGATAGACG

Ppp1ca hF GTGCCAGCATCAACCGCATCT

Ppp1ca hR GCAGGCAGTTGAAGCAGTCAG

Ppp1ca mF CATCTGCAGAGCACATCAGGTT

Ppp1ca mR CATCATGGCACCAGCATTGTCA

Ppp2ca hF GGTGCCATGACCGGAATGTAG

Ppp2ca hR GAGGTGCTGGGTCAAACTGCA

Ppp2ca mF ACCAGCTGGTGATGGAGGGAT

Ppp2ca mR

TTGCAGCTTGGTTACCACAACGTP53INP1 hF CCACCCGTGGGACTGATGAAT

TP53INP1 hR GAGCAGCAAGAGCTGCAACATA

Tp53inp1 mF AACTCAAGTGGTCCCAGAATGG

Tp53inp1 mR GGGCGAAAACTCTTGGGTTGTT

MLN51 hF TAATCCCAGTTACCCTTATGCTCCA

MLN51 hR GTTATAGTAGGTCACTCCTCCATATACCTGT

ActinehF TCCCTGGAGAAGAGCTACGA

ActinehR AGGAAGGAAGGGTGGAAGAG

18smFGTAACCCGTTGAACCCCATT

18smR CCATCCAATCGGTAGTAGC

-actinmF GGCTGTATTCCCCTCCATCG

-actinmR CCAGTTGGTAACAATGCCATGT


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QuantitativePCRwasperformedon96wellplateusingABI7600.Theamplifiedproductlength

rangesfrom100to120bp.

Celllysis,proteinquantificationandWesternBlot

The cellswere harvested and lysedbyELB buffer (250mMNaCl, 0.1%Nonidet P-40,50mMHEPES [pH7.0], 5mMEDTA) containing proteinase inhibitorcocktail (complete tablet-Roche).

The cell lysate was kept on ice for 30 min and centrifuged at 13,000 rpm for 10 min. The

supernatantcontainingtotalproteinofthecellswasstoredat-80oCafterproteinconcentration

beingquantified.

For westernblotting, 50g protein (in supernatant) was denatured byNuPAGE loading buffer

(Invitrogen)at70oCfor10min.WesternBlotswereprobedwiththefollowingantibodies:GCK

(MAP4K2)(CellSignaling);PPP2R1B(Abcam);SP1(SantaCruz)andCBFB(Abcam).

FACSanalysis.Infected32DCl3ER(5x105

-1x106

)cellsafter5daysremovalofIL3andadditionofGCSF were washed in 3 ml of 2% FBS PBS and then resuspended in 50 uL of 2% FBS PBS

containinganti-mouseCD11b coupledwithPE-Cy7 atratio of1:100. The stainingmixturewas

incubatedatroomtemperaturefor30minutesindarkandthenwashedagainin2%FBSPBSto

removeallofspareantibodies.Afterthat,thestainedcellswereresuspendedin500uL2%FBS

PBSandanalysedusingFluorescenceactivatedcellsortingFACSLSRII(BDBiosciences).Unstained

andstaineduntransfected32DCl3ERcellswereusedasacontrol.

Morphologicalanalysis.Infected32DCl3cells(1x105)atday5ofGCSFinductionwerespunusingthecytospin(SHANDON)at500rpmfor5minutesonaglassslideandthendriedfor20minutes

atroomtemperature.CytospuncellslideswerethenstainedwithMay-Grunwaldsolution(Sigma

Aldrich)for 5minutesandwashedwithwater threetimes. Afterthat,the slideswerestained

withGiemsasolution(Sigma-Aldrich) (1:20 solution inwater) for15minutesandwashedwith

water again. Complete drying of the slideswas followedbefore visualizingundermicroscope

AxioCamMRc(Zeiss).


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RESULTSANDDISCUSSION

Confirmationofdifferentiationblockagein32DCl3cellsbymiR-125b

In the previouswork,BousquetM.etal. demonstrated that theNB4 cell lines ATRA-induced

differentiationwassuppressedbymiR-125b,transientlytransfectedintothecells. Inthisstudy,weconfirmedthatmiR-125bhadthesameeffectonthemurinecell line32DCl3.Notethatas

miR125bgeneisincorporatedintothegenomeof32DCl3cellsastheresultofretroviralvector

infection the expressionofmiR-125b is constitutiveandmore physiological than inNB4 cells,

which were transiently tranfected with microRNA mimics. Figure 4 shows FACS analysis on

32DCl3ER XZ and 32DCl3ER XZmiR-125b atday 5 after induction with G-CSF (as described in

Materials and Methods). In this histogram, the horizontal axis represents the intensity of

chromophorePE(Comp-PE-A),thusthelevelofCD11bastheyareconjugated.Thesecondpeak

oftheredcurveindicatesaCD11bpositivepopulationinthe32DCl3ERXZwhilethereisnosuch

peak in32DCl3ERXZmiR-125b.BecauseCD11b isamarker ofdifferentiatedmyeloidcells, theresultmeanstherearemorematurecellsin32DCl3ERXZ(red)thanthatin32DCl3ERXZmiR-125b

(green).

Figure 4. Confirmationof differentiation blockageof

32DCl3ER XZmiR-125b. Histogram FACS analysis of

CD11b(coupledwithPE)expressiononNB4transfected

withmiR-control (red) andwith miR-125b (green) (A)

and changes in morphology of May-Grunwald and

Giemsa stained cells at day 5 of induction ofdifferentiation in 32DCl3ER XZmiR-125b (C) compared

to32DCl3ERXZemptyvector(B)

A


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Figure4BandCshowtheresultsofcellstaining(May-GrunwaldandGiemsa)ofinfectedcellsat

day5afterinduction.Terminallydifferentiatedmyeloidcellsaretypicallycharacterizedby

multilobulatedandsegmentedshapeoftheirnucleiandcytoplasmicgranules(Sung,Gaoetal.2006).Itisobviousthat32DCl3ERXZ(Figure4B)ismoredifferentiatedthan32DCl3ERXZmiR-

125b(Figure4C).Takentogether,miR-125binhibitsgranulocyticmaturationinthemurinecell

line32DCl3.ThisresultsupportsourdataofthepreviousworkonthehumancelllineNB4and

hencestrengthensthehypothesisthatmiR-125boverexpressionblocksmyeloiddifferentiation.

ApoptosispreventionbymiR-125binHL60and32DCl3

In our study, we also addressed the question of whether miR-125b is a causative factor of

leukemiaor inotherwords,whethermiR-125bis anoncogene.A gene isconsideredtobean

oncogeneifwhenmutatedorinincreasedexpressionitcancause(1)differentiationblockage(2)apoptosissuppressionand/or(3)cellproliferationstimulation.InthecaseofmiR-125b,thefirst

evidence has been confirmedwith three different cell lines, NB4, HL60 and 32DCl3. To next

examinewhethermiR-125bpreventsapoptosisandpromotescellgrowth,HL60ERand32DCl3ER

cells expressingmiR-125bat stable and high levelswere forced toundergo programmed cell

death by adding Camptothecin and removing IL3 from their culture, respectively. In HL60ER,

Camptothecin promotes apoptosis by inhibiting Topoisomerase I,which is necessary for DNA

synthesisInIL3-dependent32DCl3ERcells,theremovalofIL3resultsinDNAfragmentationand

hencecelldeath(Pommier,Pourquieretal.1998).After24hofinductionforHL60ERand4days

ofinductionfor32DCl3ER,thecellswerestainedwithonlyAnnexinVorAnnexinVand7AAD(seeMaterialsandMethodsfordetails),whichdetectapoptosisandnecrosisatanearlystage.Ascan

beseeninFigures5A1-2intwoindependentexperimentstheproportionofAnnexinVnegative

cellsinHL60ERXZmiR-125bissignificantlygreaterthanthatinHL60XZ,indicatingthatthecells

overexpressingmiR-125baremoreresistanttoapoptosisthanthecontrolcells.Aclearerpicture

ofthis canbeseeninthecaseof 32DCl3ERcellswhichwerestainedwithbothAnnexinV and

7AAD,whichdistinguishescelldeathbyapoptosis(AnnexinVpositive)andnecrosis(AnnexinV

BC


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and7AADdoublepositive).Figures5B1-4showthatthepercentageofdoublenegativecellsin

32DCl3ERXZmiR-125b(livecells)wasmorethantwiceasmuchasthosein32DCl3ERXZ(60.81%

vs27.15%and60.98%vs30.36%).

A1

B1

A2

B2

B3 B4

AnnexinV


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Figure5.ApoptosissuppressionbymiR-125binHL60(A1andA2)and32DCl3(B1-B4)

A1-A2: FACS histogram of two independentapoptosisassays onHL60ERXZ125b (Red) andHL60ERXZ

(Green).ApoptosiswasinducedbyaddingCamptothecin(SeeMaterialsandMethods)andafter24h,they

were stainedwith AnnexinV coupled to PE.Thehorizontal axes areAnnexinV-PE intensity indicating

apoptosis and the vertical ones are the number of cells counted. B1-B4: FACS dot plots of twoindependentapoptosisassayson32DCl3ERXZmiR-125band32DCl3ERXZ.Thehorizontalaxesrepresent

AnnexinV-PEandtheverticalaxes,7AAD-PE-Cy-5-5whichindicatesnecrosis.B1andB3areduplicatesof

thecellsinfectedwithcontrolretroviralvectorsXZ.B2andB4areduplicatesofthecellsinfectedwith

retroviralvectorXZmiR-125b.

Inthefirstinternship,IusedmRNA-seqandRT-PCRtofindputativetargetgenesofmiR-125b.

Thisresultedintheidentificationofsixcandidategenes,namelyDUSP6,ETS1,MAP4K2,PPP2R1B,

SP1 and CBFB. These genes are involved in cell proliferation, apoptosis and myeloid

differentiation (Okudela, 2009;Zhang, 2010;Hauses, 1998; Yang, 2003;Tamaki, 2004;Kundu,

2003). In the following sections, validation of these putative targets by luciferase assay andwesternblotwillbepresentedanddiscussed.

Bindingsitescloningintothepsicheck2vector

Tobeabletodoluciferaseassays,whichallowtestingwhetheraputativegeneisadirecttarget

ofmiR-125b, we first constructed vectors carrying a part of 3UTR sequence of the putative

targetscontainingbindingsitesformiR-125b.Fragmentsofthegenesflankingthebindingsites

were cloned into 3UTR of the Renilla gene in psicheck2 (chk2) vector (Promega) using the

primersmentioned inMaterials andMethods.Selectedplasmidswere confirmed tohave the

bindingsitesbydigestionwithtworestrictionenzymeswhoserestrictionsequencesareatthe

ends ofeach insertion, followedbysequencingof thefragments.DUSP6/chk2,CBFB/chk2and

SP1.2/chk2carryonemiR-125bbindingsitein3UTRofthecorrespondinggenes. ETS1/chk2and

PPP2R1B/chk2containallthreeandtwobindingsites,respectivelyintheir3UTR.M4K2-1/chk2

andM4K2-2/chk2carrythefirstandthesecondbindingsite,respectively,intheORFofMAP4K2.


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Lowerbandsinallthelanesinagarosegel(Figure6),exceptemptypsicheck2vector(thebandat

lowerthan100bpinthislaneprobablyisthefragmentbetweentworecognitionsitesofXhoIand

NotI)indicatetheinsertedfragmentswiththeexpectedsizessuggestingthatallofthedesired

bindingsiteswereclonedsuccessfully.

Generationofbindingsitemutants

Deletionand/orsubstitutionmutationsofthebindingsiteswerecreatedthroughsite-directed

mutagenesis(seeMaterialsandMethods)usingprimersbearing2-4mismatchednucleotidesin

thebindingsitescomplementarytotheseedsequenceofmiR-125b.Thepurposeofgenerating

mutatedbindingsiteswastoprovidenecessaryandsufficientevidencethatthesesitesaredirect

targetsofmiR-125b,thereforethemutantswereexpectedtoabolishtheeffectofmiR-125bin

thecells.Inthisstudy,weattemptedtocreatemutantsofsevensubstitutionordeletionmutants

of five putative targets DUSP6, M4K2, PPP2R1B, SP1 and CBFB; however, due to difficulties

aroundthebindingsites,IwasunsuccessfulinmakingmutantsofM4K2.Figure7showstheside-

by-sidealignmentbetweenmutatedandwild-typebindingsites(Blat).

DUSP6Del

tgtgctaaacagtatattacctctgtataaaattct......agtgtcac

|||||||||||||||||||||||||||||||||||| ||||||||

tgtgctaaacagtatattacctctgtataaaattcttcagggagtgtcac

PPP2R1BMut

aagccacgtgttaatgtagtggttgttttggtttttatttgctcaattttgtc

|||| | ||||||||||||||||||||||||||||||||||||||||||||

aagctcagggttaatgtagtggttgttttggtttttatttgctcaattttgtc

SP1.2Del

tttacccagaacagtaacaacccacaccgtcttcctt.......ttccaactgg

||||||||||||||||||||||||||||||||||||| ||||||||||

tttacccagaacagtaacaacccacaccgtcttccttcagggatttccaactgg

SP1.2Mut

tttacccagaacagtaacaacccacaccgtcttcctcttaagctttccaactg

Figure6.CloningmiR-125bbindingsitesintothe3UTRofluciferasegeneinthepsicheck2vector.Leftto

right:DUSP6XhoI/NotI;ETS1XhoI/NotI;M4K2-1XhoI/NotI;M4K2-2XhoI/NotI;PPP2R1BXhoI/NotI;DNA

ladder1Kbplus;SP1.2XhoI/NotI;CBFBXhoI/NotI;psicheck2XhoI/NotIandDNAladder1Kbplus.


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Figure8.DUSP6isnotadirecttargetofmiR-125b.

A. FoldchangeinmRNAlevel(mRNA-seqandRQ-PCRdatafrommyfirstinternship)intransiently

transfectedNB4cellline.Thebarsrepresentmeansofrelativeexpressionmeasuredfromthree

independentexperiments.Theerrorbarsindicatestandarddeviationofthemeans.

B. FoldchangeinmRNAlevel(quantifiedbyRQ-PCR)instablymiR-125b-expressingHL60ER.Datais

representedwithmean+/-SE,n=2.

C. Luciferaseassayof3UTRofDUSP6carryingonebindingsiteofmiR-125bandmutatedbinding

site.Chk2isemptypsicheck2vectorandperfectM125bischk2containingasequenceperfectly

B

C


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complementarywithmiR-125b.Datarepresentsmeansofrelativeluminescenceunits+/-SEof

twoindependentexperiments(n=2).

ETS1

Figure9.ETS1isnotadirecttargetofmiR-125b.

A. Fold change in ETS1 mRNA level (mRNA-seq and RQ-PCR data from the first internship) in

transientlytransfectedNB4cell line.Thebarsrepresentmeansof relativeexpressionmeasured

fromthreeindependentexperiments.Theerrorbarsindicatestandarddeviationofthemeans.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Q-PCR mRNA-seq Q-PCR mRNA-seq

undifferenated differenated

RelagveETS1expression

miRctl

miR125b

A

B


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B. Luciferaseassaywiththe3UTRofETS1,carryingthreemiR-125bbindingsites.Chk2ispsicheck2

withoutan insertandperfectM125bis chk2containinga sequenceperfectlycomplementaryto

miR-125b. Data represents means of relative luminescence units +/- SE of two independent

experiments(n=2).

MAP4K2

MAP4K2wasreportedtobeup-regulatedaboutthreefoldduringnormalNB4cellmaturationto

granulocytes (Yang, Zhao etal. 2003). Itwas observedtobedecreased atmRNA levelduring

differentiation of NB4 transfected with miR-125bmimics in our previous study. Our RQ-PCR

resultsofthisgeneinstablymiR-125b-overexpressedHL60ERat0and2hoursafterinductionof

apoptosisalsoshowadecreaseinthemRNAlevel.Thissupportsthehypothesisthat MAP4K2,

whichplaysanimportantroleinsignalingpathwaysandcellresponsestostress,ismodulatedby

miR-125b.

InordertocheckwhetherornotMAP4K2couldbeadirecttargetofmiR-125b,twobindingsites

intheORFofthegenewereinsertedseparatelyintopsicheck2andco-transfectedwithmiR-125b

mimicsinto293Tcells.LuciferaseassaysrevealedthattheybothrespondedtomiR-125bandthat

the firstbinding site (closer to5UTR) showed a greateffect (luciferaseactivity decreased by

morethan50%incomparisontotheChkcontrol)thanthesecondsite(Figure10C)

Wenext examinedthe protein levelchangesdue tomiR-125boverexpression indifferent cell

lines,namelyNB4transfectedwithmimics,HL60ERXZmiR-125b(humancell line)and32DCl3ER

XZmiR-125b(mouse cell line). Thewesternblot resultsare shown inFigure 11. Itcanbeseen

fromtheFigure,therewasalmostnosignificantandconsistentchangeinMAP4K2proteinlevels

in transfected NB4 and HL60ER XZ125b compared to the controls. Oddly, there was a cleardecreaseofthisproteinin32DCl3ERXZ125bcomparedto32DCl3ERXZcells(emptyvector).Itis

hard to findabiological explanationfor this result having inmind thatbothbinding sitesare

conservedinhumanandmouse.Becauseofalackofconsistentevidenceatproteinlevel,weare

unabletoconcludewhetherornotMAP4K2isadirecttargetofmiR-125b.


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A

0

0.2

0.4

0.6

0.8

1

1.2



RelagveMAP4K2expression

miRctl

miR125b

B


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A. Figure10.ValidationofMAP4K2asapotentialtargetofmiR-125b.A. Fold change inMAP4K2 mRNA level (mRNA-seq and RQ-PCR data from the first internship) in

transientlytransfectedNB4cell line.Thebarsrepresentmeansof relativeexpressionmeasured

fromthreeindependentexperiments.Theerrorbarsindicatestandarddeviationofthemeans.

B. Fold change inMAP4K2 mRNA level (quantifiedby RQ-PCR) in constantlymiR-125b-expressing

HL60ERbeforeandduringapoptosisinduction.Dataisrepresentedwithmean+/-SE,n=2.

C. Luciferase assay of luciferase constructs containing two binding sites located in the ORF of

MAP4K2(M4K2-1andM4K2-2).Chk2isemptypsicheck2withoutaninsertandperfectM125bis

chk2containingasequenceperfectlycomplementarywiththeseedsequenceofmiR-125b.Data

representsmeansofrelativeluminescenceunits+/-SEoftwoindependentexperiments(n=2).

C


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Figure11.MAP4K2proteinquantification

A. WesternBlotofMAP4K2inthreecelllinesNB4,HL60ERand32DCl3ER.

B. RelativefoldchangeofMAP4K2proteininmiR-125btransfected/infectedcellscomparedto

controls.GAPDHwasusedasaloadingcontrol.Datawasobtainedusingdensitometryonblot

filmsusingPhotoShopCS4.0.Thebarsarethemeansofdensityofthebandsfromtwo

independentexperiments.Theerrorbarsrepresentthestandarddeviationofthemeans.

0

0.5

1

1.5

2

2.5

NB4-RA HL60 32D

ctl

miR125b

D2oftransfectionD2oftransfection

A

B


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PPP2R1B

Thenexttargetcandidatetobevalidatedattheproteinlevelis PPP2R1B,whichwasfoundtobe

down-regulated at mRNA level in NB4 cells after transfection with miR-125b. This gene is a

componentofSerine/ThreonineProteinphosphotase2(PP2A),animportantenzymethatdown-

regulatesthemitogen-activatedproteinkinase(MAPK)cascade,involvedincellcyclecontroland

growth factor signaling. PPP2R1B gene encodes the beta isoform of the constant regulatory

subunitAofPP2Aandhasbeenfoundtobemutated,inactivatedordeletedinmanytypesof

cancers,suchascolorectalcancer,cervicalcancer,lungcancerandbreastcancer(Tamaki,Goiet

al. 2004; Esplin, Ramos et al. 2006; Yeh, Hsieh et al. 2007). Relative PPP2R1B mRNA level in

HL60ER XZ125b was also dramatically decreased by more than 50% and further dropped by

approximately70-80%(2hand4h)comparedtothecontrolsafterapoptosiswasinducedinthe

cellsbyCamptothecin.

Inthenextstepofvalidation,luciferaseassayperformedwithafragmentfoundinthe3UTRof

thegenecontainingbothmiR-125bbindingsites(7mer)showedareductionofluciferaseactivity

byabout55%.TheeffectofmiR-125bwasabolishedwhenthehighlyconservedsite(oneofthe

twosites)wasmutated(Figure12C),indicatingthatPPP2R1BispotentiallyadirecttargetofmiR-

125b.WesternBlotinHL60ERXZ125batday0providedmoreevidencefor PPP2R1Bbeingareal

target (Figure 12D);however, thewesternblot inNB4 transfectedwithmiR-125bmimics and

HL60ERXZmir-125bduringapoptosis(Figure12D).Apparently,thedegreeofproteinreduction

didnotmatchwiththedegreeofmRNAlevelobservedinNB4andHL60ER.GiventhatmiR-125b

overexpressioninNB4wasachievedbytransienttransfectionwhileinHL60ERitwasobtained

throughinfection,itcouldbethatlowtransfectionefficiencyandunstablelevelofthemimicsin

thecellsledtoasmallreduction(about30%)ofmRNAresultinginundetectablechangeofthe

protein.However, that couldnot explainwhy a significant reduction inprotein levelwas not

detectedinHL60ERXZ125bafterinductionofapoptosiswhenPPP2R1BmRNAdippedat30-20%,

comparedtothecontrol.ItisnecessarytorepeatthesamevalidationinanotherconstantlymiR-

125b-expressedcell line, for example 32DCl3, tobeable todraw a satisfactory conclusion of

whetherPPP2R1BisadirectmiR-125btarget.


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Figure12.ValidationofPPP2R1BasapotentialtargetofmiR-125b.

A. FoldchangeinPPP2R1BmRNAlevel(mRNA-seqandRQ-PCRdatafromthefirstinternship)in

transientlytransfectedNB4cellline.

B. FoldchangeinPPP2R1BmRNAlevel(quantifiedbyRQ-PCR)inconstantlymiR-125b-expressing

HL60ERbeforeandduringapoptosisinduction.Dataisrepresentedwithmean+/-SE,n=2.

C. Luciferaseassayof3UTRofPPP2R1Bwithtwobindingsitesand3UTRwithonemutatedbinding

site(PPP2R1BMut1).Chk2isemptypsicheck2withoutaninsertandperfectM125bischk2

0

0.2

0.4

0.6

0.8

1

1.2



RelagvePPP2R1

Bexpression

miRctl

AB

C


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containingasequenceperfectlycomplementarywith5strandofmiR-125b.Datarepresents

meansofrelativeluminescenceunits+/-SEoftwoindependentexperiments(n=2).

D. PPP2R1BproteinlevelsinHL60ERXZ125bcomparedtothecontrol.NoreductionwasobservedinNB4transfectedwithmiR-125bmimicsandHL60ERXZ125bafter4hofapoptosisinduction.

Validationofputativetargetsinvolvedinmyeloiddifferentiation

SP1

Inmypreviousstudy,SP1wasfoundtobeapromisingmiR-125btarget.First,itwasshowntobe

decreased at mRNA level by miR-125b overexpression in NB4 cells during RA-induced

differentiation. Second, we observed the consequential down-regulation of down-stream SP1

targets,namelyCD11bandHCK,whoseregulationoftranscription istypicallyachievedthrough

binding of SP1 to the promoter regions. Note that these two genes are essential for cell

differentiation.Third,thereareseveralbindingsitesofmiR-125balongthe SP1mRNAsequence,

including4sitesintheORF(onesite8mer,onesite7merandtwosites6mer)and2othersinthe

3UTR(onesite7merAandonesite6mer).

D2oftransfection D2oftransfection


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TofurtherexaminethepossibilitythatSP1isarealtargetofmiR125bandtoindentifywhichof

the6bindingsiteshasthemainresponseelementofmiR-125b,afragmentcontainingallbinding

sitesintheORFwasclonedinthevectorpsicheck2.Thetwobindingsitesin3UTRwereputinto

twoseparatedclones.LuciferaseassaysontheORFbindingsitesaltogetherandthesecondsitein

3UTRshowednoeffect(datanotshown)whereasthefirst3UTRsite,SP1.2(7merA)responded

strongly tomiR-125b leading to a reduction of about 45% of luciferase activity (Figure 13B).

Furthermore, luciferase expressionwas recovered significantly when the site was mutated or

deleted(Figure13B).

However, the verification byWestern Blotting did not support conclusively the results from

mRNAquantificationandluciferaseassay(Figure13CandD).Nochangeintheproteinlevelof

SP1wasobservedinNB4treatedwithRAwhilethereductionobservedin32DCl3ERXZ125band

HL60ERXZ125bwasnotsignificant.WethereforeconcludethatSP1isnotadirecttargetofmiR-

125binpromyelocyticcelllines.

0

0.2

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0.6

0.8

1

1.2



RelagveSP1expression

miRctl

miR125b

A


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0

0.2

0.4

0.6

0.8

1

1.2

1.4

NB4+RA HL60 32D

ctl

miR125b

B

C

D


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subsequently leading to a decrease in protein levels. Because CBFB is essential for myeloid

differentiation(asdiscussedinthefirstreport),thereductioninCBFBproteinmayexplainthe

blockageofdifferentiationobservedinNB4and32DCl3cells.WeproposethatCBFBisadirect

targetofmiR-125binpromyelocyticcelllines.

Figure 15. mRNA levels of CBFB measured by mRNA-seq and Q-PCR before and after induction of

differentiation(normalizedtoActineandMLN51).Datarepresentsmeans+/-standarderrorofthemean

(SE)normalizedtocontrol.mRNAseqdata:n=2;Q-PCRdata:n=3.

0

0.2

0.4

0.6

0.8

1

1.2

1.4



RelagveCBFBexpression

miRctl

A


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Figure16.(A)Dual-luciferasereporterassayofHEK293Tcellsco-transfectedwithLuc-CBFB3UTR/chk2

orLuc-CBFB3UTR-MutandmiR-125bormircontrolmimics.Relativeluciferaseactivityispresentedas

means+/-SEofn=2independentexperimentsinrelationtocontrols(n=1).Emptyvector(chk2)andvector

containingperfectmatchsequencewithmiR-125b(PerfectM125b)areusedascontrols.(B)Alignmentofwild-typeCBFB3UTRandmutatedCBFB3UTRwithmiR-125b.Replacednucleotidesarein red.

B

A

D2oftransfection D2oftransfection D2oftransfection


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Figure17.ReductioninCBFBproteinlevelundertheeffectofmiR-125bwasobservedinthreedifferent

celllines.(A)DatafromWesternBlotinthreedifferentcelllines,NB4atday2oftransfection(triplicate),

NB4transfectedwithmiR-125batday2afterdifferentiationinductionwithretinoidacid(RA),HL60ER

infectedwithXZorXZmiR-125batday0(triplicate),32DCl3ERinfectedwithXZorXZmiR-125batday0,

day1andday4afterinductionofdifferentiationbyGCSF.(B)Proteinquantificationbydensitometry(B).

Datarepresentsmeansofrelativedensityofthebandsonwesternblottingfilms+/-SEofthree

independentindependentexperimentswithNB4(D2oftransfection)andHL60(D0),andoftwo

independentexperimentswithallothergroups.Statisticalsignificanceofthedifferencebetweenthe

controlandtheexperimentalcellswereperformedusingT-testwithp-valuesonthetopofthebarsof

eachgroup.

GENERALDISCUSSIONANDCONCLUSION

We I achieved two goalsof the project, firstobtainingmore invitro evidence tosupport the

hypothesis thatmiR-125bis the causative factor of leukemia (AMLandMDS)which carry the

chromosomal translocation t(2;11)(p21;q23) (Bousquet, Quelen et al. 2008) and second,

validatingsixputativetargetsofmiR-125bwhichwerefoundinthefirstinternship: DUSP6,ETS1,

MAP4K2,PPP2R1B,SP1andCBFB.

0

0.2

0.4

0.6

0.8

1

1.2

RelagveCBFBexpression

toGAPDH

ctl

miR125b

B

P=0.003 P=0.11 P=0.013 P=0.17 P=0.28 P=0.11


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MiR-125bhasbeenproventopossesstypicalfeaturesofanoncomir.Intheprimarystudy,when

up-regulatedinNB4cells,miR-125bpreventedcellsfromdevelopingintomaturegranulocytes.

Later,inunpublisheddatabyBousquetM.(2009)itwasshowntoblockapoptosisinthiscellline

aswell.NotethatthemeansofoverexpressingmiR-125bintheseexperimentswasthroughthe

introduction of an immense amount of mature microRNA directly into the cells through

electroporation,thereforealthoughthephenotypeswereundoubtedandimpressive,thereisstill

araisingcontroversyofwhetherthisisphysiologicallyrelevant.Thehurdleinaccomplishinga

physiologicaloverexpressionliesintheinabilityoftheNB4celllinetoharboraretroviralvector

carryingmiR-125b.OurattempttolookforasystemthatstablyexpressesmiR-125binamore

physiological degree led to themodification of two cell lines, HL60and 32DCl3 into the ones

bearingmurineecotopicreceptorsontheircellularmembrane(namelyHL60ERand32DCl3ER),

whichallowthemtobeinfectablebymurineretrovirus.Anotheradvantageofthismethodisthat

the vectors also contain GFP, bywhich it is possible to select cells expressingmiR-125b. The

modification of the cell lines and the selectionwere done andwill be described in detail by

BousquetM.,2010(unpublished).ThedatafromFACSanalysisandmorphologicalobservationon

32DCl3ER XZ125b and HL60ER XZ125b presented here strengthens our conclusion from the

previousstudythatmiR-125binhibitsmaturationofmyeloidprogenitorsandapoptosis.

In this study, all miR-125b target sequences in the six gene candidates were tested in the

luciferase reporter assay and protein quantification by immunoblotting. DUSP6 has been

demonstrated to be a member of the ERK signaling pathway which controls cell growth

(Furukawa,Sunamuraetal.2003;Okudela,Yazawaetal.2009)and ETS1involvedinavarietyof

cellularprocessesincludingcellproliferationandapoptosis.Interestingly,arecentstudyrevealed

thatETS1actuallyregulatestheexpressionofDUSP6(Zhang,Kobayashietal.;Okudela,Yazawaetal. 2009). Furthermore, theywere predicted tobemiR-125btargetswith highly conserved

binding sites. All of this, together with the evidence that mRNA levels of these two genes

decreasedbyinductionofthemicroRNAleadustothinkthatthesegenesweremiR-125bdirect

targets(seeResultsandDiscussion).However,ourresultssuggestthattheyarenotrealdirect

targetsbecausewedidnotobservechangesintheproteinlevels.Iproposethatthedecreasein

mRNAlevelsofboth DUSP6andETS1 couldbethe resultoftheregulationofsomeup-stream

directtargetsofmiR-125b.

The next three putative targets,MAP4K2, PPP2R1B and SP1 all passed the validation by the

luciferase reporterassay.However,when it came to protein quantification in vivo the results

were ambiguous. It is possible that in the case of MAP4K2 and SP1, the reduction of the

messengerswasnot large enough tobesignificantlydifferentfromthecontrol.However,this

maynotbethecaseofPPP2R1B,ofwhichmRNAdatafromNB4indicatedthatabout40%ofits

total transcripts were degraded by miR-125b. In HL60ER where miR-125b was expressed

constantly, the decrease was greater (approximate 45% at day 0) and became even more


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dramaticwhenapoptosiswasinduced(about65%after2hand75%after4hofCamptothecin

addition). Such a tremendous reduction in mRNA was expected to result in a drop in the

translationyieldofPPP2R1B,butthequantificationbywesternblotdidnotsupportthis.Itcould

bearguedthatthereareotherfactorsaffectingtranslation,forinstance,thecellsarenotina

high demand of the protein soonly a smallproportionof the synthesizedmRNAare used to

produce a basal level of PPP2R1B and therefore, even a huge decrease of mRNA makes no

difference.Anotherexplanationtoalltheresultsobservedinthesethreeputativetargetsisthe

technical limitationsofwesternblot(nonspecificityof theantibodiesor lowsensitivity),asmall

differencebetweentheexperimentalandthecontrolsamplescouldnotbedetected.Infact,the

correlationbetweenmRNAandproteinabundancedatawasreportedtobeloworlimitedforall

threegenes(Greenbaum,Colangeloetal.2003).Thisreviewalsopointedoutonereasonwhich

was that evenwith the significantdevelopment in the technologies used tomeasure protein

quantityoverthepast fewyears, proteinquantification andidentificationstill lagsbehind the

advanced and high-throughput techniques used for mRNA level determination. Even though

mRNAlevelshavebeenproventobeveryusefulinthediagnosisofdiseases(e.g.cancers),they

arecorrelativenotcausative.Itismostlytheconcentrationofproteinsandtheinteractionamong

themthatarethetruecausativefactorsandtherefore,theirquantitiesshouldbestudiedinmuch

greater detail (Greenbaum, Colangelo et al. 2003). To the best of our knowledge, immune-

blottinghasbeentheonlychoiceusedinthemajorityofthemicrornatargetsresearch,asthe

finalstepofdeterminingtruetargets.Nevertheless,arecentstudyhasfoundanalternativeway,

flow cytometry using chromophore-coupled antibodies, which seems to be more sensitive

(Gururajan,Hagaetal.2010).

There is another important point that needs to be discussed in microRNA target studies ingeneralandthisworkinparticular.Ingeneral,ifthedetecteddownregulationinproteinlevelis

smallbutstatisticallysignificant,thequestioniswhatmagnitudeofdown-regulationinprotein

levelcanleadtoachangeinphenotype.Forexample,ifonlyhalfofthenormallevelofsome

proteinisenoughforthecelltoperformitsroleinacell,a50%reductionofthatproteinwould

unlikelytohaveabiologicaleffect.Ontheotherhand,aphenotypecausedbyoverexpressionof

amicroRNAcouldbethecollectiveoutcomeofsmallchangesinmanyofitstargets.Takenthese

points into account when considering CBFB as a real target of miR-125b, it is necessary to

generatethesamephenotypeofblockageofmyeloiddifferentiationinthetestedcelllinesby

siRNAinordertodrawafirmconclusion.Nevertheless,thecorrelationbetweenourdataandthephysiologicalunderlyingsthathavebeendemonstratedfor CBFB(Sasaki,Yagietal.1996;Blake,

Adyaetal.2000;Huang,Shigesadaetal.2001;Kunduand Liu2003)supports thenotion that

CBFBmightbeadirecttargetofmiR-125bandalongthiswayinvolvedintheoriginofleukemias

characterizedbyat(2;11)(p21;q23)translocation.


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ACKNOWLEDGMENT

IthankNuffic(Netherlandsorganizationforhighereducation)andVrijeUniversiteitAmsterdam

for the studentship. I thankDr. Jan Kooter for helpful lectures, advice and commentson the

report.My special thanks to Prof. Harvey Lodish for providingme with the stipend, general

laboratory support and a great supervision, to Dr. Marina Bousquet for all the techniques

teaching,dailysupervisingontheproject,commentsonthereportandagreatfriendship.Ithank

Sumeet Guptaand all the staff inGenomic Core facility,Whitehead Institute for helpingwith

mRNA-seqdataanalysis, BingbingYuanandGeorgeBellwith theirhelpongene functionand

networkanalysis.IthankalltheLodishlabmembersfortechnicalsupport,VictorYehandVioleta

Rayonfortheirproofreadingthisreportandfruitfuldiscussion.

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APPENDIX

Searchingforotherputativetargets

AnumberofothercandidateschosenfromboththelistofmRNA-sequencingdataandliterature

reviewwerealsotestedbyRQ-PCRandluciferaseassays,asshowninfigure18A-18C.FormRNAsequencingdataandbiologicalpathwaysofthesegenes,seeinternshipreport1.


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Figure18A.Foldchangeofexpressionoffourgenes,APAF1,ETV6,STAT3andZNF148,undermiR-125b

overexpression,measuredbyRQ-PCRintransfectedNB4andinfectedHL60ER. Theblueandgreenbars

indicate the expression of corresponding genes inNB4 and HL60ER transfected or infected withmiR-

control,respectively,whichisconsideredas1or100%.The redandvioletbarsrepresenttheexpressionof

corresponding genes inNB4andHL60ER, transfectedorinfectedwithmiR-125b, respectively.Thedata

correspondtothemeanfromatleasttwoindependentexperiments.

Figure18B.Foldchangeofexpressionofthreegenes,PPP1CA,PPP2CAandTP53INP1,allinvolvedin

apoptosis, under miR-125b overexpression at different time points during apoptosis induction,

measuredbyRQ-PCRintheinfectedHL60ERand32DCl3ER. Thedatacorrespondtothemeanfromtwo

independentexperiments.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

APAF1 ETV6 STAT3 ZNF148

Relagveexpress

ion

HL60miRctl

HL60miR125b

NB4miRctl

NB4miR125b

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Figure18C.Luciferaseactivityisreducedsignificantlywiththebindingsitesinmostofthegenestested

byRQ-PCRinfigure18AandB.ThebindingsiteofETV6wasnotsuccessfullyclonedintopsicheck2,thus,

noresultofluciferaseassayisavailableforithere.Emptyvector(chk2)andvectorcontainingperfect

matchsequencewithmiR-125b(PerfectM125b)areusedascontrols.

diu internship report2 jk reviewed

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