Investigating the role of microRNAs in hypertrophic and keloid scar formation Dominic Guanzon Bachelor of Biomedical Science (First Class Honours) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biomedical Sciences Faculty of Health Queensland University of Technology 2017
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hypertrophic and keloid scar formation
Dominic Guanzon
Bachelor of Biomedical Science (First Class Honours)
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the best
of my knowledge and belief, this thesis contains no material previously published or
written by another person except where due reference is made.
Signature: QUT Verified Signature
ii
Acknowledgments
Firstly I would like to acknowledge my primary supervisor Tony Parker. I can’t
thank you enough for all the support you have given me, I wouldn’t have been able to
complete my PhD without your help. I enjoy the conversations we have, and I really
appreciate the feedback you have given me regarding my thesis and project. I would
also like to thank my former primary supervisor David Leavesley for his guidance and
support throughout my honours and half way through my PhD, the experience was
enjoyable. I would also like to acknowledge my current and former associate
supervisors Ana Pavasovic, Brett Hollier and Kerry Manton for their support.
Furthermore, I would like to thank the Genomics laboratory at CARF for their
excellent service regarding NGS, with specific mention to Vincent Chand, Kevin
Dudley and Sahana Manoli. This PhD project would have not been possible without
your help. I would also like to acknowledge the Optical and electron microscopy
laboratory for their help regarding TEM and imaging of exosomes, with specific
mention to Rachel Hancock and Jamie Riches.
I would like to acknowledge current and former members of Tissue repair and
translational physiology (TRTP) group, as well as friends which I have made during
my PhD journey, including Parvathi Haridas, Lucas Wager, Uyen Than, Jacky Sit,
Dario Stupar, Andrew Lai, Ali Shokoohmand, Leo Leung, Yee Chng and Lipsa
Mohanty. Furthermore, I would like to thank my family for always being there for me.
Most importantly, I would like to thank my partner Lancy Iljas, I would have never
made it this far without your love and support.
I would finally like to thank my funding bodies, QUT and Wound Management
Innovation Cooperative Research Centre (WMI-CRC) for the APA scholarship and
WMI-CRC top up scholarship, respectively.
iii
Abstract
Hypertrophic and keloid cutaneous scars result from abnormal extracellular
matrix (ECM) production by dermal fibroblasts of the skin. However, the cellular and
molecular mechanisms which contribute to hypertrophic and keloid scarring are still
unclear. A potential under researched mechanism may involve the regulation of
fibroblast gene expression by small regulatory RNA molecules known as microRNAs
(miRNAs). Therefore, this study identified and investigated miRNAs which were
found to be differentially expressed in keratinocytes and fibroblasts derived from
hypertrophic and keloid scars, when compared to normal skin. These results revealed
that hsa-miR-10a-5p expression was elevated in keloid fibroblasts and reduced in
hypertrophic fibroblasts, thus this miRNA could potentially be a biomarker to
differentiate between these two scar types. Additionally, the results described in this
thesis indicate that hsa-miR-10a-5p has a regulatory role in non-collagenous protein
synthesis rate in normal fibroblasts, but not in hypertrophic or keloid scar fibroblasts.
Further investigation as to why the regulation of these ECM associated proteins by
hsa-miR-10a-5p is absent in hypertrophic and keloid scar fibroblasts, will lead to a
better understanding of hypertrophic and keloid scar pathophysiology.
In addition, previous studies have also demonstrated that the communication
between fibroblasts and keratinocytes is important in hypertrophic and keloid
cutaneous scar pathology. However, the role that miRNAs play in the regulation of /
or the regulation by keratinocyte-fibroblast communication has not been previously
reported. Therefore, this study has identified miRNAs which are regulated by
keratinocyte-fibroblast communication, which included hsa-miR-146a-5p. This
miRNA is well described in the literature for its role in the regulation of inflammation,
which is a contributing factor to hypertrophic and keloid cutaneous scarring.
Additional experimentation described herein, indicated that the inflammatory
signalling proteins NF-κB and potentially IκBα; the gene expression of chemokine
CCL5; and hsa-miR-146a-5p expression in normal fibroblasts were regulated by
keratinocyte-fibroblast communication. Importantly, it was also demonstrated that this
regulation decreased proportionally relative to scar severity. These results suggest that
hypertrophic and especially keloid fibroblasts are not responsive to the regulatory
signals produced by keratinocyte-fibroblast communication. Modulation of this
iv
alleviate the formation of these severe scar phenotypes.
v
Acknowledgments ..................................................................................... ii
Abstract .................................................................................................... iii
Chapter 1: Literature Review .................................................................... 1
1.1 Introduction ........................................................................................................ 2
1.2 MicroRNAs ........................................................................................................ 2
1.3.1 Semiconductor technology .......................................................................... 7
1.3.3 Bioinformatic and statistical analysis of NGS data. .................................... 8
1.4 Wound repair ...................................................................................................... 9
1.4.1 Haemostasis phase ....................................................................................... 9
1.4.2 Inflammation phase ..................................................................................... 9
1.4.3 Proliferation phase ..................................................................................... 10
1.4.4 Remodelling phase ..................................................................................... 11
1.5 Hypertrophic Scars ........................................................................................... 11
1.5.2 MicroRNAs in hypertrophic scars ............................................................. 14
1.5.3 Hypertrophic scar conclusion .................................................................... 18
1.6 Keloid Scars ..................................................................................................... 18
1.6.2 MicroRNAs in keloid scars ....................................................................... 21
1.6.3 Keloid scar conclusion ............................................................................... 25
1.7 Project outline ................................................................................................... 26
2.1 Introduction ...................................................................................................... 28
2.2 Samples ............................................................................................................. 28
2.3 Harvesting and culture of skin cells ................................................................. 28
2.3.1 Composition of FGM and FBGM cell culture media ................................ 29
2.3.2 Skin collection ........................................................................................... 29
2.3.4 Normal keratinocyte isolation .................................................................... 30
2.3.5 Normal fibroblast isolation ........................................................................ 30
2.3.6 Cryopreservation of keratinocytes and fibroblasts .................................... 31
2.4 Construction of Transwell® co-culture system ................................................. 31
2.4.1 Fibroblast seeding ...................................................................................... 32
2.4.2 Keratinocyte seeding .................................................................................. 33
2.5 RNA extraction ................................................................................................. 35
2.5.2 Small RNA and total RNA quality analysis .............................................. 36
2.6 Sequencing utilising the Ion Proton™ ............................................................... 36
2.6.1 Constructing small RNA libraries .............................................................. 36
2.6.2 Template preparation of small RNA libraries ............................................ 38
2.6.3 Sequencing libraries on the Ion Proton™ ................................................... 39
2.7 Sequencing utilising the NextSeq 500 .............................................................. 39
2.7.1 Constructing small RNA libraries .............................................................. 40
2.7.2 Sequencing libraries on the NextSeq 500 .................................................. 41
2.8 Analysis of sequencing data ............................................................................. 42
2.8.1 Pre-processing and identification of miRNAs ........................................... 42
2.8.2 Rarefaction analysis ................................................................................... 42
2.8.3 Normalisation and statistical analysis of raw miRNA counts ................... 43
2.8.4 Identification of miRNA regulated genes and gene ontology pathway
analysis ................................................................................................................ 43
2.9.1 Reverse transcription ................................................................................. 44
2.9.2 Real-time PCR ........................................................................................... 44
2.10.1 Reverse transcription ............................................................................... 45
2.10.2 Real-time PCR ......................................................................................... 46
2.10.3 Statistical analysis .................................................................................... 46
2.11 Exosome isolation .......................................................................................... 47
2.13 Transfection of miRNA mimics ..................................................................... 48
2.14 Scar in a jar model .......................................................................................... 49
2.15 Collagen and non-collagenous protein assay ................................................. 49
2.15.1 Procedure for collagen and non-collagenous protein assay ..................... 49
2.15.2 Calculations and statistics ........................................................................ 50
2.16 Conditioning of the Transwell® co-culture system ........................................ 51
2.17 Immunoblotting .............................................................................................. 53
2.17.2 Procedure for immunoblotting ................................................................. 53
2.17.3 Densitometry and statistical analysis ....................................................... 54
Chapter 3: Identifying differences in microRNA profiles between
normal, hypertrophic and keloid scar derived keratinocytes and
fibroblasts ................................................................................................ 57
3.2.2 Harvesting and culture of skin cells ........................................................... 62
3.2.3 RNA extraction .......................................................................................... 62
3.2.5 Analysis of sequencing data ...................................................................... 63
3.2.6 MicroRNA analysis using real-time PCR .................................................. 64
3.3 Results .............................................................................................................. 65
3.3.1 Composition and bioinformatic processing of Illumina NextSeq 500 NGS
data ...................................................................................................................... 65
3.3.3 Verification and correlation of NGS results .............................................. 85
3.3.4 MicroRNA gene targets and gene ontology pathway analysis .................. 87
3.4 Discussion ........................................................................................................ 91
between keratinocytes and fibroblasts .................................................... 97
4.1 Introduction ...................................................................................................... 98
4.2.2 Harvesting and culture of skin cells ......................................................... 102
4.2.3 Construction of Transwell® co-culture system ........................................ 102
4.2.4 RNA extraction ........................................................................................ 103
4.2.6 Analysis of sequencing data..................................................................... 104
4.3 Results ............................................................................................................ 106
4.3.2 Differential expression and statistical analysis of miRNAs .................... 112
4.3.3 Verification and correlation ..................................................................... 125
4.3.4 MicroRNA gene targets and gene ontology pathway analysis ................ 128
4.4 Discussion ....................................................................................................... 132
microRNAs of interest with relation to scar pathology ........................ 139
5.1 Introduction .................................................................................................... 140
5.2.2 Harvesting and culture of skin cells ......................................................... 144
5.2.3 Transfection of miRNA mimics............................................................... 144
5.2.6 Conditioning of the Transwell® co-culture system .................................. 146
5.2.7 RNA extraction ........................................................................................ 147
5.2.8 MicroRNA and messenger RNA analysis using real-time PCR .............. 147
5.2.9 Immunoblotting........................................................................................ 148
5.3.1 The role of hsa-miR-10a-5p in collagen and non-collagenous protein
production in normal and scar fibroblasts ......................................................... 149
5.3.2 Regulation of hsa-miR-146a-5p and hsa-miR-205 (-5p/-3p) expression by
cell-cell communication in normal and scar fibroblasts ................................... 153
ix
cell-cell communication in normal keratinocytes cultured with either normal or
scar fibroblasts. ................................................................................................. 158
5.3.4 Association between hsa-miR-146a-5p and the inflammatory response
within normal and scar fibroblasts in mono-culture and co-culture conditions 160
5.3.5 Association between hsa-miR-146a-5p and the inflammatory response
within normal keratinocytes in mono-culture and co-culture conditions ......... 171
5.3.6 The role of hsa-miR-146a-5p in regulating the inflammatory response within
normal and scar fibroblasts ............................................................................... 179
5.4 Discussion ...................................................................................................... 187
5.4.1 Regulation of collagen and non-collagenous protein synthesis by hsa-miR-
10a-5p ............................................................................................................... 187
communication ................................................................................................. 189
5.4.3 Regulation of inflammatory signalling (TRAF6, IκBα and NF-κB) by
keratinocyte-fibroblast communication ............................................................ 194
5.4.4 Regulation of cytokine/chemokine expression (CCL5, IL-6 and TNFα) by
keratinocyte-fibroblast communication ............................................................ 196
by hsa-miR-146a-5p ......................................................................................... 198
Appendices ............................................................................................ 214
References ............................................................................................. 223
List of Figures
Figure 1.1 Diagram depicting the miRNA epigenetic mechanisms which regulate gene
expression. .................................................................................................................... 6
Figure 2.1 Diagrams and timeline for Transwell® co-culture and mono-culture
systems. ...................................................................................................................... 34
.................................................................................................................................... 52
Figure 3.1 Overall flowchart of methods used for the experiments described in chapter
3. ................................................................................................................................. 61
Figure 3.2 Analysis of the read distribution revealed that NGS data contains miRNAs,
tRNA derived fragments and tRNA halves. ............................................................... 66
Figure 3.3 The bioinformatic pipeline utilised can filter and discard reads
appropriately, leaving a population of reads that are enriched for miRNAs. ............. 68
Figure 3.4 Rarefaction analysis revealed that small RNA samples sequenced using
NextSeq 500 NGS technology should have a minimum of 4,900,000 raw reads to
capture 50% of the miRNA species within the sample. ............................................. 70
Figure 3.5 Euclidean distance of miRNA profiles revealed that keratinocytes and
fibroblasts can be separated based on normal or scar origin, with the exception of
normal scar fibroblasts. .............................................................................................. 72
between normal scar fibroblast analysis, hypertrophic scar fibroblast analysis and
keloid scar fibroblast analysis. ................................................................................... 74
Figure 3.7 A total of 32 miRNAs were differentially expressed between normal scar
fibroblasts compared to normal fibroblasts, where hsa-miR-10a-3p, hsa-miR-10a-5p
and hsa-miR-196a-2-3p are all elevated in normal scar fibroblasts. .......................... 76
Figure 3.8 A total of 35 miRNAs were differentially expressed between hypertrophic
scar fibroblasts compared to normal fibroblasts, where hsa-miR-10a-3p, hsa-miR-10a-
5p and hsa-miR-196a-2-3p are all reduced in hypertrophic scar fibroblasts. ............ 78
Figure 3.9 A total of 19 miRNAs were differentially expressed between keloid scar
fibroblasts compared to normal fibroblasts, where hsa-miR-10a-3p, hsa-miR-10a-5p,
hsa-miR-196a-2-3p, hsa-miR-143-3p and hsa-miR-143-5p are all elevated in keloid
scar fibroblasts. ........................................................................................................... 80
xi
Figure 3.10 A total of 145 miRNAs were differentially expressed between keloid scar
keratinocytes compared to normal keratinocytes, where hsa-miR-143-3p and hsa-miR-
143-5p are all reduced in keloid scar keratinocytes. .................................................. 82
Figure 3.11 Hierarchal clustering analysis of abundance levels for differentially
expressed miRNAs, can separate samples based on normal and scar origin, for both
keratinocytes and fibroblasts. ..................................................................................... 84
Figure 3.12 Real-time PCR analysis confirmed NGS expression results for miRNAs
of interest hsa-miR-10a-5p and hsa-miR-143-3p, which are differentially expressed
between normal and scar conditions. ......................................................................... 86
Figure 3.13 Gene ontology analysis revealed that miRNAs which are differentially
expressed between normal and scar conditions, regulate biological processes such as
toll-like receptor signalling. ....................................................................................... 88
Figure 3.14 hsa-miR-10a-5p targets genes such as TFAP2C, SFRS1, NCOR2, SFRS10
and ACTG1, and can potentially regulate biological processes such as metabolism. 90
Figure 4.1 Overall flowchart of methods used for the experiments described in chapter
4. ............................................................................................................................... 101
Figure 4.2 Analysis of the read distribution revealed that NGS data contained miRNAs,
tRNA derived fragments and tRNA halves. ............................................................. 107
Figure 4.3 The bioinformatic pipeline utilised is able to filter and discard reads
appropriately, leaving a population of reads which were enriched for miRNAs. .... 109
Figure 4.4 Rarefaction analysis revealed that small RNA samples sequenced using Ion
Proton™ NGS technology should have a minimum of 1,000,000 raw reads to capture
50% of the miRNA species within the sample......................................................... 111
Figure 4.5 Euclidean distance of miRNA profiles revealed that keratinocyte-fibroblast
communication regulated different miRNA populations in normal keratinocytes and
normal fibroblasts..................................................................................................... 113
Figure 4.6 hsa-miR-146a-5p expression is common between normal fibroblasts mono-
culture/co-culture analysis and normal keratinocytes mono-culture/co-culture
analysis. .................................................................................................................... 115
Figure 4.7 A total of 11 miRNAs were differentially expressed between normal
fibroblasts in co-culture with normal keratinocytes compared to normal fibroblasts in
mono-culture, where hsa-miR-146a-5p expression is elevated in normal fibroblasts in
co-culture.................................................................................................................. 117
xii
Figure 4.8 A total of 10 miRNAs were differentially expressed between normal
keratinocytes in co-culture with normal fibroblasts compared to normal keratinocytes
in mono-culture, where hsa-miR-146a-5p expression is elevated in normal
keratinocytes in co-culture. ...................................................................................... 119
Figure 4.9 A total of 236 miRNAs were differentially expressed between normal
keratinocytes and normal fibroblasts in mono-culture, where hsa-miR-203a-3p and the
miR-200 family are elevated while hsa-miR-199a-1-5p is reduced in normal
keratinocytes. ............................................................................................................ 121
expressed miRNAs, can separate normal keratinocytes and normal fibroblasts based
on mono-culture and co-culture conditions, as well as distinguish between these cell
types. ........................................................................................................................ 124
Figure 4.11 Real-time PCR analysis confirmed the NGS expression results for
miRNAs hsa-miR-203a-3p, hsa-miR-141-3p and hsa-miR-146a-5p, where the latter is
regulated be keratinocyte-fibroblast communication. .............................................. 127
Figure 4.12 Gene ontology analysis revealed that miRNAs of interest which are
differentially expressed by keratinocyte-fibroblast communication, regulate biological
processes such as toll-like receptor signalling. ........................................................ 129
Figure 4.13 hsa-miR-146a-5p targets genes such as TRAF6 and IRAK1, and can
potentially regulate biological processes such as toll-like receptor signalling. ....... 131
Figure 5.1 Overall flowchart of methods used for the experiments described in chapter
5. ............................................................................................................................... 143
Figure 5.2 Transfection of hsa-miR-10a-5p into normal, hypertrophic or keloid scar
fibroblasts does not alter collagen or non-collagenous protein concentration at 4, 24
and 48 hours. ............................................................................................................ 150
reduction in non-collagenous protein synthesis rate, for normal fibroblasts. .......... 152
Figure 5.4 hsa-miR-146a-5p is regulated by paracrine signalling in normal fibroblasts,
while hsa-miR-205-5p and hsa-miR-205-3p are regulated by juxtacrine signalling in
normal, hypertrophic and keloid scar fibroblasts. .................................................... 155
Figure 5.5 In normal fibroblasts, hsa-miR-146a-5p is regulated by paracrine signalling
between keratinocytes and fibroblasts, which was not dependent on whether
conditioned media was derived from normal or scar co-culture conditions. ........... 157
xiii
Figure 5.6 hsa-miR-146a-5p is regulated by juxtacrine signalling in normal
keratinocytes cultured with normal fibroblasts. ....................................................... 159
Figure 5.7 NF-κB and potentially IκBα in normal fibroblasts were regulated by the
communication between keratinocytes and fibroblasts............................................ 162
Figure 5.8 TRAF6 in hypertrophic scar fibroblasts is regulated by the communication
between keratinocytes and fibroblasts. .................................................................... 164
Figure 5.9 NF-κB, IκBα and TRAF6 are not regulated by keratinocyte-fibroblast
communication within keloid scar fibroblasts. ........................................................ 166
Figure 5.10 The communication between keratinocytes and fibroblasts regulates CCL5
in normal fibroblasts. ............................................................................................... 168
in normal fibroblasts. ............................................................................................... 170
regulates NF-κB within normal keratinocytes. ........................................................ 172
Figure 5.13 NF-κB, IκBα and TRAF6 are not regulated by the communication between
normal keratinocytes and hypertrophic scar fibroblasts........................................... 174
Figure 5.14 The communication between normal keratinocytes and keloid scar
fibroblasts regulates NF-κB in normal keratinocytes............................................... 176
Figure 5.15 TNFα, CCL5 and IL-6 are not regulated by the communication between
normal keratinocytes co-cultured with either normal, hypertrophic or keloid scar
fibroblasts. ................................................................................................................ 178
Figure 5.16 hsa-miR-146a-5p may potentially reduce TRAF6 expression in normal
fibroblasts. ................................................................................................................ 180
Figure 5.17 hsa-miR-146a-5p is able to reduce TRAF6 expression in hypertrophic scar
fibroblasts. ................................................................................................................ 182
Figure 5.18 hsa-miR-146a-5p reduced TRAF6 expression in keloid scar fibroblasts.
.................................................................................................................................. 184
Figure 5.19 hsa-miR-146a-5p does not regulate CCL5, IL-6 or TNFα expression in
normal, hypertrophic or keloid scar derived fibroblasts. ......................................... 186
Figure 5.20 Schematic diagram of inflammatory signalling regulated by hsa-miR-
146a, and transcriptional regulation mediated by NF-κB ........................................ 193
Figure 6.1 Summary of the experimental results investigating hsa-miR-10a-5p
expression. ................................................................................................................ 205
expression. ................................................................................................................ 211
Table 1.1 Tabulation of literature investigating miRNAs in hypertrophic scars. ...... 15
Table 1.2 Tabulation of literature investigating miRNAs in keloid scars.................. 22
Table 2.1: miRNA targets analysed using real-time PCR.......................................... 45
Table 2.2: mRNA targets analysed using real-time PCR. .......................................... 46
Table 2.3: Antibodies used for immunoblotting. ....................................................... 54
xvi
CO2 carbon dioxide
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
miRNA microRNA
mL millilitre
mm millimeter
mM millimolar
mRNA messenger RNA
NaOAc Sodium acetate
NaOH Sodium hydroxide
N-Fb Normal fibroblasts
RIPA Radio-Immunoprecipitation Assay
TEM transmission electron microscopy
v/v volume per volume
w/v weight per volume
Other conference papers
An Analysis of Exosomes from Keratinocytes and Fibroblasts
Than TTU, Guanzon D, Wager L, Manton KJ, Hollier B, Leavesley D.
5th International Conference on Biomedical Engineering in Vietnam, pp 137-141,
(2015).
Characterization of exosomal mirnas present in plasma from women with
gestational diabetes mellitus
Placenta, pp 68, (2016)
Conference paper has been published.
Identification of exosomal mirna biomarkers at early gestation (< 18 Weeks) in
asymptomatic women at early gestation (< 18 Weeks) who subsequently develop
spontaneous preterm birth
Kinhal V, Guanzon D, Scholz-Romero K, Longo S, Fortunato S, Menon R, Rice GE,
Salomon C.
Other publications
diabetes mellitus
Iljas JD, Guanzon D, Elfeky O, Rice GE, Salomon C.
Placenta (2016).
Oxygen tension regulates the miRNA profile and bioactivity of exosomes
released from extravillous trophoblast cells – Liquid biopsies for monitoring
complications of pregnancy
Troung G, Guanzon D, Kinhal V, Elfeky O, Lai A, Longo S, Nuzhat Z, Palma C,
Menon R, Rice GE, Salomon C.
PLOS ONE (2017).
Article is published.
Than TTU, Guanzon D, Leavesley D, Parker T.
International Journal of Molecular Sciences (2017).
Article is published.
Placenta-derived exosomes as early biomarker of preeclampsia - Potential role of
exosomal microRNAs across gestation
Salomon C, Guanzon D, Scholz-Romero K, Longo S, Correa P, Illanes SE, Rice GE
The Journal of Clinical Endocrinology & Metabolism (2017).
Article is published.
Other book chapters
Using a next generation sequencing approach to profile microRNAs from human
origin.
Methods in molecular biology (2017).
Book chapter is published.
Chapter 1: Literature Review
1.1 Introduction
The development of the wound repair process was an important evolutionary
mechanism to aid in organism survival. As organisms increased in complexity, so did
the process of wound repair. Understanding the process of wound repair and why
wounds fail to heal remains a challenge. At present, it is understood that the wound
repair process in mammals involves a cascade of overlapping events, namely
haemostasis, inflammation, proliferation and remodelling [1]. Each stage is
orchestrated by the interplay of cells, growth factors and cytokines which result in
wound closure [1]. Dysregulation of the wound repair process in skin leads to
conditions such as hypertrophic and keloid scars. [2]. Every year, more than 100
million people suffer from hypertrophic and keloid scars, which often cause
psychological and physical distress, and can affect their quality of life [3]. Currently,
there are no reliable treatments for hypertrophic scars and keloids [2]. Furthermore,
the treatments that do exist are often accompanied by side effects, thus there is an
urgent need for improved therapies [4]. The emerging role of microRNAs (miRNAs)
as important regulators in biological processes may provide new therapeutic avenues
for treatment of hypertrophic and keloid scars [5].
1.2 MicroRNAs
Completion of the Human Genome Project revealed for the first time the
nucleic acid sequence composition of the human genome [6]. Surprisingly, only 1.2%
of the human genome appears to encode protein, which raises questions regarding the
biological significance of the remaining genome [7]. It has been estimated that
approximately 80% of the human genome serves at least one biochemical function
within the cell [8, 9]. Within this 80% are a family of non-coding regulatory
ribonucleic acids (RNA), one important class being miRNAs [8].
miRNAs are small RNA fragments that are approximately 22 nucleotides in
length [8]. These…
Dominic Guanzon
Bachelor of Biomedical Science (First Class Honours)
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the best
of my knowledge and belief, this thesis contains no material previously published or
written by another person except where due reference is made.
Signature: QUT Verified Signature
ii
Acknowledgments
Firstly I would like to acknowledge my primary supervisor Tony Parker. I can’t
thank you enough for all the support you have given me, I wouldn’t have been able to
complete my PhD without your help. I enjoy the conversations we have, and I really
appreciate the feedback you have given me regarding my thesis and project. I would
also like to thank my former primary supervisor David Leavesley for his guidance and
support throughout my honours and half way through my PhD, the experience was
enjoyable. I would also like to acknowledge my current and former associate
supervisors Ana Pavasovic, Brett Hollier and Kerry Manton for their support.
Furthermore, I would like to thank the Genomics laboratory at CARF for their
excellent service regarding NGS, with specific mention to Vincent Chand, Kevin
Dudley and Sahana Manoli. This PhD project would have not been possible without
your help. I would also like to acknowledge the Optical and electron microscopy
laboratory for their help regarding TEM and imaging of exosomes, with specific
mention to Rachel Hancock and Jamie Riches.
I would like to acknowledge current and former members of Tissue repair and
translational physiology (TRTP) group, as well as friends which I have made during
my PhD journey, including Parvathi Haridas, Lucas Wager, Uyen Than, Jacky Sit,
Dario Stupar, Andrew Lai, Ali Shokoohmand, Leo Leung, Yee Chng and Lipsa
Mohanty. Furthermore, I would like to thank my family for always being there for me.
Most importantly, I would like to thank my partner Lancy Iljas, I would have never
made it this far without your love and support.
I would finally like to thank my funding bodies, QUT and Wound Management
Innovation Cooperative Research Centre (WMI-CRC) for the APA scholarship and
WMI-CRC top up scholarship, respectively.
iii
Abstract
Hypertrophic and keloid cutaneous scars result from abnormal extracellular
matrix (ECM) production by dermal fibroblasts of the skin. However, the cellular and
molecular mechanisms which contribute to hypertrophic and keloid scarring are still
unclear. A potential under researched mechanism may involve the regulation of
fibroblast gene expression by small regulatory RNA molecules known as microRNAs
(miRNAs). Therefore, this study identified and investigated miRNAs which were
found to be differentially expressed in keratinocytes and fibroblasts derived from
hypertrophic and keloid scars, when compared to normal skin. These results revealed
that hsa-miR-10a-5p expression was elevated in keloid fibroblasts and reduced in
hypertrophic fibroblasts, thus this miRNA could potentially be a biomarker to
differentiate between these two scar types. Additionally, the results described in this
thesis indicate that hsa-miR-10a-5p has a regulatory role in non-collagenous protein
synthesis rate in normal fibroblasts, but not in hypertrophic or keloid scar fibroblasts.
Further investigation as to why the regulation of these ECM associated proteins by
hsa-miR-10a-5p is absent in hypertrophic and keloid scar fibroblasts, will lead to a
better understanding of hypertrophic and keloid scar pathophysiology.
In addition, previous studies have also demonstrated that the communication
between fibroblasts and keratinocytes is important in hypertrophic and keloid
cutaneous scar pathology. However, the role that miRNAs play in the regulation of /
or the regulation by keratinocyte-fibroblast communication has not been previously
reported. Therefore, this study has identified miRNAs which are regulated by
keratinocyte-fibroblast communication, which included hsa-miR-146a-5p. This
miRNA is well described in the literature for its role in the regulation of inflammation,
which is a contributing factor to hypertrophic and keloid cutaneous scarring.
Additional experimentation described herein, indicated that the inflammatory
signalling proteins NF-κB and potentially IκBα; the gene expression of chemokine
CCL5; and hsa-miR-146a-5p expression in normal fibroblasts were regulated by
keratinocyte-fibroblast communication. Importantly, it was also demonstrated that this
regulation decreased proportionally relative to scar severity. These results suggest that
hypertrophic and especially keloid fibroblasts are not responsive to the regulatory
signals produced by keratinocyte-fibroblast communication. Modulation of this
iv
alleviate the formation of these severe scar phenotypes.
v
Acknowledgments ..................................................................................... ii
Abstract .................................................................................................... iii
Chapter 1: Literature Review .................................................................... 1
1.1 Introduction ........................................................................................................ 2
1.2 MicroRNAs ........................................................................................................ 2
1.3.1 Semiconductor technology .......................................................................... 7
1.3.3 Bioinformatic and statistical analysis of NGS data. .................................... 8
1.4 Wound repair ...................................................................................................... 9
1.4.1 Haemostasis phase ....................................................................................... 9
1.4.2 Inflammation phase ..................................................................................... 9
1.4.3 Proliferation phase ..................................................................................... 10
1.4.4 Remodelling phase ..................................................................................... 11
1.5 Hypertrophic Scars ........................................................................................... 11
1.5.2 MicroRNAs in hypertrophic scars ............................................................. 14
1.5.3 Hypertrophic scar conclusion .................................................................... 18
1.6 Keloid Scars ..................................................................................................... 18
1.6.2 MicroRNAs in keloid scars ....................................................................... 21
1.6.3 Keloid scar conclusion ............................................................................... 25
1.7 Project outline ................................................................................................... 26
2.1 Introduction ...................................................................................................... 28
2.2 Samples ............................................................................................................. 28
2.3 Harvesting and culture of skin cells ................................................................. 28
2.3.1 Composition of FGM and FBGM cell culture media ................................ 29
2.3.2 Skin collection ........................................................................................... 29
2.3.4 Normal keratinocyte isolation .................................................................... 30
2.3.5 Normal fibroblast isolation ........................................................................ 30
2.3.6 Cryopreservation of keratinocytes and fibroblasts .................................... 31
2.4 Construction of Transwell® co-culture system ................................................. 31
2.4.1 Fibroblast seeding ...................................................................................... 32
2.4.2 Keratinocyte seeding .................................................................................. 33
2.5 RNA extraction ................................................................................................. 35
2.5.2 Small RNA and total RNA quality analysis .............................................. 36
2.6 Sequencing utilising the Ion Proton™ ............................................................... 36
2.6.1 Constructing small RNA libraries .............................................................. 36
2.6.2 Template preparation of small RNA libraries ............................................ 38
2.6.3 Sequencing libraries on the Ion Proton™ ................................................... 39
2.7 Sequencing utilising the NextSeq 500 .............................................................. 39
2.7.1 Constructing small RNA libraries .............................................................. 40
2.7.2 Sequencing libraries on the NextSeq 500 .................................................. 41
2.8 Analysis of sequencing data ............................................................................. 42
2.8.1 Pre-processing and identification of miRNAs ........................................... 42
2.8.2 Rarefaction analysis ................................................................................... 42
2.8.3 Normalisation and statistical analysis of raw miRNA counts ................... 43
2.8.4 Identification of miRNA regulated genes and gene ontology pathway
analysis ................................................................................................................ 43
2.9.1 Reverse transcription ................................................................................. 44
2.9.2 Real-time PCR ........................................................................................... 44
2.10.1 Reverse transcription ............................................................................... 45
2.10.2 Real-time PCR ......................................................................................... 46
2.10.3 Statistical analysis .................................................................................... 46
2.11 Exosome isolation .......................................................................................... 47
2.13 Transfection of miRNA mimics ..................................................................... 48
2.14 Scar in a jar model .......................................................................................... 49
2.15 Collagen and non-collagenous protein assay ................................................. 49
2.15.1 Procedure for collagen and non-collagenous protein assay ..................... 49
2.15.2 Calculations and statistics ........................................................................ 50
2.16 Conditioning of the Transwell® co-culture system ........................................ 51
2.17 Immunoblotting .............................................................................................. 53
2.17.2 Procedure for immunoblotting ................................................................. 53
2.17.3 Densitometry and statistical analysis ....................................................... 54
Chapter 3: Identifying differences in microRNA profiles between
normal, hypertrophic and keloid scar derived keratinocytes and
fibroblasts ................................................................................................ 57
3.2.2 Harvesting and culture of skin cells ........................................................... 62
3.2.3 RNA extraction .......................................................................................... 62
3.2.5 Analysis of sequencing data ...................................................................... 63
3.2.6 MicroRNA analysis using real-time PCR .................................................. 64
3.3 Results .............................................................................................................. 65
3.3.1 Composition and bioinformatic processing of Illumina NextSeq 500 NGS
data ...................................................................................................................... 65
3.3.3 Verification and correlation of NGS results .............................................. 85
3.3.4 MicroRNA gene targets and gene ontology pathway analysis .................. 87
3.4 Discussion ........................................................................................................ 91
between keratinocytes and fibroblasts .................................................... 97
4.1 Introduction ...................................................................................................... 98
4.2.2 Harvesting and culture of skin cells ......................................................... 102
4.2.3 Construction of Transwell® co-culture system ........................................ 102
4.2.4 RNA extraction ........................................................................................ 103
4.2.6 Analysis of sequencing data..................................................................... 104
4.3 Results ............................................................................................................ 106
4.3.2 Differential expression and statistical analysis of miRNAs .................... 112
4.3.3 Verification and correlation ..................................................................... 125
4.3.4 MicroRNA gene targets and gene ontology pathway analysis ................ 128
4.4 Discussion ....................................................................................................... 132
microRNAs of interest with relation to scar pathology ........................ 139
5.1 Introduction .................................................................................................... 140
5.2.2 Harvesting and culture of skin cells ......................................................... 144
5.2.3 Transfection of miRNA mimics............................................................... 144
5.2.6 Conditioning of the Transwell® co-culture system .................................. 146
5.2.7 RNA extraction ........................................................................................ 147
5.2.8 MicroRNA and messenger RNA analysis using real-time PCR .............. 147
5.2.9 Immunoblotting........................................................................................ 148
5.3.1 The role of hsa-miR-10a-5p in collagen and non-collagenous protein
production in normal and scar fibroblasts ......................................................... 149
5.3.2 Regulation of hsa-miR-146a-5p and hsa-miR-205 (-5p/-3p) expression by
cell-cell communication in normal and scar fibroblasts ................................... 153
ix
cell-cell communication in normal keratinocytes cultured with either normal or
scar fibroblasts. ................................................................................................. 158
5.3.4 Association between hsa-miR-146a-5p and the inflammatory response
within normal and scar fibroblasts in mono-culture and co-culture conditions 160
5.3.5 Association between hsa-miR-146a-5p and the inflammatory response
within normal keratinocytes in mono-culture and co-culture conditions ......... 171
5.3.6 The role of hsa-miR-146a-5p in regulating the inflammatory response within
normal and scar fibroblasts ............................................................................... 179
5.4 Discussion ...................................................................................................... 187
5.4.1 Regulation of collagen and non-collagenous protein synthesis by hsa-miR-
10a-5p ............................................................................................................... 187
communication ................................................................................................. 189
5.4.3 Regulation of inflammatory signalling (TRAF6, IκBα and NF-κB) by
keratinocyte-fibroblast communication ............................................................ 194
5.4.4 Regulation of cytokine/chemokine expression (CCL5, IL-6 and TNFα) by
keratinocyte-fibroblast communication ............................................................ 196
by hsa-miR-146a-5p ......................................................................................... 198
Appendices ............................................................................................ 214
References ............................................................................................. 223
List of Figures
Figure 1.1 Diagram depicting the miRNA epigenetic mechanisms which regulate gene
expression. .................................................................................................................... 6
Figure 2.1 Diagrams and timeline for Transwell® co-culture and mono-culture
systems. ...................................................................................................................... 34
.................................................................................................................................... 52
Figure 3.1 Overall flowchart of methods used for the experiments described in chapter
3. ................................................................................................................................. 61
Figure 3.2 Analysis of the read distribution revealed that NGS data contains miRNAs,
tRNA derived fragments and tRNA halves. ............................................................... 66
Figure 3.3 The bioinformatic pipeline utilised can filter and discard reads
appropriately, leaving a population of reads that are enriched for miRNAs. ............. 68
Figure 3.4 Rarefaction analysis revealed that small RNA samples sequenced using
NextSeq 500 NGS technology should have a minimum of 4,900,000 raw reads to
capture 50% of the miRNA species within the sample. ............................................. 70
Figure 3.5 Euclidean distance of miRNA profiles revealed that keratinocytes and
fibroblasts can be separated based on normal or scar origin, with the exception of
normal scar fibroblasts. .............................................................................................. 72
between normal scar fibroblast analysis, hypertrophic scar fibroblast analysis and
keloid scar fibroblast analysis. ................................................................................... 74
Figure 3.7 A total of 32 miRNAs were differentially expressed between normal scar
fibroblasts compared to normal fibroblasts, where hsa-miR-10a-3p, hsa-miR-10a-5p
and hsa-miR-196a-2-3p are all elevated in normal scar fibroblasts. .......................... 76
Figure 3.8 A total of 35 miRNAs were differentially expressed between hypertrophic
scar fibroblasts compared to normal fibroblasts, where hsa-miR-10a-3p, hsa-miR-10a-
5p and hsa-miR-196a-2-3p are all reduced in hypertrophic scar fibroblasts. ............ 78
Figure 3.9 A total of 19 miRNAs were differentially expressed between keloid scar
fibroblasts compared to normal fibroblasts, where hsa-miR-10a-3p, hsa-miR-10a-5p,
hsa-miR-196a-2-3p, hsa-miR-143-3p and hsa-miR-143-5p are all elevated in keloid
scar fibroblasts. ........................................................................................................... 80
xi
Figure 3.10 A total of 145 miRNAs were differentially expressed between keloid scar
keratinocytes compared to normal keratinocytes, where hsa-miR-143-3p and hsa-miR-
143-5p are all reduced in keloid scar keratinocytes. .................................................. 82
Figure 3.11 Hierarchal clustering analysis of abundance levels for differentially
expressed miRNAs, can separate samples based on normal and scar origin, for both
keratinocytes and fibroblasts. ..................................................................................... 84
Figure 3.12 Real-time PCR analysis confirmed NGS expression results for miRNAs
of interest hsa-miR-10a-5p and hsa-miR-143-3p, which are differentially expressed
between normal and scar conditions. ......................................................................... 86
Figure 3.13 Gene ontology analysis revealed that miRNAs which are differentially
expressed between normal and scar conditions, regulate biological processes such as
toll-like receptor signalling. ....................................................................................... 88
Figure 3.14 hsa-miR-10a-5p targets genes such as TFAP2C, SFRS1, NCOR2, SFRS10
and ACTG1, and can potentially regulate biological processes such as metabolism. 90
Figure 4.1 Overall flowchart of methods used for the experiments described in chapter
4. ............................................................................................................................... 101
Figure 4.2 Analysis of the read distribution revealed that NGS data contained miRNAs,
tRNA derived fragments and tRNA halves. ............................................................. 107
Figure 4.3 The bioinformatic pipeline utilised is able to filter and discard reads
appropriately, leaving a population of reads which were enriched for miRNAs. .... 109
Figure 4.4 Rarefaction analysis revealed that small RNA samples sequenced using Ion
Proton™ NGS technology should have a minimum of 1,000,000 raw reads to capture
50% of the miRNA species within the sample......................................................... 111
Figure 4.5 Euclidean distance of miRNA profiles revealed that keratinocyte-fibroblast
communication regulated different miRNA populations in normal keratinocytes and
normal fibroblasts..................................................................................................... 113
Figure 4.6 hsa-miR-146a-5p expression is common between normal fibroblasts mono-
culture/co-culture analysis and normal keratinocytes mono-culture/co-culture
analysis. .................................................................................................................... 115
Figure 4.7 A total of 11 miRNAs were differentially expressed between normal
fibroblasts in co-culture with normal keratinocytes compared to normal fibroblasts in
mono-culture, where hsa-miR-146a-5p expression is elevated in normal fibroblasts in
co-culture.................................................................................................................. 117
xii
Figure 4.8 A total of 10 miRNAs were differentially expressed between normal
keratinocytes in co-culture with normal fibroblasts compared to normal keratinocytes
in mono-culture, where hsa-miR-146a-5p expression is elevated in normal
keratinocytes in co-culture. ...................................................................................... 119
Figure 4.9 A total of 236 miRNAs were differentially expressed between normal
keratinocytes and normal fibroblasts in mono-culture, where hsa-miR-203a-3p and the
miR-200 family are elevated while hsa-miR-199a-1-5p is reduced in normal
keratinocytes. ............................................................................................................ 121
expressed miRNAs, can separate normal keratinocytes and normal fibroblasts based
on mono-culture and co-culture conditions, as well as distinguish between these cell
types. ........................................................................................................................ 124
Figure 4.11 Real-time PCR analysis confirmed the NGS expression results for
miRNAs hsa-miR-203a-3p, hsa-miR-141-3p and hsa-miR-146a-5p, where the latter is
regulated be keratinocyte-fibroblast communication. .............................................. 127
Figure 4.12 Gene ontology analysis revealed that miRNAs of interest which are
differentially expressed by keratinocyte-fibroblast communication, regulate biological
processes such as toll-like receptor signalling. ........................................................ 129
Figure 4.13 hsa-miR-146a-5p targets genes such as TRAF6 and IRAK1, and can
potentially regulate biological processes such as toll-like receptor signalling. ....... 131
Figure 5.1 Overall flowchart of methods used for the experiments described in chapter
5. ............................................................................................................................... 143
Figure 5.2 Transfection of hsa-miR-10a-5p into normal, hypertrophic or keloid scar
fibroblasts does not alter collagen or non-collagenous protein concentration at 4, 24
and 48 hours. ............................................................................................................ 150
reduction in non-collagenous protein synthesis rate, for normal fibroblasts. .......... 152
Figure 5.4 hsa-miR-146a-5p is regulated by paracrine signalling in normal fibroblasts,
while hsa-miR-205-5p and hsa-miR-205-3p are regulated by juxtacrine signalling in
normal, hypertrophic and keloid scar fibroblasts. .................................................... 155
Figure 5.5 In normal fibroblasts, hsa-miR-146a-5p is regulated by paracrine signalling
between keratinocytes and fibroblasts, which was not dependent on whether
conditioned media was derived from normal or scar co-culture conditions. ........... 157
xiii
Figure 5.6 hsa-miR-146a-5p is regulated by juxtacrine signalling in normal
keratinocytes cultured with normal fibroblasts. ....................................................... 159
Figure 5.7 NF-κB and potentially IκBα in normal fibroblasts were regulated by the
communication between keratinocytes and fibroblasts............................................ 162
Figure 5.8 TRAF6 in hypertrophic scar fibroblasts is regulated by the communication
between keratinocytes and fibroblasts. .................................................................... 164
Figure 5.9 NF-κB, IκBα and TRAF6 are not regulated by keratinocyte-fibroblast
communication within keloid scar fibroblasts. ........................................................ 166
Figure 5.10 The communication between keratinocytes and fibroblasts regulates CCL5
in normal fibroblasts. ............................................................................................... 168
in normal fibroblasts. ............................................................................................... 170
regulates NF-κB within normal keratinocytes. ........................................................ 172
Figure 5.13 NF-κB, IκBα and TRAF6 are not regulated by the communication between
normal keratinocytes and hypertrophic scar fibroblasts........................................... 174
Figure 5.14 The communication between normal keratinocytes and keloid scar
fibroblasts regulates NF-κB in normal keratinocytes............................................... 176
Figure 5.15 TNFα, CCL5 and IL-6 are not regulated by the communication between
normal keratinocytes co-cultured with either normal, hypertrophic or keloid scar
fibroblasts. ................................................................................................................ 178
Figure 5.16 hsa-miR-146a-5p may potentially reduce TRAF6 expression in normal
fibroblasts. ................................................................................................................ 180
Figure 5.17 hsa-miR-146a-5p is able to reduce TRAF6 expression in hypertrophic scar
fibroblasts. ................................................................................................................ 182
Figure 5.18 hsa-miR-146a-5p reduced TRAF6 expression in keloid scar fibroblasts.
.................................................................................................................................. 184
Figure 5.19 hsa-miR-146a-5p does not regulate CCL5, IL-6 or TNFα expression in
normal, hypertrophic or keloid scar derived fibroblasts. ......................................... 186
Figure 5.20 Schematic diagram of inflammatory signalling regulated by hsa-miR-
146a, and transcriptional regulation mediated by NF-κB ........................................ 193
Figure 6.1 Summary of the experimental results investigating hsa-miR-10a-5p
expression. ................................................................................................................ 205
expression. ................................................................................................................ 211
Table 1.1 Tabulation of literature investigating miRNAs in hypertrophic scars. ...... 15
Table 1.2 Tabulation of literature investigating miRNAs in keloid scars.................. 22
Table 2.1: miRNA targets analysed using real-time PCR.......................................... 45
Table 2.2: mRNA targets analysed using real-time PCR. .......................................... 46
Table 2.3: Antibodies used for immunoblotting. ....................................................... 54
xvi
CO2 carbon dioxide
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
miRNA microRNA
mL millilitre
mm millimeter
mM millimolar
mRNA messenger RNA
NaOAc Sodium acetate
NaOH Sodium hydroxide
N-Fb Normal fibroblasts
RIPA Radio-Immunoprecipitation Assay
TEM transmission electron microscopy
v/v volume per volume
w/v weight per volume
Other conference papers
An Analysis of Exosomes from Keratinocytes and Fibroblasts
Than TTU, Guanzon D, Wager L, Manton KJ, Hollier B, Leavesley D.
5th International Conference on Biomedical Engineering in Vietnam, pp 137-141,
(2015).
Characterization of exosomal mirnas present in plasma from women with
gestational diabetes mellitus
Placenta, pp 68, (2016)
Conference paper has been published.
Identification of exosomal mirna biomarkers at early gestation (< 18 Weeks) in
asymptomatic women at early gestation (< 18 Weeks) who subsequently develop
spontaneous preterm birth
Kinhal V, Guanzon D, Scholz-Romero K, Longo S, Fortunato S, Menon R, Rice GE,
Salomon C.
Other publications
diabetes mellitus
Iljas JD, Guanzon D, Elfeky O, Rice GE, Salomon C.
Placenta (2016).
Oxygen tension regulates the miRNA profile and bioactivity of exosomes
released from extravillous trophoblast cells – Liquid biopsies for monitoring
complications of pregnancy
Troung G, Guanzon D, Kinhal V, Elfeky O, Lai A, Longo S, Nuzhat Z, Palma C,
Menon R, Rice GE, Salomon C.
PLOS ONE (2017).
Article is published.
Than TTU, Guanzon D, Leavesley D, Parker T.
International Journal of Molecular Sciences (2017).
Article is published.
Placenta-derived exosomes as early biomarker of preeclampsia - Potential role of
exosomal microRNAs across gestation
Salomon C, Guanzon D, Scholz-Romero K, Longo S, Correa P, Illanes SE, Rice GE
The Journal of Clinical Endocrinology & Metabolism (2017).
Article is published.
Other book chapters
Using a next generation sequencing approach to profile microRNAs from human
origin.
Methods in molecular biology (2017).
Book chapter is published.
Chapter 1: Literature Review
1.1 Introduction
The development of the wound repair process was an important evolutionary
mechanism to aid in organism survival. As organisms increased in complexity, so did
the process of wound repair. Understanding the process of wound repair and why
wounds fail to heal remains a challenge. At present, it is understood that the wound
repair process in mammals involves a cascade of overlapping events, namely
haemostasis, inflammation, proliferation and remodelling [1]. Each stage is
orchestrated by the interplay of cells, growth factors and cytokines which result in
wound closure [1]. Dysregulation of the wound repair process in skin leads to
conditions such as hypertrophic and keloid scars. [2]. Every year, more than 100
million people suffer from hypertrophic and keloid scars, which often cause
psychological and physical distress, and can affect their quality of life [3]. Currently,
there are no reliable treatments for hypertrophic scars and keloids [2]. Furthermore,
the treatments that do exist are often accompanied by side effects, thus there is an
urgent need for improved therapies [4]. The emerging role of microRNAs (miRNAs)
as important regulators in biological processes may provide new therapeutic avenues
for treatment of hypertrophic and keloid scars [5].
1.2 MicroRNAs
Completion of the Human Genome Project revealed for the first time the
nucleic acid sequence composition of the human genome [6]. Surprisingly, only 1.2%
of the human genome appears to encode protein, which raises questions regarding the
biological significance of the remaining genome [7]. It has been estimated that
approximately 80% of the human genome serves at least one biochemical function
within the cell [8, 9]. Within this 80% are a family of non-coding regulatory
ribonucleic acids (RNA), one important class being miRNAs [8].
miRNAs are small RNA fragments that are approximately 22 nucleotides in
length [8]. These…
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