[1]

Determination of the role of receptor

silencing micro RNAs in the regulation of

retinal epithelial cell fate: New insights into

therapeutic reprogramming

Emer Shelly (12355486)

Core Techniques in Biomolecular and Biomedical Research, 4th

Year Project, 2015/2016

BMOL40100

Supervisor: Dr.John Crean

BSc (Neuroscience)

[2]

Table of contents Page

Abstract 3

Introduction 4

Materials and Methods 8

Results 11

Discussion 19

Acknowledgements 26

References 27

[3]

Abstract

The TGFβ signalling pathway has been widely shown to be linked to the pathobiology of

diabetic retinopathy, the progression of which is accepted to feature epithelial differentiation.

Recent therapeutics have therefore focused on targeting this pathway by interacting with its

receptors and changing the fate of the cell by creating a pluripotent stem cell. Ever since the

rise of iPSCs and the four Yamanaka factors (Oct3/4, Sox2, Klf4, c-myc), the need for an

efficient method of generating these stem cells from within the adult tissue has been a major

focus. With the discovery of microRNAs and their ability to regulate cell fate and generate

pluripotency, came a new and promising avenue for cell reprogramming in the treatment of

many diseases such as diabetic retinopathy. In this investigation we focused on the mir-302

cluster of microRNAs and investigated its role in the regulation of retinal epithelial cell fate.

We identified the TGFβ type II receptor as a direct target of mir-302 and demonstrated that

mir-302 promotes pluripotency within ARPE cells in vitro, through the regulation of signal

transduction and epigenetic changes. We also investigated the role of small molecules

DZNEP and SB431542 in inducing pluripotency and found that they also cause attenuation of

the aberrant signalling pathways involved in cell differentiation. Our results show evidence

that supports the belief that microRNAs will continue to be at the forefront of regenerative

medicine and with more research, small molecules will also contribute to this new and

exciting field.

[4]

Introduction

Diabetes mellitus is a group of chronic conditions which results from the inability of the

pancreas to produce insulin (Type I) or the inability of the body to utilise it (Type II).

Diabetes Ireland estimates that ~200,000 people suffer from diabetes in the 20-79 age group.

The injurious effects of hyperglycemia in diabetes can manifest as macrovascular

complications such as coronary artery disease, peripheral arterial disease and stroke, or

microvascular complication such as nephropathy and retinopathy. Diabetic retinopathy is

currently the leading cause of blindness in adults in the developed world and according to the

U.S Centers for Disease Control and Prevention the number of cases of diabetic retinopathy

will rise to ~16 million by 2050. Diabetic retinopathy is a progressive disease predominantly

affecting the integrity of the microscopic blood vessels of the retina(Williams et al. 2004).

Damage to these blood vessels causes them to leak blood and other fluids which cause

swelling of the retinal tissue and clouding of vision. Current therapies for the later stages of

the disease include using corticosteroids, photocoagulation, and using anti-angiogenic

factors(Ciulla et al. 2003). While these treatments are effective in delaying and reducing

vision loss they are not a cure for the disease. Recently however new therapies are beginning

to emerge on the idea that populations of cells within the eye have the ability to self renew

and by utilising this ability, new treatments could aim to reverse the effects of this

debilitating disease.

The exact mechanism by which Diabetic Retinopathy (DR) occurs is not fully understood

however Gerhardinger et al showed that the retinal vessels of diabetic rats showed differential

expression of 20 genes of the transforming growth factor-beta (TGFβ) pathway in addition to

genes involved in oxidative stress, inflammation, vascular remodelling and apoptosis. TGFβ

superfamily is an important group of cytokines and regulates a wide variety of functions

within the majority of multi-cellular organisms. This pathway has been shown to be involved

in the pathobiology of the eye by many different groups and is currently being investigated as

a potential therapeutic target for the reprogramming of cells in diseases such as DR. When a

TGFβ receptor is bound by a ligand, a heterotetrameric complex forms, consisting of two

type I and two type II serine/threonine kinase receptors (demonstrated in Fig 1). The type I

receptor is then phosphorylated by a type II receptor which then recruits and phosphorylates

R-Smad. R-Smad then dimerizes with a common mediator (co)Smads to form a

[5]

herterodimeric complex which then translocates to the nucleus with a DNA binding partner

(DBP), where it acts as a transcription factor for various genes (Massagué et al. 2005). Other

than smad-mediated transcription, TGFβ can activate other signalling pathways like the MAP

kinase pathways. Some MAPK pathways can interact with smad activation and there is much

evidence of cross-talk between the two signalling cascades (Javelaud & Mauviel 2005).

Fig 1. Simplified diagram of the key downstream signaling pathways activated by the TGFβ

receptor. Adapted from: A model of Smad-dependent signalling pathway activated by TGF-β.

Motifolio, Biomedical Poweroint Toolkit for Presentations. (Online source)

TGFβ signalling cascades as mentioned have been associated with playing a central role in

the pathomechanisms responsible for the development of ocular diseases like DR. TGFβ

signalling pathways can induce EMT (epithelial to mesenchymal transition), a process by

which an epithelial cell undergoes multiple biochemical changes to allow it to assume a

mesenchymal cell fate. Polarized epithelial cells normally interact with the basement

membrane via their basal surface. Once EMT occurs, the cell loses its interaction with the

basement membrane therefore has increased migratory capacity, invasiveness, elevated

[6]

resistance to apoptosis and increased production of extracellular matrix components (Kalluri

& Weinberg 2009). The first sign of EMT is a loss of epithelial cell adhesion proteins such as

e-cadherin, which is a calcium-dependent protein located at junctions between epithelial cells

(Pećina-Slaus 2003). This protein is suppressed by TGFβ induced EMT which leads to the

breakdown of tight junctions. The next stage of EMT is the expression of α-smooth muscle

actin (α-SMA) and actin reorganisation, allowing cells to migrate and contract. The cells

intermediate filaments also change from a keratin rich network which connects to adherens

junctions to a vimentin-rich network connecting to focal adhesions (Kokkinos et al 2007). In

order for EMT to reach completion, activation of transcription factors, expression of cell

surface proteins, reorganization and expression of cytoskeletal proteins and changes in the

specific microRNAs occurs(Kalluri & Weinberg 2009).

Fig 2. Simplified diagram of epithelial to mesenchymal transition. Adapted from

Douglas S. Micalizzi, Susan M. Farabaugh, Heide L. Ford (2010) Epithelial-Mesenchymal

Transition in Cancer: Parallels Between Normal Development and Tumor Progression.

Journal of Mammary Gland Biology and Neoplasia. Volume 15, Issue 2, pp 117-134

Current research is looking at reversing EMT by means of MET (mesenchymal to epithelial

transition) by the reprogramming of the cell to ESC-like pluripotency. The new era of

reprogramming began with induced pluripotent stem cells (iPS cells). iPS cells are adult cells

that have been genetically reprogrammed to an embryonic stem cell-like pluripotency and can

be manipulated into proliferating and dedifferentiating into the cell type required. The first

[7]

iPS cells were generated by Takashashi and Yamanaka in 2006 , by first introducing four

embryonic factors Oct3/4, Sox2, c-Myc and Klf4 into mouse and adult fibroblasts and then

into human fibroblasts in 2007. However iPSCs produced by the four factor method tend to

be tumorigenic, making them unsafe for clinical application(Kelley & Shi-Yung 2012). In the

last few years there has been much interest and promising evidence in the field of using

microRNAs to induce this reprogramming of the cell. MicroRNAs are ~22 nucleotide small

non-coding RNAs and are highly conserved among species. In mammals, miRNAs act as

post transcriptional regulators to reduce expression of target genes by destabilizing mRNAs

or blocking their translation. Numerous reports have shown their ability to reprogram cells to

iPSC.

Studies by Chen et al looked at human miRNA expression profiles using microarrays. 304

miRNAs were found to be differentially expressed in TGFβ induced EMT in human Retinal

Pigment Epithelial Cells (RPEs). Of these, 183 miRNAs were downregulated and 119

upregulated at least 2-fold in TGFβ-treated samples. Yang et al showed that by using

specific groups of microRNA clusters, they can interfere with EMT and reverse it through

means of MET. They found that introducing the mir-302 cluster caused an enhancement of

epithelial properties and prevented TGFβ induced EMT(Yang & Rana 2013). This kind of

development in reprogramming the fate of cells is extremely important in getting closer to

developing a therapeutic strategy for multiple diseases including DR.

The aim of this project therefore was to investigate the phenotypic, signalling and epigenetic

effects of miR-302 when it attenuates the TGFβ pathway and its aberrant signalling in ARPE

cells in vitro. We will look at how miR-302 and also how pharmacological small molecules,

DZNEP and SB431542, can regulate the TGFβ signal and what implications this has for the

fate of these cells. Through this we will investigate the role of miR-302 in the reprogramming

of cells in disease and examine its implications for future therapeutics.

[8]

Materials and Methods

1. Making plasmids and isolating plasmid DNA

Plasmids were made by growing DH5-alpha E.coli containing the plasmids pCMV,

pGipz, pMir302 and pMD2G on agar plates.

Viral plasmids were purified by isolating a single colony and using the Qiagen Maxi

Prep kit as per normal protocol.

2. Cell culture-ARPEs and HEKs

Primary human apical retinal epithelial cells were cultured in DMEM F-12 Hams

media (Sigma) supplemented with 10% fetal bovine serum (FBS), 100 IU/µl

penicillin, 100µg stremtomycin and 2mmol L-glutamine (all from Invitrogen, Paisley,

UK). HEX-293T, cells were cultured in DMEM (Lonza) supplemented with FBS,

PenStrep, and L-Glutamine also. Cultures were maintained at 37°C in an environment

of 5% CO2/95% air and were serum restricted (0.2% FBS) 24 hours prior to all

transfections.

3. Transfection, Cell stimulation and Viral transduction

Scrambled and miR-302 were made by adding pCMV and pMD2G (packaging

vectors) to both pGipz for the scrambled virus or pMir for the mir-302 virus

ARPE-19 cells were transfected with the viral DNA (scrambled and mir-302). 2 days

post transfection, the virus was centrifuged, filtered and 1 part virus supernatant, 3 part

media were transduced onto ARPE cells at ~70% confluency for 5 days.

ARPE cells were stimulated with TGFβ for 24 and 48 hour time points.ARPE cells 7

day post transduction were stimulated with TGFβ for 72 hours. ARPE cells 7 day post

transduction were stimulated with DZNEP at 5μm (Abcam) or SB431542 at 5μm

(Tocris) both at 1:1000 dilution.

[9]

4. Protein isolation, quantification, SDS polyacrylamide gel electrophoresis and

western blotting

Total protein was extracted from cells using RIPA lysis buffer (Tris-HCl, 10% NP-40,

100mM EDTA, 10% Na-deoxycholate, supplemented with protease/phosphatase

inhibitor cocktails on day of use).

The protein concentrations of the samples were determined using the Bradford

method, using Bovine serum albumin for the standard assay and the absorbance

measured at 595nm.

Protein samples were run in 10% polyacrylamide SDS gels using H2O, 30%

acrylamide mix, 1.5M Tris (loading gel pH 8.8, stacking gel pH6.8), 10% ammonium

persulfate and TEMED (all from Sigma), transferred to polyvinylidene fluoride or

nitrocellulose membrane and probed for β-actin, TGFβ, Smad2/3, Phospho-Smad2/3,

fibronectin, α-SMA, E-cadherin, EZH2, and Vimentin using ECL, WestDura or Sirius

detection kit. The table below shows the dilutions that were used for the primary and

secondary antibodies:

[10]

5. Immunofluorescence

Confluent ARPE cells were transfected with non-transduced, scrambled or miR-302

virus for 24 hours and serum restricted prior to being treated with TGFβ, DZNEP and

SB431542 for 48 hours. Cells were fixed with 4% paraformaldehyde(EMS, Fort

Washingtn,PA), permeabilised with 0.1% Triton X-100 (Sigma), blocked with 5%

goat serum (Sigma) and stained for ZO1(1:200 primary dilution, 1:500 secondary

dilution) and Alexa fluor 488 (phalloidin-1:200 primary, 1:500 secondary dilution) and

counterstained with DAP1 (1:1000 dilution). Images were acquired using an Axiovert

200M or Imager.MI microscope and processed with Axiovision 4.0 (Carl Zeiss, Jena,

Germany).

[11]

Results

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

Discussion

Stem cell generation in regenerative medicine has faced many issues, some of which have

been overcome since the rise of iPS cells. The need for a constant supply of pluripotent stem

cells for replication into different cell types that can be used in tissues of the body was a

major issue. Adult somatic multipotent stem cells whilst shown to have the ability to

differentiate into many different cell types(Hanna et al. 2008), they tend to contain more

mutations and are less adaptable than pluripotent stem cells. ESCs are clearly much more

adaptable and diverse, however there are ethical issues surrounding these types of cells as

they must be taken directly from the unborn embryo. With the development of iPS cells, cells

that have the properties of ESCs can be generated from an adults own tissue and are as

pluripotent and diverse without all of the surrounding ethical issues. The generation of these

iPS cells however has been another major challenge, with retroviruses producing some

properties that make iPSCs improper for cell therapy and reprogramming factors in some

cases promoting tumour development (Medvedev et al. 2010). The need for a method of

generating iPS cells that does not cause these problems has led to the recent focus on

investigating the role of microRNAs in inducing stem cell pluripotency. This is because

members of the miR-302 family have been shown to be expressed uniquely in ESCs and to be

a direct target of Oct4 and Sox2 which are critical transcription factors involved in

maintaining the pluripotent ESC phenotype(Rodda 2005). Inparticular it was discovered by

Faherty et al that the miR-302 family of microRNAs acts on the TGFβ type II receptor and

inhibits it, which causes decreased TGFβ-induced EMT in renal HKC8 cells(Faherty et al.

2012). Subsequently, the aim of this research project was to investigate the role of miR-302

in the regulation of pluripotency, where TGFβ is overexpressed in cells of the epithelium of

the retina in vitro. We have shown that miR-302 mimic targets and knocks down the TGFβ

type II receptor (Fig 4), which promotes the generation and maintenance of stem cell

pluripotency.

First we wanted to look at how overexpression of TGFβII affects the phenotype, signalling

and epigenetics of ARPE cells (Fig 2). We expected to see responses in the cell due to TGFβ

aberrant signalling such as cell migration and EMT, which are critical during embryogenesis

but also in the development of fibrotic diseases (Lee et al. 2013). We observed increased

expression of the widely accepted mesenchymal markers in vitro; fibronectin, α-sma and

EZH2. Fibronectin binds extracellular matrix proteins such as collagen and fibrin and is an

[20]

established marker of EMT. α-sma plays a role in upregulating fibroblast contractile

activity(Hinz et al. 2001). EZH2 (enhancer of zeste 2 polycomb repressive complex 2

subunit) is a catalytic subunit involved in gene silencing through the methylation of H3K27

on the chromatin of DNA(Yamaguchi & Hung 2014), and is activated by the transcription

factor Snail1(Herranz et al. 2008). It is upregulated in cells undergoing EMT and in cancer

initiation, development and metastasis (Yamaguchi & Hung 2014). Overexpression of TGFβ

also increased the phosphorylation of Smad-2 and Smad-3 in the ARPE cells. This indicates

that the TGFβ canonical pathway is being activated and the signal is travelling downstream to

the nucleus via the phosphorylation of Smads (canonical pathway) and subsequently their

interaction with EZH2 to switch off epithelial genes. Total Smad 2 and total Smad 3 showed

a slight decrease due to the fact that some were becoming phospho-Smads. E-cadherin was

down-regulated in this experiment, which is another hallmark of EMT(Larue & Bellacosa

2005). E-cadherin plays an important role in maintaining epithelial integrity in cells. TGFβ

therefore induces the differentiation of a cell from epithelial to less epithelial and towards a

more mesenchymal resembling cell in vitro.

Next we wanted to see the effects of miR-302 on healthy ARPE cells. MiR-302 treated cells

showed rescue of the epithelial marker e-cadherin (Fig 3.1) and decreased activation of EZH2

compared to scrambled virus which caused complete loss of e-cadherin. This indicates that

miR-302 rescues the cell from the loss of e-cadherin by inhibiting EZH2 and therefore

inhibiting its transcriptional repression at the chromatin. The chromatin is no longer

methylated and transcription factors that turn on the gene for e-cadherin can access the DNA.

We expected to see a decrease in the expression of fibronectin in this experiment when in fact

we saw an upregulation of this marker. However on further inspection, it is believed that

fibronectin needs cooperative signalling between TGFβ and other signalling

pathways(Margadant & Sonnenberg 2010), which is possibly why we did not see its

downregulation when cells were treated with miR-302.The immunofluorescence imaging

(Fig 3.2) showed that miR-302 caused the cells to demonstrate a more epithelial-like

phenotype when compared to cells transfected with scrambled virus. We examined the

expression of two established cell type specific markers. ZO-1 is a tight junction protein

found in epithelial cells and would not be found in fibroblasts. F-actin(filamentous actin) is a

component of the cytoskeleton important for mobility and contraction of cells during cell

division and would be found during actin remodelling. MiR-302 transduced cells show a

redistribution of f-actin and junctional associated staining of ZO-1. Cells not treated with

[21]

miR-302 show less of an epithelial phenotype with loss of cobblestone morphology, which is

an epithelial morphological characteristic in vitro (Davis et al. 1995). These results indicate

that when ARPE cells are transduced with miR-302 in vitro, it maintains the cells at a

pluripotent state and prevents the loss of tight junctions and the induction of mobility and

contraction in cells.

We next wanted to investigate whether transduction of TGFβ treated cells with miR-302

could induce plasticity in the cells so that they would be rescued from progressing into

fibrosis. After the ARPE cells were transduced with miR-302, scrambled virus or control

(non-transduced) for 48 hours prior to being treated with TGFβ, the cells were assessed by

Western blot analysis. We hypothesized that miR-302 would cause downregualtion of

mesenchymal markers and upregulation of epithelial markers in the cells treated with TGFβ

and no change would be seen in the cells with TGFβ containing no miR-302. The cells

transduced with scrambled virus and treated with TGFβ showed very little change (Fig 4).

Epithelial marker e-cadherin was completely absent from scrambled virus and α-SMA was

increased. The levels of p-Smad 2 were also increased due to the activation of the TGFβ

signalling pathway. These cells demonstrated a mesenchymal phenotype due to the over

expression of TGFβ and the activation of the pathway through the TGFβ type II receptor.

Successful knockdown of the TGFβ type II receptor was reflected in the cells pre-treated with

miR-302, confirming that the receptor was a true target of miR-302. This was proven by the

observation that e-cadherin was rescued in the miR-302 + TGFβ cells, indicating that the

transcriptional repression of this marker was being repressed. We also were interested in

whether the knockdown of TGFβ type II receptor was reflected in altered signalling

pathways. Phosphorylated smads by the activated TGFβ heterotetrameric complex translocate

to the nucleus where they can regulate gene expression. We propose that by knocking out the

TGFβ type II receptor, the activation of smads would be inhibited and importantly

differentiation by EMT would be reversed. From our results (Fig 4), p-smad2 levels were

decreased in cells transfected with miR-302 compared to cells that did not have miR-302,

proving that miR-302 has a knock on effect downstream in the signalling pathway of TGFβ.

MiR-302d interestingly however offered no protection against fibronectin. Fibronectin is a

key matrix protein accumulated in excess in diabetic retinopathy. As mentioned previously,

fibronectin may need cooperative signalling for it to be regulated. We would expect EZH2

levels to be decreased in cells with miR-302 + TGFβ, due to the fact that e-cadherin was

upregulated. However in this case the levels were not lower when compared with scrambled

[22]

and non-transduced. This may be due to many factors such as the cells could have suffered in

cell culture if they were not fed correctly which may have disrupted their

epigenetics(Villeneuve & Rama 2010),(Elder & Dale 2010).Looking at our results

collectively however miR-302 alters signalling and transcriptional responses which seem to

cause the cell to become more pluripotent and reverse specification by EMT. Because these

cells have been pushed to a more plastic state, they now have the ability to dedifferentiate

into healthy ARPE cells. This finding demonstrates the ability of miR-302 in the

reprogramming of cells in many diseases. Other members of the miR-302 family, such as

miR-302s, have also been proven to be effective in reprogramming cancer cells into an ES-

like pluripotent state and maintaining this, even under a feeder-free culture condition(Lin et

al. 2008), which again shows that this type of therapeutic holds much promise in the

reprogramming of cells in many conditions.

Next we wanted to investigate the effects of two small molecules on the TGFβ type I/II

receptors and if they could enhance iPSC generation. We expected that if these small

molecules were effective, they could be used therapeutically in the same way as miR-302 in

regulating TGFβ induced EMT by blocking the TGFβ type I/II receptor and reversing the

mesenchymal phenotype to more a more pluripotent cell. Cells were treated with either

SB431542 or DZNEP for 1 hour prior to stimulating with TGFβ. SB431542 is a specific

inhibitor of TGFβ-type I receptor(Inman et al. 2002) and DZNEP globally inhibits histone

methylation and returns histones back to their original state, allowing transcription factors to

access all of the genes for their expression(Miranda et al. 2009).When cells were left for 48

hours, we found that in cells treated with the TGFβ only, there was an induction of EMT as

expected (Fig 5). This was observed by the upregulation of mesenchymal markers

fibronectin, α-SMA and the phosphorylation of Smad-3. It was also observed that e-cadherin

levels were decreased. In the plates treated with the SB431542 only, there was no indication

of a mesenchymal phenotype. However when cells were pre-treated with the SB431542 drug

and then stimulated with TGFβ, there was a decrease in the expression of mesenchymal

markers fibronectin and α-SMA compared to cells that just had TGFβ. The phosphorylated

levels of Smad3 were also decreased. However there was no rescue of the e-cadherin by

SB431542. These results allowed us to evaluate the effectiveness of the drug SB431542 in

altering the mesenchymal phenotype of ARPE cells in vitro. SB431542 whilst an effective

downregulator of fibronectin, α-SMA and p-Smad3, did not rescue the epithelial marker e-

cadherin. This is because SB431542 does not act at an epigenetic level so does not inhibit the

[23]

H3K27 methylation by EZH2 so that the e-cadherin gene can be turned back on. This may be

an important observation in the future as the use of this molecule comes into focus for the

induced pluripotency of ARPE cells in diseases like diabetic retinopathy. SB431542 from

our results and from other groups seems to be an effective inhibitor of the TGFβ pathway

upstream. Inman et al showed that SB431542 blocked the phosphorylation and nuclear

translocation of Smads and there was decreased TGFβ mediated transcription. In this

investigation human glioma cells were used and treatment with SB431542 offered inhibited

proliferation, TGFβ mediated morphological changes and cellular motility. We believe that

this molecule could be efficient in improving the efficiency of human iPSC generation;

however the experiment would need to be repeated for more conclusive results.

In the plates treated with DZNEP+TGFβ, a downregulation of α-SMA was seen and a rescue

of e-cadherin (Note: E-cadherin only and TGFβ only were loaded the wrong way around-

DZNEP only: has increased expression of E-cad and TGFβ only: has no expression of E-cad).

However there was no change in the level of phosphorylated Smad-3, and there was a

reduction in the levels of EZH2, which indicates that DZNEP does not alter the upstream

TGFβ signalling pathway but prevents the expression of mesenchymal markers and allows

for the transcription of epithelial markers by acting downstream at the polycomb repressive

complex which contains the catalytic subunit EZH2. Other groups have proven that

pharmacological therapy that uses DZNEP to inhibit EZH2 in the growth of cancer cells in

the lung may be a novel approach to treating human malignancies (Kikuchi et al. 2012). From

our findings, DZNEP works similarly to miR-302 in that it prevents the cells from

specification (to a mesenchymal cell), and gives them a more pluripotent phenotype.

Importantly DZNEP seems to work at an epigenetic level which is very important in the

reprogramming of cells to induce ESC-like pluripotency, as it allows the access of the entire

genetic material for transcription factors (Liang & Zhang 2013). This stemness is therefore

not a one way mechanism but means that the cells can be manipulated in any direction which

is desired, which displays great therapeutic benefit.

We also wanted to investigate the effects of treating ARPE cells with a combination of miR-

302 and DZNEP. ARPE cells were transduced with miR-302/scrambled virus and stimulated

with DZNEP (Fig 6.1). Cells with miR-302 and DZNEP demonstrated increased expression

of e-cadherin and decreased expression of α-SMA compared to scrambled virus. We know

that miR-302 blocks the TGFβ type II receptor, causing e-cadherin to be restored and α-SMA

to be downregulated. DZNEP by inhibiting EZH2 from catalyzing the methylation of the

[24]

H3K27 mark allows the cell to express e-cadherin once again because it is not being

repressed by EZH2. Therefore DZNEP increased the expression of e-cadherin even more

when used in combination with miR-302. Immunofluorescence imaging also showed that

cells with miR-302 and DZNEP showed decreased staining for filamentous actin (Fig 6.2).

Filamentous actin is characteristic of a mobile, contracting cell. MiR-302 and DZNEP

completely abolish f-actin from the cells when used in combination, which like the previous

results, show that they bring the cell to a stem cell-like phenotype. However in this

experiment the final immunofluorescence image in figure 6.2, showed that there were very

little cells visible, therefore it was difficult to confirm the effects of miR-302 and DZNEP on

the cells. This lack of cells may have been due to the amount of time that the cells were

transduced for (14 days), or the washing of the cells was too vigorous, causing them to die, or

possibly their lack of adhesion to the slide. This investigation showed that a therapeutic that

targets the epigenetics of the cell, allows for a more desirable result than one that doesn’t. In

order for e-cadherin to be expressed in a previously differentiated mesenchymal cell, there

must be alterations made to its epigenetics so that the gene for e-cadherin can was switched

back on. Other groups have also shown the promise of small molecules in stem cell

regeneration where recently it was shown that the use of these small molecules in conjunction

with microRNAs greatly increased the efficiency of direct reprogramming and could even

replace transcription factors to induce reprogramming in some cases(Lewis et al. 2015).

From our investigations, it is evident that miR-302 has a crucial role to play in the future of

regenerative medicine. MiR-302 has shown promising results in the reprogramming of a cell

through its regulation of the TGFβ signalling pathway through interaction with the TGFβ

type II receptor, its induction of MET and its ability to induce stem-cell pluripotency in

ARPE cells. MiR-302 is involved in the activation of embryonic stem cell-specific gene

expression, inhibition of developmental signaling and prevention of stem cell tumorigenicity

(Sell 2013). How miR-302 specifically interacts with transcription factors involved in

embryonic stem cell differentiation is also interesting because core factors, Oct4 and NR2F2

are pivotal for maintaining the undifferentiated state. Rosa et al showed that miR-302 is

linked to these factors through regulatory circuitry that critically regulates pluripotency and

differentiation in human ESCs. This further demonstrates that miR-302 can be used to

promote an undifferentiated state in cells.

DZNEP and SB431542 also appear to be promising in the field of reprogramming as they

also inhibit the TGFβ pathway and therefore contribute to the induced pluripotency of a cell.

[25]

Our findings suggested that the polycomb mediated repression is a major aspect of epithelial

cell differentiation as de-repression caused the restoration of aspects of epithelial de-

differentiation. Better understanding of the role of small molecules like DZNEP in the

regulation of epigenetics will no doubt open up more opportunities for treating this disease

through reprogramming. Future work in this field will therefore focus on using in vivo

models of disease for better accuracy of results, understanding more about the role of miR-

302 used in combination with pharmacological small molecules in cellular reprogramming,

investigating its mechanisms of transcriptional and epigenetic regulation, and how its abilities

can be translated into being used to as a medication that manipulates cell fate in conditions

such as DR

[26]

Acknowledgements

I would like to firstly thank Dr. John Crean for giving us the opportunity to undertake this

project and for his continued guidance and support over the last couple of months.

I would secondly like to thank Darrell Andrews and Mary Doran for their continuous help in

carrying out the experiments and in their support throughout the whole project.

Finally thank you also to Thomas Dodd and Hayley Beaton for their support and knowledge

while carrying out the project.

I would like to wish everyone involved in the continuous research in this exciting field in

UCD Conway Institute of Biomolecular and Biomedical Science all the best in the future.

[27]

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Assessment Submission Form

Student Name Emer Shelly

Student Number 12355486

Assessment Title Determination of the role of receptor silencing microRNAs in the regulation of retinal epithelial cell fate: New insights into therapeutic reprogramming

Module Code BMOL40100

Module Title Core Techniques in Biomolecular Research

Module Co-ordinator Dr. John Crean

Tutor (if applicable) Darrell Andrews

Date Submitted 27/11/15

Date Received

Grade/Mark

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Declaration of Authorship

I declare that all material in this assessment is my own work except where there is clear acknowledgement and

appropriate reference to the work of others.

Signed…………Emer Shelly……………………………………. Date ………………27/11/15……………………………

Assessment submission form_modular