This document summarizes a study investigating the role of microRNA-302 in regulating retinal epithelial cell fate by targeting the TGF-β type II receptor. The study demonstrates that microRNA-302 promotes pluripotency in ARPE cells in vitro by regulating TGF-β signaling and epigenetic changes. It also shows that small molecules DZNEP and SB431542 can induce pluripotency by attenuating pathways involved in cell differentiation, supporting the potential for microRNAs and small molecules in regenerative medicine and therapeutics for diseases like diabetic retinopathy.

1. [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. [2]
Table of contents Page
Abstract 3
Introduction 4
Materials and Methods 8
Results 11
Discussion 19
Acknowledgements 26
References 27

3. [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. [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. [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. [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. [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. [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. [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. [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. [11]
Results

12. [12]

13. [13]

14. [14]

15. [15]

16. [16]

17. [17]

18. [18]

19. [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. [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. [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. [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. [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. [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. [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. [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. [27]
References
CIULLA, T.A., AMADOR, A.G. & BERNARD, Z., 2003. Diabetic Retinopathy and
Diabetic. , 26(9), pp.2653–2664.
Davis, a et al., 1995. Human Retinal Pigment Epithelial Cell Line That etains Epithelial
Characteristics After Prolonged Culture. Investigative Ophthalmology, 36(5), pp.955–
964.
Elder, K. & Dale, B., 2010. In vitro fertilization third edition,
Faherty, N. et al., 2012. CCN2/CTGF increases expression of miR-302 microRNAs, which
target the TGF type II receptor with implications for nephropathic cell phenotypes.
Journal of Cell Science, 44(0), pp.5621–5629.
Hanna, J., Carey, B.W. & Jaenisch, R., 2008. Reprogramming of somatic cell identity. Cold
Spring Harb Symp Quant Biol, 73, pp.147–155.
Herranz, N. et al., 2008. Polycomb Complex 2 Is Required for E-cadherin Repression by the
Snail1 Transcription Factor. Molecular and Cellular Biology, 28(15), pp.4772–4781.
Hinz, B. et al., 2001. Alpha-smooth muscle actin expression upregulates fibroblast contractile
activity. Molecular biology of the cell, 12(9), pp.2730–41.
Inman, G.J. et al., 2002. SB-431542 is a potent and specific inhibitor of transforming growth
factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4,
ALK5, and ALK7. Molecular pharmacology, 62(1), pp.65–74.
Javelaud, D. & Mauviel, A., 2005. Crosstalk mechanisms between the mitogen-activated
protein kinase pathways and Smad signaling downstream of TGF-beta: implications for
carcinogenesis. Oncogene, 24(37), pp.5742–5750.
Kalluri, R. & Weinberg, R. a, 2009. Review series The basics of epithelial-mesenchymal
transition. Journal of Clinical Investigation, 119(6), pp.1420–1428.
Kelly, K. & Shi-Yung, L., 2012. Chapter 4 – Induction of Somatic Cell Reprogramming
Using the MicroRNA miR-302. Progress in molecular biology and translational
science, 111, pp.83–107.
Kikuchi, J. et al., 2012. Epigenetic therapy with 3-deazaneplanocin A, an inhibitor of the
histone methyltransferase EZH2, inhibits growth of non-small cell lung cancer cells.
Lung Cancer, 78(2), pp.138–143.
Kokkinos, M Wafa, R Wong, M.K. Newgreen, D.F. Thompson, E.W.a, W.M., 2007.
Vimentin and Epithelial-Mesenchymal Transition in Human Breast Cancer –
Observations in vitro and in vivo. Cells, Tissues, Organs, 185(1-3), pp.191–203.
Larue, L. & Bellacosa, A., 2005. Epithelial–mesenchymal transition in development and

28. [28]
cancer: role of phosphatidylinositol 3′ kinase/AKT pathways. Oncogene, 24(50),
pp.7443–7454.
Lee, J., Choi, J.-H. & Joo, C.-K., 2013. TGF-β1 regulates cell fate during epithelial-
mesenchymal transition by upregulating survivin. Cell death & disease, 4(7), p.e714.
Lewis, C. et al., 2015. Direct Reprogramming Facilitated by Small Molecules. Journal os
stem cell and transplantation biology, 1(1), p.103.
Liang, G. & Zhang, Y., 2013. Embryonic stem cell and induced pluripotent stem cell: an
epigenetic perspective. Cell Research, 23(1), pp.49–69.
Lin, S.-L. et al., 2008. Mir-302 reprograms human skin cancer cells into a pluripotent ES-
cell-like state. RNA (New York, N.Y.), 14(10), pp.2115–24.
Margadant, C. & Sonnenberg, A., 2010. Integrin-TGF-beta crosstalk in fibrosis, cancer and
wound healing. EMBO reports, 11(2), pp.97–105.
Massagué, J., Seoane, J. & Wotton, D., 2005. Smad transcription factors. , pp.2783–2810.
Medvedev, S.P., Shevchenko, a I. & Zakian, S.M., 2010. Induced Pluripotent Stem Cells:
Problems and Advantages when Applying them in Regenerative Medicine. Acta
naturae, 2(2), pp.18–28.
Miranda, T.B. et al., 2009. DZNep is a global histone methylation inhibitor that reactivates
developmental genes not silenced by DNA methylation. Molecular cancer therapeutics,
8(6), pp.1579–88.
Pećina-Slaus, N., 2003. Tumor suppressor gene E-cadherin and its role in normal and
malignant cells. Cancer cell international, 3, p.17.
Rodda, D.J., 2005. Transcriptional Regulation of Nanog by OCT4 and SOX2. Journal of
Biological Chemistry, 280(26), pp.24731–24737.
Sell, S., 2013. Stem Cells Handbook,
Villeneuve, L.M. & Rama, N., 2010. The role of epigenetics in the pathology of diabetic
complications. American Journal of Physiology - Renal Physiology, 299(1), pp.14–25.
Williams, R. et al., 2004. Epidemiology of diabetic retinopathy and macular oedema: a
systematic review. Eye, 18(10), pp.963–983.
Yamaguchi, H. & Hung, M.C., 2014. Regulation and role of EZH2 in cancer. Cancer
Research and Treatment, 46(3), pp.209–222.
Yang, C.-S. & Rana, T.M., 2013. Learning the molecular mechanisms of the reprogramming
factors: let’s start from microRNAs. Molecular biosystems, 9(1), pp.10–17.

29. [29]
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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the University’s Policy on Late Submission of Coursework, (http://www.ucd.ie/registrar/)
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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