Teza final braicu ovidiu leonard

DOCTORAL SCHOOL
UNIVERSITY OF MEDICINE AND PHARMACY “IULIU HAŢIEGANU” CLUJ-NAPOCA
CLUJ-NAPOCA 2017
Ion-
Radu
Badea
Digitally signed
by Ion-Radu
Badea
Date: 2017.10.30
15:18:25 +02'00'

PhD Thesis
Molecular profiling for
endometriosis and related
malignancies
PhD student Braicu Ovidiu-Leonard
Scientific supervisor Prof.dr. Irimie Alexandru

I dedicate this work to my family for their endless support and understanding, to my
beloved daughters Amalia and Diana…

2 Braicu Ovidiu-Leonard
PUBLICATION LIST
Articles published in extenso as a result of doctoral research
1. Braicu OL, Budisan L, Buiga R, Jurj A, Achimas-Cadariu P, Pop LA, Braicu C,
Irimie A, Berindan-Neagoe I, miRNA expression profiling in formalin-fixed
paraffin-embedded endometriosis and ovarian cancer samples, 2017:10,
Pages 4225—4238, doi.org/10.2147/OTT.S137107. ISI Impact Factor 2.6
(Study included in the chapter 4)
2. Gherman C, Braicu OL, Zanoaga O, Jurj A, Pileczki V, Maralani M, Drigla F,
Braicu C, Budisan L, Achimas-Cadariu P, Berindan-Neagoe I. Caffeic acid
phenethyl ester activates pro-apoptotic and epithelial-mesenchymal
transition-related genes in ovarian cancer cells A2780 and A2780cis. Mol Cell
Biochem. 2016 Feb;413(1-2):189-98. doi: 10.1007/s1 1010-015-2652-3;
*Gherman C and Braicu OL co-first authors with equal contribution; ISI Impact
Factor 2. 66 (Study included in the chapter 5)
3. Braicu O, Pileczki V, Braicu C, Achimas-Cadariu P, Irimie A, Berindan-Neagoe I.
p53 siRNA - a therapeutic tool with significant implication in the modulation
of apoptosis and angiogenic pathways. Clujul Med. 2015;88(3):333-7. doi:
10.15386/cjmed-434. B+ journal (Study included in the chapter 6)

3
Molecular profiling for endometriosis and related malignancies
PUBLICATION LIST
Articles published in extenso as a result of doctoral research
1. Braicu OL, Budisan L, Buiga R, Jurj A, Achimas-Cadariu P, Pop LA, Braicu C,
Irimie A, Berindan-Neagoe I, miRNA expression profiling in formalin-fixed
paraffin-embedded endometriosis and ovarian cancer samples, 2017:10,
Pages 4225—4238, doi.org/10.2147/OTT.S137107. ISI Impact Factor 2.6
(Study included in the chapter 4)
2. Gherman C, Braicu OL, Zanoaga O, Jurj A, Pileczki V, Maralani M, Drigla F,
Braicu C, Budisan L, Achimas-Cadariu P, Berindan-Neagoe I. Caffeic acid
phenethyl ester activates pro-apoptotic and epithelial-mesenchymal
transition-related genes in ovarian cancer cells A2780 and A2780cis. Mol Cell
Biochem. 2016 Feb;413(1-2):189-98. doi: 10.1007/s1 1010-015-2652-3;
*Gherman C and Braicu OL co-first authors with equal contribution; ISI Impact
Factor 2. 66 (Study included in the chapter 5)
3. Braicu O, Pileczki V, Braicu C, Achimas-Cadariu P, Irimie A, Berindan-Neagoe I.
p53 siRNA - a therapeutic tool with significant implication in the modulation
of apoptosis and angiogenic pathways. Clujul Med. 2015;88(3):333-7. doi:
10.15386/cjmed-434. B+ journal (Study included in the chapter 6)

5
SUMMARY
INTRODUCTION 9
STATE OF THE ART
1. Introduction in gynecological disorder 15
1.1. Pathogenesis of Endometriosis 15
1.2. Endometriosis as multifactorial disease 18
1.3. Malignant transformation of endometriosis 20
1.4. Ovarian and endometrial cancer 21
1.5. Molecular features of endometrioid and ovarian cancer 23
1.6. Clinical implication of coding and non-coding genes 24
1.6.1. RNA interference mechanism 26
1.6.2. Methods used for molecular profiling studies on
coding and non-coding RNAs
30
PERSONAL CONTRIBUTION
1. Working hypothesis/Objectives 37
2. General metodology 39
3. Study 1. miRNA expression proffiling in formalin-
fixed paraffin-embeded endometriosis and ovarian
cancer samples
41
3.1. Context of the study 41
3.2. Working hypothesis and objective 43
3.3. Materials and methods 43
3.4. Results 47
3.4.1. Relative miRNA expression level in endometriosis
and ovarian cancer
47
3.4.2. Generation of miRNA based networks for the
altered miRNA profile in endometriosis versus control,
ovarian cancer versus control, respectively ovarian cancer
versus endometriosis
51
3.4.3. qRT-PCR data validation 53
3.5. Discussions 56
3.6, Conclusions 62
4. Study 2. Targeting epithelial to mesenchymal transition
in ovarian cancer using natural compunds
63
4.1. Context of the study 63
4.2. Working hypothesis and objective 64

4.3. Materials and methods 64
4.4. Results 66
4.4.1. Evaluation of the antiproliferative effect 66
4.4.2. Apoptosis assessment by Flow Cytometry 66
4.4.3 Gene expression pattern in A2780 and A2780cis
cells
67
4.4.4. TP53 and TGFB1 protein validation 68
4.4.5. Gene network interaction effect to CAPE
treatment
70
4.5. Discussions 73
4.6. Conclusions 74
5. Studiu 3. Therapeutic implication of RNA interference in
gynaecological cancer
75
5.1. Implication of transforming growth factor β siRNA in ovarian
cancer
5.1.1. Context of the study 75
5.1.1. Working hypothesis and objective 75
5.1.1. Materials and methods 76
5.1.2. Results 77
5.1.3. Discussions 79
5.1.4 Conclusions 81
5.2. siRNA a therapeutic tool for modulating the expression level
for mutated p53 gene in cervical cancer
81
5.2.1. Context of the study 81
5.2.2. Working hypothesis and objective 81
5.2.3. Materials and methods 82
5.2.4. Results 83
5.2.5. Discussions 85
5.2.5.Conclusions 87
7. General discussions 89
8. General conclusions 91
9. Originality and inovative contributions of the thesis 93
REFERENCES 95

7
ABREVIATION LIST
CAPE caffeic acid phenethyl ester
circRNA circular RNA
EAOC endometriosis associated ovarian cancer
EGF epidermal growth factor
EMT epithelial to mesenchymal transition
FFPE formalin fixed paraffin embedded
HPV human papilloma virus
IPA ingenuity Patways Analysis
lncRNA long non-coding RNA
miRNA microRNA
mRNA messanger RNA
ncRNA non-coding RNAs
ncRNAs non-coding RNAs
PI propidium iodine
PR progesterone receptor
RISC RNA-induced silencing complex
RISC RNA-induced silencing complex
RNAi RNA interference
ROC receiver–operator characteristic
siRNA short intefering RNA
ssRNA single strand sequences
TGFβ1 transforming growth factor β
VEGF vascular endothelial growth factor

9
INTRODUCTION
Endometriosis is a common condition in women of reproductive age. According
to several epidemiological studies endometriosis may be associated with increased
risk for various malignancies, including ovarian and uterine tumors. Therefore there is
an urgent demanding for identification of those markers related to malignant
transformation and prognostic role. Despite all the improvements in therapeutic
management of the gynecological malignancies and the emergence of targeted
molecular therapies and immunotherapy, the prognosis of ovarian and uterine tumors
still remain unfavorable.
The rapid advancements in molecular biology techniques have permitted the
discovery of non-coding RNAs (ncRNAs) as key regulators of physiological processes
but also as important players not only for physiological mechanisms but also for
pathological mechanisms, especially in those related to cell-to-cell communication. As
their name suggest, ncRNAs are sequences that do not translate into a target protein,
but are able to simultaneously regulate the expression of multiple genes by targeting
the complementary sequence within the genes and to stop the translational process,
affecting physiological and pathological processes. ncRNAs were demonstrated to have
an important role in endometriosis and its transformation and carcinogenesis related
processes, invasion and metastasis of solid tumors, including the case of gynecological
cancers.
From what has been compiled, the deregulation of biogenesis and functional
roles of ncRNAs were, as expected, at the crossroads for different human pathologies
ranging from genetic or not genetic diseased or those related to environmental factors.
Finally, the continued understanding of the molecular mechanisms and signaling
pathways where ncRNAs participate, should offer new insights to define new
diagnostic strategies and open new avenues for therapies.
Presently, numerous ncRNAs have been characterized in different malignant
pathologies as possible biomarkers for clinical management of a wide range of
diseases, but there is still a need for additional research activities in order to elucidate
the complex roles for these transcripts in organism, homeostasis and pathological
conditions in endometriosis and its related malignancies.

The oncologic research in the last years has been focused on the study of short
ncRNAs also called microRNA (miRNA) profiles, with important significance in
biomarkers discovery. Discovering the multifaceted role of the miRNA role may reveal
novel translational opportunities for biomarkers development and therapeutic
targeting for a wide range of pathologies, including endometriosis or related
malignancies.
By understanding the role of miRNAs in intercellular communication in
endometriosis and related malignancies, will lead to new insights in the complex
biology of these malignancies. As struggles pursue towards the transition of miRNA
profiling from bench to bedside, PCR-array panels offer a perfect instrument to sustain
our present applied research and the resulting clinical processes of diagnostic and
prognostic of endometriosis and related malignancies.
The most important results of this thesis refer to the fact of highlighting the
relationship between frequently deregulated transcripts in endometriosis and the
derived malignancies followed by identification of a common miRNA signature in
order to discriminate from normal, endometrioid or malignant tissue, with an
implication for biomarkers for prevention, early diagnostic and response to therapy.
Our research direction consists in the process of fusion between two mostly
individually studied non-coding transcripts: miRNAs and siRNA, in order to correlate
the activity of these two important participants in tumor development. Another major
research interest is to develop novel therapeutic strategies using small natural
molecules or siRNA targeting oncogenes like p53 or immune response modulators,
leading to an increased therapeutic response.
The present study comprises of several innovative aspects and approaches to a
difficult challenge faced by both patients and clinicians – molecular profiling studies.
This makes our study of great importance from technological point of view, since it
brings together many different and yet complementary techniques, from PCR-array,
bioinformatics data analysis, to cell culture based studies and functional studies
evaluating the therapeutic effect of siRNA. All this is done for the patients’ benefit, for
improving health status of the patients diagnosed with endometriosis and related
malignancies.
The integration of transcriptomics pattern will provide a more complete
understanding of how single cells and different tissue types are organized and
controlled and how they are altered in a specific pathological state, thus offering the
opportunity of tailored therapeutics, using different combinatorial strategies for
chemotherapeutics with siRNA or small natural or synthetic compounds. Our study
is focused on filling some gaps regarding the role of these miRNA and siRNA in
pathological status for gynecological diseases.
miRNA and siRNA can be used to target a specific ‘signature’ that is able to
increase the precision of diagnosis, deciding prognosis and as therapeutic targets for
therapy. Presently are available as oligonucleotide sequences miRNA inhibitors or

11
mimics, respectively as siRNA, able to target a specific gene. All these facts have an
important role in personalized care. In spite of all these, there is still much work to do
starting from molecular profiling, validation of the miRNA/siRNA target mechanism in
preclinical studies before a clinical trial can be done. All these need to be done
rigorously in order to assure a success and to prevent the failure, alike the situation
registered for miR-34, where the clinical trial was a fiasco.

13
STATE OF THE ART

15
1. Introduction in gynecological disorders
Gynecological disorders are diagnosed and treated by gynecologists, this specialty
being involved in the cure of diseases and issues of the reproductive organs (breast
and organs in the abdominal and pelvic area including the uterus, ovaries, fallopian
tubes, vagina and vulva).
Most of gynecological pathologies are curable, but some pathologies may have
devastating effects - impacting women capacity to have children and might be even life
threatening.
The gynecological disorders are classified as:
 pathologies that cause infertility (Pelvic inflammatory disease, infectious
disease- chlamydia, endometriosis, menstrual disorder)
 female cancer (breast, endometrial, ovarian cancer, cervical cancer, vulva
and vaginal cancer), the incidence is increasing annually (1).
Gynecological pre-cancer and cancers are substantial illnesses in women
through the world (2). All these gynecological disorders are caused by the genetic
background correlated with life styles factors, leading to diverse biopsychosocial
issues.
1. 1. Pathogenesis of Endometriosis
Endometriosis is a pathology with a wide range of facades regarding life
quality (3). Endometriosis is a prevalent hormone-dependent gynaecological disorder
leading to serious menstrual disturbance and/or prolonged pelvic discomfort
with/without infertility. The prevalence of endometriosis is estimated to be 10% in
women at reproductive age (4, 5).
In spite of its first characterization as pathology three centuries ago by
Sampson, since 1920 the problem of the correct characterization of endometriosis as a
distinct pathology remains a subject of debate nowadays. Endometriosis is a chronic
benign gynaecological disorder in which endometrial-like tissue conducts to the
presence of the lesions in the exterior of uterine cavity.

From the histological point of view the endometriosis is defined by the
presence of endometrial glands and stroma in ectopic sites like ovaries, uterine
ligaments, recto- and vesicovaginal septae, pelvic peritoneum, cervix, labia, and vagina
(4, 6).
A number of theories have emerged to summarize the wide range of individual
observations concerning pathogenesis and these are classified as those suggesting that
engrafts result from uterine endometrium and those suggesting that implants originate
from other tissues than the uterus. In parallel with these suppositions, a connection of
a wide range of environmental agents with endometriosis was observed (7). The
involvement of these agents in predisposing to endometriosis (endocrine, immune,
stem/progenitor cells, epigenetic modifications) must be taken into account in the
circumstances of genetic background but also as stimulus-driven reprogramming of
the female reproductive tract (7).
The most accepted hypothesis for the acquisition of endometriosis consists in
retrograde menstruation phenomenon, where endometrial cells migrates and
stabilizes on peritoneal surfaces (8-10). Even so, there are a number of unique theories
regarding the origin of endometriosis that unifies under two major hypothesis: one
that is proposing the uterine origin of the implants and the second one that highlights
the non-uterine origin of disease (9). Although the general proposal for the
development of endometriosis harmonies in retrograde menstruation phenomenon,
this observation being supported by the clinical observations, there are many factors
that contribute to the development of endometrioid implants. The undergoing steps
consist in escape from the immune system, stabilization to peritoneal area, invasion of
the epithelium and angiogenesis that supports the growth and survival of the cells (9).
The clinical and pathological aspects or the evolution of this disease are
unpredictable; in various situations, the disease can remain as a minimal/mild disease
or even might disappear. In other situations it is associated with a severe
symptomatology based on the invasion and tissue infiltration, expansion of
endometrioses or “chocolate cysts,” or presenting pelvic adhesions, or pelvic blockage
affecting other internal organs. These steps require numerous molecular processes
that are currently unclear and non-specific.
At the time being, the etiology of endometriosis is uncertain, numerous studies
being made for the purpose to determine the exact factors that can lead to the
acquisition of this benign gynecological disease (10). Endometriosis remains an
enigmatic illnesses of the females considering there is a significant lack of information
for the cause that activate this pathology or the complex mechanism, where is the limit
between benign and malign (11). In malignant transformation of endometriosis, the
most prevalent site is the ovary (5, 11).
Therefore it is required a deeper knowledge of the altered cellular molecular
processes that are currently unclear and non-specific for developing this disease and

17
moreover for its malignant transformation in three gynecological cancers
(endometrial, cervical and ovarian cancers) (12).
The malignant transformation is based on the interconnection among the (13):
1. endocrine,
2. immunologic
3. genetic components
Diagnosis of endometriosis. Endometriosis is generally diagnosed by
visualization of lesions by laparoscopy or laparotomy. Taking into consideration its
invasive nature, surgery frequently causes a delay in the process of diagnostic and
treatment of this disease, particularly in young women. Nevertheless the early
diagnosis of endometriosis is of importance in decreasing the incidence of the disease
and infertility problems. In laparoscopy, the superficial endometriosis and ovarian
endometriosis are diagnosed due to the appearance of bleeding. There are available
also noninvasive imaging technologies, such as high resolution transvaginal ultrasound
or magnetic resonance imaging to explore large ovarian endometrioid cysts and deep
endometrioid nodules; but they do not permit precise staging of endometriosis;
superficial lesions, small ovarian endometriosis and endometriosis-related adhesions
cannot be discerned.
Many studies are therefore focusing on identifying biomarkers for the diagnosis
and for assuring the follow-up of patients with endometriosis. Even if the ‘‘gold
standard” for the identification of endometriosis is the laparoscopy, various scientific
articles have mentioned that whole blood serum/plasma, peritoneal fluid and tissue
biomarkers can be correlated with endometriosis (14).
In fact, the detection of more sensitive and specific markers of endometriosis
should facilitate the development of accurate and non-invasive techniques for
diagnosis and prognosis, where the coding and non-coding genes play an important
role. Moreover, the inheritable susceptibility to endometriosis supports the
exponentially interest in transcriptomic pattern and/or genetic polymorphisms that
might conduct to an augmented risk of disease. By detecting these alterations, may
lead to their application as biomarkers for endometriosis or its related disease (14).
The present instruments for diagnosis and treatment demand to be developed
to certify reliable diagnosis based on molecular profiling leading to an efficacious
treatment. In addition, endometriosis is connected with enlarged risk of ovarian cancer
and therefore, the differential evaluation among the benign and malignant transition is
essential.

1.2. Endometriosis as a multifactorial disease
Endometriosis is a multifactorial disease considering the diverse molecular
mechanisms identified to be participating in the pathophysiology of this disease (5). In
a recent study Burney et al., 2012, displayed the main molecular hallmarks of this
pathology:
 estrogenic dependence
 progesterone resistance
 inflammation
 genetic predisposition
Estrogen is an important factor that contributes to the installation and
development of endometriosis outside the uterus, abnormalities in signaling pathways
of this hormone being associated with pathological status (15, 16). Abnormal estrogen
metabolism increases the proliferation of endometrioid cells (17).
Progesterone is normally involved in suppression of oestrogen dependent
proliferation of epithelial cells, secretory evolution of the glands and conversion of
stromal cells into specialized cells. In endometriosis the status of progesterone
resistance is installed and genes responsible for the events mentioned above are
altered, conducting to the dysregulations of these critical events (18-20). Also, the
progesterone resistance can be acquired during inflammation, event that take place in
endometriosis, due to the competition with proinflammatory transcriptional factors
that alter the progesterone signalling pathway (21). An altered expression pattern for
progesterone receptor (PR) isotypes (PR-A and PR-B) at both eutopic and ectopic sites
of endometrial growth was observed (22). But the biological origin of diminished
endometrial progesterone responsiveness between women with endometriosis
remains to be completely clarified (23).
The inflammatory medium inside the pelvis lead to the pathophysiology of
pain perception in symptomatic patients with endometriosis (24). This implies the
participation of estradiol, growth factors, prostaglandins and additional inflammatory
molecules in the pathogenesis of pain. It is speculated that nerve fibers in
endometriosis implants impact dorsal root neurons inside the central nervous system,
expanding pain recognition in endometriosis sufferers (24, 25).
It is generally agreed that endometriosis is an inflammatory state connected
with alteration in immune response, this being a proclivity for early carcinogenic
events. The presence of foreign tissue in the peritoneal cavity triggers the
inflammation response characterized by the over secretion of prostaglandins,
cytokines and chemokines. Also, the macrophages invading the endometriosis lesions
are promoting the extension of the lesions and also the angiogenetic process (26-29).
Modified immunity lead to the activation of factors involved in adhesion, invasion, and

19
angiogenesis, as well as proliferation and disabled apoptosis are indispensable in the
developing of the lesions. Chronic inflammation has a proeminent function in the
modulation of various pathophysiological mechanisms e.g. angiogenesis, estrogen
metabolism and oxidative stress (30).
Another important panel of proteins is the one regulating epithelial to
mesenchymal transition. Low expression of E-cadherin in epithelial cells in peritoneal
endometriosis was observed, in special in advanced stages of endometrioid
lesions(31). In this context, the treatment strategies for endometriosis focused on this
alteration, in particular toward ovarian function suppression and indirect control of
cell growth and activity of endometriosis remain insufficient (30).
Comprehension of the inflammatory origin, diminished progesterone activity
at the level of the endometrium mucosa or the neuronal sensitization of endometrioid
injuries will lead to improvement of new therapeutic methods, in particular to reduce
pain and infertility and to prevent malignant transition (32).
An alteration of genetic profile for the endometrial cells is persuading their
predisposition to insert the hereditary component as key component of this pathology.
A higher risk of endometriosis for first degree relatives was observed. The presence of
genomic alterations illustrates a probable origin for a deliberated survival benefit to
sloughed endometrial cells in the case of endometrioid grafts (17, 32).
Endometriosis can be considered a consequence for the presence of several
genetic and epigenetic alterations; ovarian cancer and endometrioid lesions were
linked with a specific altered genetic profile (33). It is well known that mutation of
tumor suppressor genes coding protein p53 and B-cell lymphoma 2 (BCL-2) are
mutated and differentially expressed in endometrioid tissue versus normal tissue (13).
BRCA1/2 mutations were connected firstly with an increased risk for gynecological
cancers, but recently were discovered to be involved in endometriosis and malignant
transformation (34, 35). Other genetic factors have an important contribution for the
risk of endometriosis, several studies revealing some of the key risk-loci, where WNT4,
CDKN2B-AS1 and GREB1 genes are important candidates for the study of
endometriosis pathogenesis (36, 37). The WNT4 gene translates into a member of the
wingless-type family, member with important roles in the development of the
reproductive system of the female. The CDKN2B-AS1 gene is expressed through a long
noncoding RNA that encodes three proteins with tumor suppressive roles: p15, p16-
INK4a and p14ARF. The last gene, GREB1 is involved in the estrogen receptor-
regulated pathway (36, 37).
The above factors along with environmental factors can impact the
progression of this disease (11), the concomitant presence of all this complex factors
may conduct to the malignant transformation, via modulation of the transcriptomic
pattern, where non-coding RNA plays a key role.

1.3. Malignant transformation of endometriosis
Statistically, about 80 % of endometriosis associated malignancies were
identified in the ovary, while 20 % are localized in extra gonadal sites, including the
intestine, rectovaginal septum, abdominal wall, pleura and others (38).
More than 1100 PubMed publications present data concerning the connection
among endometriosis and gynecological cancers and present neoplastic phenotypes,
containing invasion of stromal tissue and lymphatic dissemination to distant organ
sites. It was clearly demonstrated that the atypical endometriosis have been related
with a premalignant transformation and from histological point of view was
demonstrated to present cytological atypia and an architecture that sustains cell
proliferation features (39).
Endometriosis associated ovarian cancer (EAOC) was demonstrated to have an
incidence of around 22%. EAOC is mainly related with clear-cell and endometriosis
histology in younger patients. The main advantage of EAOC is related to early
diagnostic that confers a better prognostic value (40).
Endometriosis was clearly demonstrated as precursor of gynecological cancer.
An increased number of investigations present that endometriosis is bracketed with an
inflated risk of ovarian cancer (the values for the relative risk being between 1.4-8.95)
and the prevalence of endometriosis in ovarian cancer ranges among 3.4 and 52.6% as
displays the literature data (4, 40, 41). The connection with the ovarian cancer, is
retrieved even for the early stages or the case of low grade stages of this pathology,
being generally related with endometrioid or clear cell carcinoma, these were the
conclusions of a metanalysis study (42).
As for the case of the endometrial cancer, the genetic investigations revealed
that endometrioid lesions are characterized by a set of genes related to neoplasms,
including p53, KRAS or PTEN or inflammatory mediators, mentioning a clear transition
for a specific profile that allow the transition from endometrioid lesions to invasive
carcinomas.
The detection of genetic and epigenetic markers is fundamental for recognizing
patients at risk for transition to neoplasia (4). In fig.1 frequent hallmarks apportioned
by endometriosis and ovarian cancer, starting with environmental exposure, alteration
of immunological pattern or the presence of some mutations or transcriptomic
alteration.
Until present, a number of frequent pathways related to endometriosis and
ovarian cancer have been deciphered, comprising the dominance of certain cytokines
(interleukins, citokines1)(43), oxidative stress related markers, estrogenic
environment, retrieved as a common picture in endometriosis and EOAC (44, 45). In
the last years the number of studies presenting the implication of endometriosis in
malignant transformation has exponentially increased. In spite of this alteration in the

21
pre-neoplastic state of endometrioid lesions are limited. There are only a few data
about the altered pathways and genetic and epigenetic events related to this
phenomenon (46).
Fig. 1. Common features shared by endometriosis and ovarian cancer
It is clear that oxidative stress caused by environmental factors or internal
factors has an important role in malignant transformation. These mechanisms are
connected with the liberation in the micro-medium of a wide range of cytokines and
soluble mediators. This stage in literature is retrieved as “atypical” endometriosis; in
this step might be accused the genetic and transcriptomic alteration that leads to
ovarian carcinoma.
1.4. Ovarian and endometrial cancer
Ovarian and endometrial carcinomas contain the majority of gynecological
carcinomas in developed countries. Endometrial carcinoma is the most frequent
with an estimated value of 500.000 new cases worldwide; however, ovarian cancer
remains as a highly fatal gynecologic pathology in women at global level due to the late

diagnostic, with around 140.000 deaths/year worldwide (1); until now the exact
reason for ovarian cancer remains predominantly unidentified, BRCA mutations are
the principal recognized hereditary factors responsible for the initiation and
progression of ovarian cancer.
Ovarian cancer has similarities with breast cancer, but also with late age at
menopause and infertility. The use of exogenous sex hormones or other variable
factors like obesity, tobacco smoke, radiation exposure, psychotropic medication and
high-level physical activity are associated with potential ovarian cancer development
(3). Other diseases regarding the reproductive system (pelvic inflammatory disease,
polycystic ovary syndrome, and endometriosis) can be associated with increased
susceptibility for cancer appearance. Separate from these risk factors, there are a
number of circumstances that can provide protection against these types of tumors,
such as use of oral contraceptives (the protection persists for at least a decade after
ceasing of use), parity, pregnancy, lactation, tubal ligation and hysterectomy.
From the histological point view, both pathologies are divided in:
endometrioid, serous, clear cell, mixed, carcinosarcomas and mucinous carcinomas
(47, 48).
Ovarian endometrioid carcinomas represent less than 10% of ovarian
carcinomas, most being retrieved as low grade with favorable prognosis. Most of the
ovarian cancer cases (70–80%) are represented by the high-grade serous carcinomas,
which have similar features with the endometrial serous carcinomas, having an
unfavorable prognostic and survival rate.
Endometrial endometrioid carcinomas arise most regularly, representing
around 70–80% of the total number of cases; these incline to be low-grade with
favorable prognosis and are in general cured by hysterectomy. The remaining percent
for the case of endometrial carcinoma are represented by serous, mixed, clear cell
carcinomas and carcinosarcomas (47).
There is a cumulating demonstration that these two gynecological
malignancies occur from transformed endometriosis, consequently, derive from
equivalent precursor endometrial epithelial cells; however, the process of molecular
pathogenesis still needs to be deciphered (48).
Generally for most of the gynecological pathologies as first-line therapy
as curative intention is represented by surgery, including the resection of the
ovaries, fallopian tubes, uterus and cervix, and for some cases depending of the
stage, the para-aortic and pelvic lymphadenectomy.
Second line of treatment is represented by the adjuvant therapy or
combination with radiotherapy. The selection of the combination between the
chemotherapy, hormonal therapy or radiotherapy is based on the disease status, the
degree of the tumor resection margins, evaluated by histological analysis that is
focused on the identification of the hormonal status (the presence/absence of the
hormone receptor /mutational pattern). In general the combination of

23
chemotherapy, radiotherapy and chemotherapy—the so-called “sandwich”
approach—is connected with enhanced overall-survival rate, even for those in
advanced stages (49). The standard chemotherapy is represented by
platinum/taxane-based and the response rate differs in function of histological
subtypes. For increasing the response rate, it is applied a combination of targeted
therapy such is the case of anti-VEGF antibody (bevacizumab) and PARP inhibitors
(Olaparib) (49).
The key of cure in these types of cancers is represented by the targeted
therapies; a special attention should be focused on the identification of particular
molecular subtypes that are predisposed to chemoresistance. This new investigation
need to overcome this issue. Future epigenetic, genomic or genetic evaluation will have
a positive impact on diagnostic and treatment application.
1.5. Molecular features of endometrioid and ovarian cancer
The most prevalent type of endometrial cancer is represented by the Type I,
defined in literature as endometrioid cancer; the molecular abnormalities comprise
mutations in PTEN, KRAS and β-catenin. For most of the cases type I precursor is
related with the endometrial intraepithelial neoplasia which shows PTEN mutations.
Type II endometrial cancer is less known, but includes serous and clear cell tumors
and has as common feature: the p53 mutation. Other frequent mutation retrieved for
this pathology is represented by ARID1A, PIK3CA, PIK3R1, CTNNB1 and KRAS (48).
Ovarian cancer has a heterogeneous biology, such is the case of the most solid
tumors having unique altered pathway (50, 51). Based on the recent investigation
taking in account the morphologic, immunohistochemical and molecular genetic
determination, there was developed a novel paradigm related to the pathogenesis and
origin of the ovarian cancer. This paradigm has allowed to be implemented as a novel
classification in two subtypes based on the dualistic model of carcinogenesis. In type I
are included the low-grade serous, low-grade endometriosis, clear cell and mucinous
carcinomas or some rare cases as is the case of Brenner tumors. Type I are commonly
less aggressive, being related with the presence of some particular mutations (KRAS,
BRAF, ERBB2, CTNNB1, PTEN PIK3CA, ARID1A, and PPPR1A), targeting particular
pathways, being relatively stable genetically. Type II tumors are comprised of high-
grade serous, high-grade endometriosis, malignant mixed mesodermal tumors (also
called as carcinosarcomas) and undifferentiated carcinomas. By opposite to type I, type
II are characterized by a high frequency of TP53 and BRCA mutation and do not have,
in generally, mutations retrieved for type I, being considered as genetically highly
unstable (50).

The advantage of BRACA1 and BRCA2 evaluation was connected with new
therapeutic agents, such is the case of poly (ADP-ribose) polymerase (PARP) DNA
repair pathway, these inhibitors have significantly increased the response to therapy
for endometrioid and ovarian cancer (52).
In the last years the management of the ovarian cancer and endometrial cancer
has considerably enhanced owing to more operative surgery and treatment, based on
the different combination with the cytotoxic agents, but the survival rate still remains
reduced, being less than 30%.
Many studies consider that additional experimental trials using combinations of
small molecules with different combination of monoclonal antibody or non-coding
RNA can be used for increasing the response rate to treatment, but not to replace
chemotherapy.
Taking in account the high heterogeneity of these two gynecological pathologies,
an increased survival rate can be obtained only based on the implementation of the
novel insights at molecular and cellular levels to personalize the treatment and to
permit an efficient method for monitoring the response to therapy. Also the attention
should be focused on early diagnostic for these pathologies.
1.6. Clinical implication of coding and non-coding genes
Even if the Human Genome Project was accomplished 10 years ago, the catalog
of human protein-coding genes has highly controversial issues (53). Presently there
are described more than 20000 of protein-coding genes (54).
The human genome (about 98%) encodes for non-coding RNAs (ncRNAs), which
are functional RNA species that coordinate all complex genetic phenomena in
eukaryotic organisms (54). These ncRNAs are subdivided into two groups, in
accordance to their size: long non-coding RNAs (lncRNAs), which have over 200
nucleotides, and short non-coding RNAs, with less than 200 nucleotides, being
represented by three main classes of short interfering RNAs (siRNAs), microRNAs
(miRNAs) and PIWI-interacting RNAs (piRNAs). In human carcinogenesis, their role is
undeniable and it stands as evidence that these molecules should be therapeutical
targets.
Long non-coding RNAs (lncRNAs) are non-protein-coding transcripts which,
in the past years, have emerged as crucial factors in normal cell physiology as well as
in disease mechanisms. Regarding their role in tumorigenesis, lncRNAs are divided
into two categories: tumor-suppressor and oncogenic. Because of their tissue-specific
expression patterns, high stability and efficient detection in bodily fluids, lncRNAs have
great potential as biomarkers for diagnostic, prognostic and monitoring schemes in
carcinogenesis. An important class of circular RNA (circRNA) was emphasized, these

25
are characterized by the capacity of a covalent bond between the 3′ and 5′ ends of
protein-coding exons produced by back-splicing. circRNA is involved in gene
expression regulation due to the fact that they act as miRNA sponge and as RNA-
binding protein sequestering agents (54).
It was observed that the lncRNA MALAT-1 has a decreased expression level
when reported to the normal or hyperplastic endometrium. Another important
lcRNA transcript is represented by OVAL, the expression level was observed to have
a reduced pattern in type II endometrioid carcinoma but an increased expression
level in type I endometrioid carcinoma (49). Other relevant ncRNAs are represented
by HOTAIR, H19 and SRA, having an increased level in endometrioid carcinoma,
being related with tumor grade and patient prognostic (49). lncRNA-TUG1 was
proved to promote endometrial carcinoma, by inhibiting miR-299 and miR-34a-5p
(55). LncRNA-GAS5 activates the expression of PTEN expression by reducing the
expression level of miR-103 in endometrial cancer cells(56)
MicroRNAs (miRNAs) are a group of short, single stranded, 20-22 nucleotide
RNA molecules that have the function of negative gene expression regulators at post-
transcriptional level. miRNAs were first described in C. elegans more than two
decades ago, were identified in vertebrates, flies, worms, plants and even viruses. In
humans, were identified over 2500 transcripts (57). miRNAs have been publicized to
have the capacity to reduce the levels of their target transcripts and thus the amount of
protein encoded by these transcripts by direct and imperfect miRNA:messenger mRNA
interaction (58). In addition, the profiling studies were exploited to identify miRNA
genes that may represent downstream targets of activated oncogenic pathways or
those targeting protein-coding genes involved in cancer, being considered as
‘hormones’ (59, 60).
As a result of the miRNA binding with imperfect complementarity to target
messenger RNA (mRNA) of protein-coding genes, both translational repression and/or
mRNA cleavage can occur, and consequently the levels of encoded proteins are
diminished. MiRNAs are able to regulate key cellular processes, including cell
proliferation control and promotion of cell survival. Also, a high number of miRNAs
were proved to be key regulators of the genes involved in the hallmarks of cancer.
MicroRNAs from body fluids are now emerging as suitable biomarkers for
cancer risk evaluation, detection and prognosis in numerous cancers, including the
case of endometriosis and its related malignancies. Tissue (fresh or paraffin
embedded), blood, plasma, serum, urine, and other fluids preserve microRNA
sequences, stable in various extreme conditions due to their size and configuration
that are able to form a specific panel with applications in discerning between healthy
and high risk patients and also in early detection of incipient cancer.
There is an increased interest to study the potential role of miRNA in
endometriosis based on the fact that miRNA are key regulators of gene expression and
are involved also in endometriosis pathogenesis, including apoptosis, cell survival,

hypoxia, angiogenesis or matrix remodeling and tissue repairing related to
endometriosis (61).
Apoptosis acts as a vital function for the pathogenesis of endometriosis. The
main characteristic retrieved in the case of endometriosis is the fact that the
endometrial cells have the capacity to avoid apoptosis when are placed outside the
uterus. In eutopic endometrium from endometrioid lesion was retrieved raised
expression of antiapoptotic factor and decreased expression of proapoptotic factors
when compared to healthy endometrium (62). miR193a, miR-29c, miR-203 and
miR200c were proved to be down-regulated in epithelium of late proliferative
endometrium (63, 64). miR-34 regulates the p53 tumor suppressor network leading
to modulated apoptosis and cellular senescence (65);this was proved to have a
reduced expression level in the case of early secretory endometrium in women with
endometriosis (66).
Latest investigation displays a new facet of endometriosis revealing that
hypoxic condition is connected to the activation of a wide range of coding or non-
coding transcripts, that finally will conduct to the implantation, cellular proliferation
and survival or the preservation of ectopic endometrioid lesions (67). Aberrant
activation of MAPK has been proved to play important roles in pathological processes
in endometriosis. However, how MAPK are constitutively activated in endometrioid
tissues remains largely unknown, but recently were proved to be regulated by miRNAs.
miR-20 was retrieved upregulated by HiF-1α in endometrioid stromal cells (68). Also
the up-regulation of miR-20 was connected to prolonged ERK phosphorylation and the
overexpression of angiogenic genes (69).
miRNAs associated to extracellular matrix remodeling and tissue repairing
were altered in the case of endometriosis. Due to its inherent nature, endometriosis is
characterized by an inflammatory microenvironment including cytokines, chemokines
but also miRNAs, where this small structure plays a key role in matrix remodeling and
tissue repairing mechanisms leading to the establishment of endometrioid lesions.
Current findings concerning endometriosis-associated matrix remodeling and tissue
molecules reveal a key role of TGFβ, where miR-200 family plays a significant role (70,
71).
1.6.1. RNA interference mechanism
RNA interference mechanism (RNAi), sometimes known as short interfering
RNA or silencing RNA (siRNA), is a class of double-stranded RNA molecules, 20-25
nucleotides in length, that play a variety of roles in biology. This phenomenon was first
described in plants, where a more intense color of petals in Petunia was obtained by
gene amplification of a pigment due to introduction of a DNA molecule containing this

27
gene (72, 73). Surprisingly the researchers have not seen a growth, but a decrease in
gene expression, the process that is called co-suppression (73).
siRNA is involved in the RNAi pathway, where it interferes with the expression
of a specific gene. RNAi has been studied on animal model by Fire et al., and they found
that introducing double stranded RNA sequences (double strand RNA, dsARN) in the
nematode Caenorhabditis elegans resulted in an inhibition of the expression of the
target gene compared to single strand sequences (ssRNA) (73). The importance of
RNAi is emphasized by the fact that for its discovery in 2006, researchers from the
United States of America: Andrew Z. Fire and Craig C. Mello were awarded with "Nobel
Prize in Physiology or Medicine" (74, 75).
siRNA mediated knockdown of genes at the messenger RNA (mRNA) level,
offers a new alternative therapeutic strategy. With this discovery began a new era in
the study of gene functions and specific gene therapy (74, 75). In this process, double-
stranded RNAs are cleaved by the cellular nuclease Dicer into short 21–22mer
fragments referred to as siRNA, which enter a ribonucleic protein complex termed the
RNA-induced silencing complex (75). Guided by the antisense strand of the siRNA, this
complex mediates a specific degradation of the corresponding mRNA. The intrinsic
ability to sequence-specifically down-regulate gene expression in a temporally- and
spatially-controlled fashion has led to heightened interest and rapid development of
siRNA-based therapeutics. siRNAs have not only been widely used as a valuable tool
for functional genomics research, but they also have demonstrated great potential in
biomedical therapeutic applications for diseases caused by abnormal gene
overexpression or mutation (76, 77).
siRNA delivery systems
One of the key problems facing the development of siRNA is the delivery to
specific cell or tissue types. Until now different delivery systems have been tested.
Depending of their structure, they can be defined as follows: viral vectors (e.g.
adenoviral, lentiviral, retroviral, herpes virus simplex, RNA viruses) and non-viral
vector systems (76, 77).
Non-viral vector systems include a variety of small molecules, lipids, peptides
and proteins, which have been examined as delivery vehicles for nucleic acids (77).
Carbon nanomaterials such as nanotubes or fullerenes can be also used as carriers for
siRNA. Modifications to the carbon surface of nanotubes for higher solubility have been
shown to reduce their toxicity. Fullerenes can also be modified to reduce the free
radical formation and also toxicity (78). The investigators injected the siRNA-nanotube
conjugated directly into tumors, which had the effect of markedly reducing the size and
weight of the tumors. The researchers noted that while the direct injection technique
might be useful with some types of cancer, a more efficient approach would be to add a
tumor-targeting agent to the surface of the carbon nanotubes (78, 79). Furthermore,

delivery systems are simple and regarded as a safe approach, and have been improved
by combining several physical techniques, for example, electroporation, gene gun,
ultrasounds and hydrodynamic pressure (80).
Viral vectors are one of the major vehicles used by scientists in gene therapy
to get their sequences expressed in the proper host. Non-viral methods present certain
advantages over viral methods: they are safer to prepare, the risks of pathogenic and
immunological complications are diminished. Nonviral vectors are the future in gene
transfer because of the limitations for viral vectors such as pathogenicity, expensive
costs for production and systemic instability (78).
Since the first description of RNAi in animals less than a decade ago, there has
been rapid progress towards its use as a therapeutic tool against human diseases.
Advances in our understanding for the mechanisms of RNAi and studies of RNAi in vivo
indicate that RNAi-based therapies might soon provide a powerful new arsenal against
pathogens and diseases for which treatment options are currently limited (78). Clinical
trials, including cancer, with siRNA are underway for a range of diseases, it has not yet
been demonstrated that delivery of siRNA can trigger RNAi in human diseases (78).
Currently, with the aid of commercially available siRNA and transfection reagents,
siRNA-mediated gene silencing has become more important in studying gene function
and identifying disease gene targets. RNAi holds great promise in gene therapy to
silence disease-causing genes (78).
Therapeutic application of siRNA
For decades the standard approach to cancer treatment typically has been a
combination of surgery, chemotherapy and radiation therapy (78). Chemotherapeutic
agents routinely produced unwanted side effects, because not only cancer cells are
destroyed but also some populations of healthy cells (78). The potential of siRNA for
cancer therapy was recognized as siRNA can regulate the gene expression in cancer
cells while leaving normal genes unperturbed.
One important factor for successful RNAi-based cancer therapy is to choose
the right target genes. Great efforts have been made to identify new molecular targets
in cancer therapy. The most common targets fall into the following categories: cellular
proliferation, apoptosis, angiogenesis, and metastasis (81). It was demonstrated that
the nanoparticles could find the target specifically in cancer cells, penetrate the cell
membranes and release their cargo of siRNA. New targeted therapies against specific
forms of cancer have been developed over the last years. More recent studies suggest
that new approaches for RNAi based therapy might be on the horizon in cancer
treatment based on selectively targeting genes involved in different molecular
mechanisms (78).
Target genes regulating apoptosis and cell cycle. siRNA-based therapeutics
can induce apoptosis of cancer cells by targeting anti-apoptotic factors. Suppression of

29
those anti-apoptotic genes by siRNAs was found to induce apoptosis in various cancers
or sensitize them to chemotherapeutic drugs. siRNA targeting anti-apoptotic factor Bcl-
2 gene can specifically down-regulate Bcl-2 expression, with a particular attention on
increasing Bax/Bcl-2 ratio expression and activation of the apoptosis monitored by
caspase-3 activity, which lead to sensitized cells to 5-FU or HCPT (82). Survivin, a
newly identified member of IAP family, is a powerful apoptosis-inhibiting factor. A
recent study suggests that the expression of survivin may be significantly decreased in
HepG2 cells after siRNA transfection. siRNA targeting survivin could induce cell
apoptosis, inhibit cell proliferation and sensitize hepatocarcinoma cells to
chemotherapy. These data provide preliminary evidence for the therapeutic use of
survivin-targeted RNA interference for human tumors that express high levels of this
molecule (83).
Target genes involved in angiogenesis. Vascular endothelial growth factor
(VEGF) plays a critical role in the pathological angiogenesis during the development of
cancer, including gynecological cancer (55). siRNA almost completely inhibited the
secretion of VEGF in endometrial carcinoma cell line and dramatically suppressed
tumor angiogenesis(55). EGFR and its ligand, epidermal growth factor (EGF), play
critical roles in the progression of ovarian cancer. EGFR is overexpressed or
dysregulated in many solid tumors. Targeting EGFR as well as other members of the
human EGFR (HER) family has proven successful using siRNA therapy. The knockdown
of the EGFR receptor with a mutant form using RNAi was related with the inhibition of
cell proliferation and apoptosis activation that have as final effect the inhibition of
tumor growth, the inhibition of the PI3K/Akt signaling pathway (84). Recent study on
endometrioid cancer using RNAi has demonstrated that Sema3E/plexin-D1 axis is a
key event in the modulation of EMT (85)
Cancer development is a gradual and complicated process, often accompanied
by accumulation of genetic alternations which lead to unregulated cancer-promoting
oncogenes or disabled tumor suppressor genes (86).
Tumor suppressor genes. The tumor suppressor p53 is absent or mutated in
more than 50% of human tumors, and abnormalities in the regulation of p53
contribute to cancer. Several therapeutic strategies have been formulated by
evaluating the function and regulation of p53 (87). In some studies the wild-type p53
(wt-p53) gene was delivered to tumor cells, causing apoptosis of the cells in response
to cytotoxic drug treatment. Another important study was focused on a mutant
adenovirus that does not express E1B, a protein that binds and inactivates p53 (88).
Thus, this mutant virus could replicate and lyse p53-deficient human tumor cells but
not cells with functional p53. Also siRNA has been used to suppress the expression of
mutated p53 and restore the function of the wild type gene. TP53 has an inverse
correlation with the amount of apoptosis. Using gene therapy, tumor regression has
been observed with the increase of apoptosis for cancer cells in patients (78).

Oncogenes are genes that are affiliated with tumorigenesis and cancer
progression – in general are mutated or overexpressed, they can help turn a normal
cell into a tumor cell (86). The oncogenes regulate the signaling pathways and sustain
the behavior of malignant tumor cells; the oncogenes are used for manipulation using
siRNA technology. siRNAs, specifically directed against the antiapoptotic HPV-E6
oncogene, restored dormant tumor suppressor pathways in HPV-positive cancer cells
that are otherwise inactive in the presence of E6 and induce apoptosis selectively in
HPV-positive tumor cells. E6 can be a promising therapeutic target to eliminate HPV-
positive tumor cells specifically by RNAi (86). Other study used siRNA targeted against
human HER2/neu, the experimental data revealed a decrease in HER2/neu mRNA and
protein levels on MDA-MB-468 cells. These data indicate that HER2/neu function is
essential for the proliferation of HER2/neu-overexpressing breast cancer cells. This
may be a valuable tool, as antiproliferative agents display activity against neoplastic
cells at very low doses (89). PI3K/Akt signaling pathway represents a key signal
cascade for cancer; it was demonstrated to be involved in the regulation of cell
proliferation and apoptosis, being a confirmed target in a specific mode in tumor cells,
including the case of endometrioid cancer (90).
1.6.2. Methods used for molecular profiling studies on coding and
non-coding RNAs
The investigation of differentially expressed transcripts as a response of the
genome at particular point to diverse environmental stimuli or
physiological/pathological situation, have been evaluated in a wide range of
situations and demonstrates the most significant evolutions and novelties of the last
years in this research field (54), being displayed in Fig. 2
Evaluation of ncRNAs expression profiles has become an important issue in a
wide range of pathological and physiological processes. Due to their higher stability
these can be detected from a wide range of biological specimens (fresh/frozen tissue,
FFPE or cell culture), including body fluids (whole blood, serum or plasma, saliva or
urine). ncRNA profiling studies have certified their potential application as screening
and early diagnosis markers for various pathologies, including endometriosis and its
related malignancies. Consequently, is mandatory the development of reliable
instruments for the assessment and quantification of these ncRNAs, which are found at
low concentrations.

31
Fig.2. Transcriptomic profiling in human diseases, emphasis the important role for biomarkers as prognostic
and diagnostic, but also as therapeutic target (54)
Presently, there is a large amount of literature data presenting the expression of
ncRNAs profiling, using a wide range of methods, each having its advantages or
disadvantages, regarding the detection accuracy, analysis work flow of the cost that
implies this determination.
The main methods used for ncRNA profiling:
 Hybridization microarrays are usually used for global microRNA
expression profiling for a wide range of biological specimen,
including fresh tissue or FFPE samples, whole blood,
serum/plasma.

 miRNA deep sequencing has become known as a novel technology
for microRNA expression profiling. This suggests the benefits of
elevated sensitivity and specificity, identification of both new and
known microRNAs, recognition of microRNA sequence
polymorphisms and editing. In the same time, this approach allows
simpler data normalization strategies for the analysed cohorts.
 PCR based approaches: there are developed several
commercial kits for sybrgreen protocol (NCodeTM miRNA SYBR
Green qRT-PCR and Stratagene High-Specificity miRNA QRT-PCR
Detection) that use the SuperScriptTM III RT, in a first strand cDNA
synthesis reaction based on specifically designed universal RT
primer, or a TaqMan based protocol using stem-loop RT primers
implemented by Applied Biosystems
Fig.3. Workflow for molecular profiling of coding and non-coding genes

33
These novel technologies allow the identification and documentation for
molecular basis for phenotypic traits, leading to the identification of novel
biomarkers, based on the integration of the omics data (54).
The enormous progresses in the evolvement of profiling approaches, not just
in the case of transcriptomics approaches, but as well as in epigenomics, proteomics
and metabolomics, have rehabilitated the molecular portrait of a disease, including
the case of endometriosis and its related malignancies, being used for patient’s
stratification and monitoring the response to therapy. In fig. 3 is presented the work
flow for miRNA determination.

35
PERSONAL
CONTRIBUTION

37
1. Working hypothesis/Objectives
Endometriosis is a well-known gynecological pathology with an increased
incidence that affects women’s life quality. The most important feature is related to the
malignant transformation, and in particular to endometrial carcinoma. Until recently,
several studies have identified either upregulated or downregulated miRNAs in
endometrial or endometroid cancer. However, there was no data presenting a
comparation of the miRNA pattern differentially expressed in endometrioid tissue
versus endometrioid cancer.
Thus, we decided to attempt to evaluate the miRNA profiling in endometriosis
and endometrioid cancer, in order to identify the most relevant altered transcript, to
identify the endometriosis miRNA portrait and those transcripts involved in
endometrial carcinogenesis and to implement those expression profiles for molecular
diagnostic and prognostic markers, particularly for identification of those
endometriosis cases that have a high risk of malignancy, data included in the first
study.
The last studies were focused on developing novel therapeutic strategies in
gynecological malignancies using small molecules or RNA interference based
therapies. A particular attention was paid for characterizing of defective signaling
pathways involved in resistance to therapy.
Two studies were focused on ovarian cancer. The justification is related to high
rates of morbidity and mortality that impose developing novel therapeutic strategies.
Despite improvements in understanding the molecular mechanisms of ovarian cancer,
therapeutic strategies still depend on surgery and various combinations of taxanes and
platinum agents; unfortunately many cases show resistance to chemotherapy.
Epidemiological studies reveal that a wide range of small molecules from
polyphenolic class are related with a reduced cancer risk, by targeting hallmarks of
cancer. These molecules were demonstrated to have the capacity to increase the
efficacy of chemotherapy and prevention of multidrug resistance. Therefore we
decided to assess the effect of a natural compound: Caffeic Acid Phenethyl Ester,
recognized in literature as NFkB inhibitor on ovarian cancer cells, as a potential agent
to counteract chemoresistance related mechanisms.
SiRNA targeted therapies are used with success for improving cancer therapy;
the utility of this therapeutic system can be demonstrated by the increased number of
clinical trials using this novel technology. siRNA can be used in gynecological
pathologies for improving the efficiency and safety of the classical treatments. In our

studies we were focused on investigating the indispensable genes ( ability to intercede
in cell response to various stresses, mainly by inducing or repressing a number of
genes involved in cell cycle arrest, senescence, apoptosis, DNA repair, and
angiogenesis.
The core elements of the transcriptional program responsible for siRNA for
these two genes are less clearly defined. For the mutated p53, the core element is the
oncogenic role, but once inhibited, might be activated compensatory mechanisms that
limit the therapeutic efficacy.
The results of this thesis were exploited in two articles with an impact factor
higher than 2 and one B+ paper. Also part of the data was presented in national and
international congresses. Presently there is under revision one important review
paper related to the relevant role of miRNA in gynecological malignancies. This is a
starting point to emphasize the important role of ncRNA, particularly in modulating
the malignant transformation, drug resistance or for prevention of metastatic disease.
This can be a good starting point for new other projects.

39
2. General metodology
The first study is a retrospective study using paraffin embedded tissue, focused
on miRNA expression profiling in endometriosis and ovarian cancer; experiments were
carried out only after the approval of the Ethics Committee of the Oncological Institute
"Prof. Dr. Ion Chiricuţă "Cluj-Napoca, all the samples were anonymized; this was based
on specific methodology. PCR-array is a technology with a powerful high-throughput
tool capable of monitoring the expression of a higher number depending of the plate
type (96 or 384 wells) of miRNA at once with tens of samples processed in parallel in a
single experiment. The protocol of the PCR-array include the RNA extraction with
appropriate method depending on biological specimens used, quality control, the cDNA
synthesis followed by qRT-PCR amplification and data analysis. The qRT-PCR reaction
is used to validate the PCR-array results in a new patient cohort, using specific miRNA
primers and a Sybrgreen based protocol. Specialized programs were used to explore
the specificity and sensitivity of selected miRNAs as biomarkers for prognostic and
diagnostic.
Functional genomics studies were used for understanding the mechanism of
cellular and molecular mechanism of action for different treatment scenarios. As cell
culture based models, was used HeLa cells, A2780 and its cisplatin-resistant cell lines;
these are two relevant models for studying drug resistance mechanisms, in the
screening of novel therapeutic agents (especially against drug resistant tumors), and in
formulating induction and salvage therapies for ovarian cancer or cervical cancer.
All the functional studies were performed at the Research Center for Functional
Genomics, Biomedicine and Translational Medicine, ”Iuliu Hatieganu” University of
Medicine and Pharmacy that have a significant experience in RNA profiling (coding and
non-coding genes) and experimental therapeutic models using siRNAs and small
molecules.
Cellular toxicological studies include the determination of IC50 (the
concentration of compound required for reducing of cell proliferation by 50%) and
relevant doses (non-cytotoxic) to study anti-tumor mechanisms of CAPE will be
established through method of detection for cell proliferation MTT [3- (4,5
dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromides]. The method is based on the
ability of mitochondrial dehydrogenase in viable cells to cleave the tetrazolium MTT
rings, product with yellow color, which will lead to the formation of formazan crystals
with blue color. The color can be quantified by a colorimetric method and the results

read with specific spectrophotometer. The cell invasion was done using xCELLigence
System. This device monitors cellular events in real time without the incorporation of
labels and provides quantitative information about the biological status of the cells,
including cell number, viability and morphology. xCELLigence System is ideal for
pinpointing optimum time points when cytotoxic compounds achieve their maximum
effect. Assessment of apoptosis will be achieved through a specific method, through
marking with Annexin-V/PI staining, followed by evaluation using flow cytometry or
fluorescence microscopy.
qRT-PCR profiling: allows the evaluation of the transcriptomic profile for
ovarian or cervical cancer cell lines with different treatment. This will highlight the
altered genes species involved in multiple cellular mechanisms involved as response to
a particular treatment.
The research was conducted in the accordance with all bioethics and biosafety
conditions, according to the current national and international legal framework and
protecting the intellectual property.

41
3. Study 1. miRNA expression proffiling in formalin-
fixed paraffin-embeded endometriosis and ovarian
cancer samples
3.1. Context of the study
Endometriosis is a benign gynecological pathology, that affects a higher
percent of premenopausal women (5-10%), generally being related with infertility and
is linked with massive pain that affect life quality. The incidence is increased at global
level, similar statistics being retrieved at local level (91). Anatomically this pathology
is connected with the presence of the ectopic endometrial implants in the pelvic area.
In the last years an increased number of investigations present a direct relationship
with the malignant transformation retrieved in literature as EAOC (44, 92, 93).
This malignant process is a gradually process characterized by successive
accumulation of mutations and alterations at genes level or in the transcriptomic
pattern (94). Therefore it is important to identify those molecular processes
responsible for the malignant transformation (94-96). In this malignant
transformation an important role play the oncogenes and tumor suppressor genes. In
physiological status these are connected to cell proliferation, but when these
oncogenes present fatal alterations, the cells in most of the cases are removed as a
protection system; once this system fails, it is initiated the first step of the
carcinogenesis.
The carcinogenic cascade is highly complex and still poorly understood in the
case of endometriosis. Signal transmission during the malignant transformation occurs
in a multistage way. In the present was identified a higher number of molecular
signaling pathways that are currently associated with the possible transition from
endometriosis to ovarian cancer, in the core of this signaling network being retrieved
well known tumor suppressor genes or oncogenes. For most of the cases these
alterations occur when the proteins are in a mutated form (97, 98), microsatellite
instability, but also related to noncoding genes. In the last years, an important role
was attributed to the miRNA, an altered pattern was related with wide ranges of
pathologies. (94).

Ovarian cancer is situated in the top of cancer list at worldwide level, according
to the latest statistic data, and for most of the cases this is incurable due to the late
diagnostic (99).
The main therapeutic strategy is represented by surgery as first line of
treatment followed by chemotherapy, where for some cases recurrence is developed.
Therefore it is important to be able to identify the cases that are predisposed for
recurrent disease (1, 94, 97, 100-103).
In this context, the international tendency is to test novel markers that can
sustain a more precise diagnosis and might have a prognostic value. Our study is in
agreement with these tendencies to use PCR-array or qRT-PCR as a screening test for
the identification of a single miRNA or a panel of miRNAs that can be implemented in
clinical practice as diagnostic/prognostic markers but also to be a starting point for
developing novel targeted therapies (104).
The recently discovered miRNAs transcripts may lead to important application
into clinical practice, due to their capacity to modulate gene expression. Presently were
identified several miRNAs transcripts related with some genes that play roles in the
installation and development of ovarian cancer or to being actively involved in the
etiology of endometriosis and its progression. The literature data is strengthening the
expectation of using miRNAs as biomarkers and therapeutic target in endometriosis
and related malignancies (97, 105).
An increased number of studies incriminate miRNA (18-20 nucleotide length
transcripts) in physiological, but also pathological status. miRNAs are presented today
as key regulators involved in numerous cellular processes (106). They control
biological pathways involved, processes that under aberrant functioning can lead to
pathological status, including malignant transformation, sustaining cells proliferation,
invasion and migration capacity. Due to their important role in cellular processes, can
be justified the interest in these biomarkers discovery (94, 107).
Formalin fixed paraffin embedded (FFPE) tissue is a key source for
translational medicine, being a valuable resource for developing and implementing
into clinic the concept of precision medicine (94).
RNA extracted from the FFPE tissue is fragmented and is chemically modified.
In spite of this inconvenient, FFPE can be used for a wide range of retrospective
analysis, including for miRNA profiling (94, 108).
Taking into consideration the crucial role of miRNA transcripts which are able
to regulate several target genes and one single gene is regulated by several miRNA
transcripts, these short sequences have an imperative role in these biomarkers
discovery processes (94).

43
3.2. Working hypothesis and objective
miRNAs are linked with the regulation of numerous signaling pathways, there
is definitely an association with the malignant transition, but this needs to be
deciphered (94). Identification of specific panel of microRNAs that are differentially
expressed in this disease, and particularly among these two pathologies, can enable the
discovery of precise biomarkers and in particular those related with the assessing of
the risk involved by the transition from the benign to malignant status.
By developing and implementing novel approaches for molecular profiling on
coding and non-coding genes for different pathologies or even for different subtypes of
the same pathologies or different stages of development, we are able to deliver a tool
with clinical application in early diagnostic, prognostic and therapy (94).
In our study, we intend to perform a miRNA profiling study using FFPE
samples of endometriosis, ovarian cancer and normal tissue in order to identify key
miRNAs that are able to distinguish among the analyzed groups. The second step of our
study is the validation on a new patient cohort using qRT-PCR. The differential
expressed miRNA analysis will be integrated on specialized bioinformatics programs
in order to assess the biological relevance, which finally might provide new therapeutic
targets in the future.
3.3. Materials and methods
Sampling procedures.
In this retrospective study we used 4-8 sections with an approximate thickness
of 10 µm for all the FFPE sections. In this study were selected the patients diagnosed
with endometriosis and ovarian cancer between the years: 2007-2014.
All samples included were macrodissected after the confirmation of the
histopathological diagnostic at the Pathological Department at the Institute of
Oncology “Prof. Dr. Ion Chiricuta” in Cluj-Napoca, Romania, after the approval of ethical
committee. The characteristic for the controls are displayed in Table I and the
information related to the endometriosis and ovarian cancer patient cohort is
displayed in table II. These include both cohorts for profiling and for validation.
For miRNA profiling study and validation patient cohort were selected only the
patients that haven’t undergone preoperative chemotherapy or radiation therapy. All
the samples were macrodissected in order to assure a high accuracy of the
determination for miRNA pattern. For the cases of ovarian cancer were included only
those pinpointed with endometrioid carcinoma, with more than 80% hyperplasic
tissue.

Table I. Clinical data for the patients with ovarian cancer incorporated in this study
Patient cohort Case Age Diagnostic
1 58 Endometrial polyps
PCR-array
2 65 Adenomyosis
3 62 Endometrial polyps
4 59 Leioma
5 60 Uterine fibroid
qRT-PCR
1 36 Leiomas
2 47 Ovarian fibroid
3 72 Leiomas
4 61 Benign endometrial polyps
5 43 Leiomas
7 41 Salpingitis
8 53 Leiomas
10 43 Leiomas
11 51 Adenomyosis
12 45 Adenomyosis
14 47 Salpingitis
15 53 Adenomyosis
16 43 Ovarian or uterine fibroid
17 57 Ovarian or uterine fibroid

45
Table II. Clinical data for the patients with ovarian cancer included in this study
Patient
cohort Case Age
Differentiation
stage Pathological diagnostic
PCR-
array
1 58 G1 pT1aNxMxL0V0
2 67 G2 pT2aNxM1L1V0
3 62 G2 pT1cN0MxL1V0
4 54 G3 pT2N1M0LoVo
5 59 G2 pT1aNxMxLoVo
6 65 G3 pT2cN1bMxL1Vo
7 45 G3 pT2N1M0LoVo
8 78 G2 pT3bNxMxLoVo
qRT-
PCR
4 67 G3 pT3cN1MxL1V1
5 51 G2 pT2bN0MxLoVo
7 56 G3 pT3cNxMxL1V1
8 42 G3 pT2aN0MxLoVo
9 54 G2 pT3cN1M1LoVo
12 45 G1 pT1cNxMxLoVo
16 41 G2 pT3cN1MxL1Vo

Total RNA isolation from FFPE samples. The extraction of total RNA was
done using a special kit adapted for low amount of genetic material from paraffin-
embedded (FFPE) tissue. The miRCURY RNA Isolation Kit FFPE (Exicon cat.no.
300115) assuring a good deparafination of the samples, assuring a good
reproducibility and reliability of the qRT-PCR reaction. The quality control for the total
RNA extracted with the Exicon kit was done using NanodropND-2000 and Bionalysor
2100 from Agilent.
miRNA profiling study using PCR array. For the case of miRNA profiling study
we started from an amount of 100 ng of total RNA from FFPE tissue, as acclaimed in
the producer protocol, using miScript HiSpec primer, in a total reaction volume of 20
μl and the following amplification program: 37ºC for 60 min, then at 95ºC for 5 min.
For the sybrgreen qRT-PCR protocol the cDNA was diluted 1:20 and all steps were
done based on the optimized protocol. For miRNA profiling was used Human Ovarian
Cancer miRNA PCR Array plate, catalog number MIHS-110Z from SABiosciences),
recognized in literature to be specific for ovarian cancer, 3 well were represented by
positive control and negative control, for monitoring the accuracy of the cDNA and
qRT-PCR amplification, for an increased accuracy of the experimental data. The
amplification steps based on producer recommended protocol was done in Roche
LightCycler480 devices and QuantiTect SYBR Green PCR kit furnished by Qiagen.
Data validation using qRT-PCR. For the validation of miRNA profiling data,
was selected a new set of biological specimens containing 3 patient cohort, comprised
by 17 normal specimens, 33 endometriosis specimens and 28 ovarian cancer
specimens. The qRT-PCR analysis was done based on two step protocol, cDNA was
generated using miScriptII-RT Kit, and used as template for qRT-PCR reaction after a
dilution step (1:10), using same sybrgreen protocol as in the case of the PCR-array
protocol.
Data analysis. The miRNA profiling analysis was done based on ΔΔct method,
as before presented by Berindan-Neagoe et al., 2012 (109), using the data analysis web
portal supplied by Qiagen. For the miRNA profiling study patient cohort, this was
comprised by five normal tissue sample, nine endometriosis samples and eight ovarian
cancer tissue. As normalization we used an artificial molecule (cel-miR-39), along with
a set of four most representative housekeeping (SNORD68, SNORD95, SNORD96A and
RNU6).
26 73 G3 p3bN0MxLoVo
28 40 G2 pT2cNoMxLoVo

47
In the case of validation study we used similar analysis, but has been done
additional statistical test, including the ROC (receiver operating characteristic curve)
representation was done using the specialized software Graphapad
(https://www.graphpad.com). In order to assess the biological relevance of the altered
genes for the case of selected groups we used Ingenuity Pathways Analysis
(www.ingenity.com).
3.4. Results
3.4.1. Relative miRNA expression level in endometriosis and ovarian cancer
The most accessible biological specimen is represented by FFPE tissue, this
represent a valuable tool for testing novel biomarkers, that can be applied for
endometriosis and endometrial cancer.
The selected endometriosis patients have an average age of 40.3±5.57 years, eight
ovarian cancer samples from patients with average age of 59.33÷10.36 years, and five
normal tissues from benign pathologies (two cases of endometrial polyps, a case of
adenomyosis, one case of uterine fibroid and a case of leiomyoma) with an average age
between 60±4.48 years.
The first step of the analysis was focused on the evaluation of the relative gene
expression, this is based on the ΔΔct method and the analysis was done for the
endometriosis or for the case of ovarian cancer versus normal tissue. An additional
analysis was done in the case of ovarian cancer versus endometrioid tissue, this has an
important practical application in order to identify those miRNAs implicated in the
processes of the malignant transformation, being a valuable source for biomarkers
discovery for prognostic but also as therapeutic targets.
The miRNA with an altered expression level in the presented analyzed groups are
summarized in Table III. The cut-off for the present analysis was set at p≤0.05 and a -
1.5>FC<+1.5, the clusterigram being presented in Fig. 4.
In the expression level for miRNAs in endometriosis tissue versus normal tissue
we found four miRNAs to be overexpressed, meanwhile in the case of analysis for
ovarian cancer versus normal tissue we found fifteen miRNAs with an altered
expression level (nine over-expressed respectively six down-regulated). What is
important to mention is the higher number of altered miRNAs for the analysis of
miRNA pattern in ovarian cancer versus endometrial tissue, this displaying fourteen
overexpressed and twenty-three downregulated miRNAs.
All information obtained from the analysis is presented in Table III and the
overlapping between the altered miRNAs across the analyzed groups being displayed
in Fig. 5, as Venny diagram.

Table III. microRNAs differentially expressed in the analyzed three groups, considering the cut-
off a value p≤0.05 and a -1.5>FC<+1.5
No Selected group for
analysis
miRNA FC p value
1 Endometrial tissue
versus normal tissue
miR-325 1.8309 0.032915
miR-492 2.0592 0.012467
miR-520e 1.5551 0.028853
miR-203a-3p -1.6384 0.043978
2 Ovarian cancer tissue
versus
normal tissue
miR-141-3p 26.5467 0.027413
miR-182-5p 37.8169 0.030483
miR-200a-3p 31.6626 0.026593
miR-200b-3p 24.3055 0.01201
miR-200c-3p 18.4169 0.000211
miR-325 2.5433 0.014251
miR-429 22.4005 0.048734
miR-492 5.6303 0.028431
miR-93-5p 5.2779 0.02893
let-7a-5p -2.2238 0.017423
let-7b-5p -3.33 0.001166
let-7c-5p -4.498 0.001597
miR-145-5p -3.4006 0.02553
miR-152-3p -2.1481 0.020069
miR-214-3p -5.0492 0.001423
3
Ovarian cancer tissue
versus endometriosis
miR-103a-3p 2.4742 0.00268
miR-106b-5p 4.9541 0.002867
miR-141-3p 154.837 0.001947
miR-155-5p 1.7668 0.045144
miR-15a-5p 4.8944 0.004837
miR-16-5p 3.2189 0.03567
miR-182-5p 27.5885 0.0041
miR-200a-3p 114.367 0.0025
miR-200b-3p 67.0795 0.000604
miR-200c-3p 50.656 0.000001
miR-205-5p 39.2156 0.02992
miR-27a-3p 2.2577 0.001569
miR-30a-5p 2.6602 0.020643
miR-30e-5p 2.5805 0.022491
miR-335-5p 6.9565 0.009211
miR-346 2.3376 0.045989

49
miR-370-3p 2.7308 0.011845
miR-429 43.266 0.007382
miR-492 2.7342 0.016271
miR-507 6.3399 0.035066
miR-514a-3p 6.1642 0.021661
miR-637 1.6775 0.028397
miR-93-5p 7.0123 0.001372
let-7a-5p -2.385 0.000186
let-7b-5p -3.9872 0.009889
let-7d-5p -1.6768 0.015271
miR-1-3p -8.3123 0.02349
miR-125b-5p -3.4177 0.002483
miR-134-5p -1.8533 0.023899
miR-143-3p -2.5183 0.007
miR-145-5p -3.7944 0.000087
miR-152-3p -1.7758 0.02794
miR-154-5p -1.8435 0.016878
miR-199b-3p -2.9882 0.043724
miR-199a-5p -2.6627 0.002688
miR-214-3p -5.6617 0.0086
miR-432-5p -1.6847 0.02067

Fig.4. Heatmap for the cases included in this study, generated using the statistic significant miRNAs. In figure
is represented by N: normal tissue; E: endometriosis tissue; O: ovarian cancer tissue.
Fig.5. Venn diagram presenting the statistically significant altered transcripts that are commonly or that are
not included in the study groups

51
3.4.2. Generation of miRNA based networks for the altered miRNA
profile in endometriosis versus control, ovarian cancer versus
control, respectively ovarian cancer versus endometriosis
Based on the altered miRNA profile for the 3 groups selected we gathered the
IPA (Ingenuity Pathway Analysis) miRNA-based networks, allowing the summarization
of their biological connotations and for defining the molecular interaction of the
altered miRNA pattern and the interconnection with the most relevant identified target
genes (Table IV).
The most relevant nodes from the altered identified miRNAs and the target
genes were represented as networks (Fig. 6, respectively Fig. 7). Also the miRNA
profile was analysed for classification based on disease and cellular and molecular
function (Table V).
In the case of the endometriosis, the core of the network is represented by miR-
291a that is interconected with miR-325 and miR-393. The most important fact that
we observed in this nework is linked to direct relathionship of miR291a and miR-492
with the TP53, the well known tumor supressor gene that regulates cell proliferation
and once mutated has an oncogenic function.
Table IV. IPA analysis for the case of the altered miRNA profile in the analyzed groups, based on
diseases
Group Selected group for
analysis
Disorder p-value Number of
molecules
involved
Molecules
1 Altered miRNA
pattern in
endometriosis tissue
versus normal tissue
Inflammatory
Response
2.35E-02-1.02E-03 2
Cancer 3.15E-02-4.24E-03 3
Organismal Injury and
Abnormalities
3.56E-02 - 4.24E-03 3
2 Altered miRNA
pattern in ovarian
cancer tissue versus
normal tissue
Cancer 4.60E-02-3.76E-11 11
Abnormalities
4.70E-02 - 3.76E-11 11
Reproductive System
Disease
3.88E-02 - 1.19E-10 10
3 Altered miRNA
pattern in ovarian
cancer versus normal
endometriosis
Cancer 4.60E-02 - 3.76E-11 11
abnormalities
4.70E-02 - 3.76E-11 11
Reproductive System
Disease
3.88E-02 - 1.19E-10 10

Table V. IPA analysis for the case of altered miRNA profile in the analyzed groups, based on the
main molecular and cellular functional roles of miRNAs
Group Selected group for
analysis
Disorder p-value #Molecules
1 Endometriosis
versus normal
tissue
Cell-To-Cell Signalling and
interaction
2.35E-02 - 1.89E-03 1
Cell Cycle 3.56E-02 - 4.07E-03 1
Cellular Movement 4.07E-03 - 4.07E-03 1
2 Ovarian cancer
versus normal
tissue
Cellular Development 4.65E-02 - 2.34E-05 7
Cellular Growth and
Proliferation
4.65E-02 - 1.08E-04 7
Cell Cycle 1.94E-02 - 1.27E-04 4
Cell Death and Survival 3.26E-02 - 5.78E-04 6
3 Ovarian cancer
versus normal
endometriosis
Cellular Development 4.65E-02 - 2.34E-05 7
Cellular Growth and
Proliferation
4.65E-02 - 1.08E-04 7
Cell Cycle 1.94E-02 - 1.27E-04 4
Cell Death and Survival 3.26E-02 - 5.78E-04 6
Fig.6. Network based on the altered miRNAs pattern for endometriosis versus benign tissue. The
overexpressed transcripts are presented as red color and those transcripts with a reduced expression level
presented as green color, those not statistically significant as grey.

53
Fig.7. Network generated based on the altered miRNAs pattern for ovarian cancer versus control. The
overexpressed transcripts are presented as red color and those transcripts with a reduced expression level
presented as green color, those not statistically significant as grey.
3.4.3. qRT-PCR data validation
qRT-PCR is a frequently used method for confirming the data obtained from
PCR-array or microarray determination. In our study qRT-PCR patient cohort is
represented by 17 benign macrodissected tissue samples, 33 of endometriosis
macrodissected samples, respectivelly 33 of ovarian cancer samples.
The demographic characteristic for the patients included in this study is
relatively similar for the endometriosis and control samples, in the case of normal
cases the average age is 48.53±9.14 years, for endometriosis is 41.68±8.78 years,
meanwhile the average age in the case of ovarian specimens is a little higher
(55.75±10.98 years).
An interesting miRNA transcript is represented by miR-93 that was
demonstrated to have an increased expression level in endometriosis, ovarian cancer
tissue and moreover the expression level being much higher in ovarian cancer samples

than in the case of endometriosis samples. The average for the expression level in the
case of the endometriosis samples was 2.42±1.63, respectively of 26.19±30.59, these
confirming the PCR-array results (Fig. 8). For ROC curves representation for this
transcript we could observe an AUC (area under the curve) value of 0.69 for
endometriosis cohort and 0.82 for the ovarian cancer cohort.
Another important miRNA transcript is represented by miR-200c that was
demonstrated to have an increased level in ovarian cancer cohort. The qRT-PCR data in
the case of validation patient cohort reveals the same expression level tendency,
therefore for normal tissue the relative expression level was 1.63±1.53, for
endometriosis we determined to be 1.27±2.91 and for ovarian cancer was
12.29±14.61.
The miR-200c AUC values for the cases of endometriosis were determined to
be 0.69, respectively 0.84 for ovarian tissue, displaying a good sensitivity and
specificity as biomarker for ovarian cancer.
A similar fold change expression level as identified for another two important
transcripts, from the same family, miR-141 and miR-429, displaying an overexpressed
level in endometriosis of 8.035±25.09 and 16.17±47.13 and ovarian cancer of
203.1±275.6, respectively 104.6±172.6, revealing a good sensitivity as displayed by the
AUC values for the ovarian cancer.
An interesting situation was observed for miR-155, this transcript has an
overexpression level for endometriosis and for ovarian cancer, but this was not proved
to be statistically significant in our patient cohort. The expression level for
endometriosis was 1.416±0.7981 (p-value=0.0507) and for ovarian cancer was
2.703±2.830 (p-value=0.0552). An overexpression level for endometriosis
(7.235±15.49) and ovarian cancer (390.2±466.3) was registered for miR-205, a highly
debated transcript. The expression fold change was confirmed also for miR-492, being
overexpressed in both selected pathologies, with high AUC values, proving a good
biomarker sensibility and sensitivity.

55

Fig.8. qRT-PCR validation in endometriosis and ovarian cancer patient cohort for the most relevant miRNAs
transcripts retrived in the case of PCR-array study. ROC curve for the expression level of miRNAs for
endometriosis and ovarian cancer group are generated for evaluating specificity and sensitivity of these
miRNA transcripts for biomarkers discovery.
The experimental data, with the exception for miR-155 was confirmed in the new
patient cohort. For the case of miR-155 we have the same expression level tendency
but this was not statistically significant for our patient cohort. Our data sustain the
utility of miRNA determination in order to develop novel prognostic or diagnostic
markers.
3.5. Discussions
The evaluation of molecular profiling in endometriosis and ovarian cancer is a
useful tool in the identification of novel biomarkers with prognostic or diagnostic
significance, in particular for the identification of those related to malignant
transformation. The miRNA profiling from FFPE normal, allowed the identification of a
panel of miRNAs that have an altered expression level for the case of analysis for
endometriosis versus normal tissue, or for the case of ovarian cancer versus normal
tissue, but what is the most important is the fact that we were able to emphasize a
panel of fourteen overexpressed and twenty-three downregulated miRNAs for the
analysis of ovarian cancer tissue versus endometrioid tissue. These can be attributed
to the process of malignant transformation (94).
Moreover, the functional pathway analyses using IPA for the target miRNA
reveals to be able to target genes involved not only in inflammatory processes but also
in apoptosis, cell proliferation, angiogenesis and invasion (94).
One of the most important set of miRNAs is represented by miR-200 family
members. We retrieved a significant overexpression level for miR-200a/b/c, miR-429,
and miR-141 for ovarian cancer specimens, being on the list as most upregulated
miRNAs from our panel of PCR-array used for evaluation. This panel was not retrieved
as overexpressed in endometriosis samples, and interesting information retrieved in a
previously study, that sustains our findings, presents the circulating miR-200a and

57
miR-141 to have a potential clinical application as novel noninvasive biomarkers for
endometriosis (110). In the case of ovarian cancer, the increased expression level was
directly correlated with the disease progress and survival rate.(111-114) Other studies
revealed that miR-141 and miR-200a have an contrasting expression level in the
endometriosis samples compared to the values found in ovarian cancer (115). These
can be proposed as non-invasive biomarkers, based on differential expression level in
tissue samples and the correlation with the values from blood samples (116).
One transcript with an increased expression level is represented by miR-325, that is
presented in literature to be associated with a decreased overall survival for patients
with hepatocarcinoma and gastric cancer, this demonstrating the implications of
HMGB1 gene in tumor promoting process (117). Despite the lack of specific data
regarding miR-325 in the context of ovarian cancer or endometriosis are limited, there
are some studies in other pathologies, proposing this as biomarker/therapeutic target
in lung cancer (118, 119). In this way, this can be also be extrapolated to ovarian
cancer and validated in higher patient cohort. miR-325 may become a clinical
parameter in view of determination for ovarian cancer invasion, being inversely
proportional with activity of HMGB1 (118), whose expression is inversely related. This
transcript being retrieved in both studied pathologies, but the greater expression value
in ovarian cancer, and being statistically significant for the case of ovarian cancer
versus endometriosis, suggesting a possible application for miR-325 as a prognostic
marker in regard to a possible role in the transition from endometriosis to malignant
pathologies (94).
In the panel with altered expression miRNAs with inflammatory role is miR-
203(120); this was observed to be downregulated in endometriosis group. Another
overexpressed transcript statistically significant overexpressed in endometrioid tissue
is represented by miR-520e. This transcript was linked with the regulation of cell
proliferation, apoptosis and metastasis in tumor pathologies like
hepatocarcinoma(121) and breast cancer (122, 123). This might represent a novel
therapeutic target for endometriosis based on the increased cell proliferation and the
migratory capacity.
In regard to expression for miR-141, it has been observed an increased
resistance for cisplatin proportionally with the grade of positive aberrancy of this
transcript, due to direct targeting of the KEAP1 gene, whose underexpression
determines the resistance to treatment (124). So, although it does not hold a
biomarker potential, miR-141 can be further used for patient’s stratification and
treatment election(94).
Members of the miR-200 family have been also identified to have a decreased
expression level in literature data, but this was not confirmed in our case.
Interestingly, there is a paradox related to the expression level in endometriosis
samples; the expression is reduced and an opposed expression was determined for the
case of ovarian cancer, being significantly downregulated in plasma samples (115).

Given the roles of miR-200 family in numerous biological and pathological processes,
especially in oxidative stress response and epithelial to mesenchymal transition, we
can assume these transcripts may be involved in the transition from benign
endometriosis to malignant pathologies such as ovarian cancer. Taking in
consideration the opposite expression values for miR-141 and miR-200a between the
two pathologies, members that are also present in our study, we can suggest the
possibility of using these two transcripts as predictive factors regarding the risk for
benign-malign transition in endometriosis patients (94).
One of the identified transcripts was overexpressed in both tissue samples
from patients with endometriosis and ovarian cancer; this is miR-325, a transcript
whose overexpression was confirmed even when comparing the degree of expression
between the two pathologies using the endometriosis samples as control probes. miR-
325 has the potential of tumor suppressor by inhibiting the expression of HMGB1 (high
mobility group box) gene whose expression is reflected by the promotion of malignant
pathology progression, increased angiogenesis, invasion and metastasis (117).
The miR-492 has been identified in the present study as overexpressed in both
pathologies, with a more noticeable level in ovarian cancer. This microRNA has been
characterized in numerous cancer pathologies, however, information on its exact role
in the acquisition and progression of endometriosis and ovarian cancer is absent from
literature. MiR-492 promotes the progression of hepatic cancer by targeting the PTEN
gene and increasing the level of AKT activation in cancer cells. Patients with
overexpression for miR-492 and an inhibition of the PTEN gene show a lower rate of
survival (125). The same oncogenic role of miR-492 is observable in breast cancer,
where it enhances malignant cell proliferation by inhibiting the tumor suppressor gene
SOX-7 (sex-determining region Y-box 7) (126). Moreover, literature data show a
possible role for miR-492 in differentiation of cells for different stages of
hepatoblastoma, whereby, the expression of the transcript increases as the cancers
advances towards metastasis. This qualifies the microRNA as a prognostic
biomarker.(127) Considering the different expression levels of miR-492 in
endometriosis and ovarian cancers, with a higher level in the later, follow-up studies
could confirm the quality of miR-492 as a prognostic biomarker in terms of transition
from benign to malignant, added with its role in abnormal cellular proliferation, which
could place this transcript on the list of potential therapeutic targets for inhibiting
aberrant cell growth.
miR-182 was proved to be overexpressed in ovarian cancer, sustaining the
present findings. This transcript is presented as a prognostic marker for ovarian
cancer(128). Further analysis need to be done in the investigation of its role in
malignant transformation, as was emphasized by Liu et al., 2012 (129). In ovarian
cancer is presented to be related to cell proliferation, invasion and migration (130).
The let-7 have 13 distinct members located on nine chromosomes (131). In our
study were identified three of them as overexpressed in ovarian cancer samples (let-

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