‫سورة االسراء ‪-‬‬
‫‪85‬‬
Circulating miRNAs profile in Hepatitis
C virus infected patients with and
without hepatocellularcancer
M.Sc. thesis by
Mohammed gamal thabet
Genetic Engineering and Biotechnology Research Institute
University of Sadat City
Department of Molecular Biology
( Molecular Immunology )
Supervisors
Prof. Dr. Omaima Khamiss
Prof. of cell and tissue culture, Genetic Engineering and
Biotechnology Research Institute, University of Sadat City.
Prof. Dr. Roba Mohamed Talaat
Prof. of Molecular Immunology, Genetic Engineering and
Biotechnology Research Institute University of Sadat City
Dr. Yasser Bastawy Mohamed Ali
Ass. Prof. of Molecular Immunology, Genetic Engineering and
Biotechnology Research Institute, University of Sadat City.
Hepatitis C virus (HCV)
 HCV is a hepatotropic enveloped positive‐sense single‐stranded RNA virus of
the genus Hepacivirus within the Flaviviridae family particles are spherical and
heterogenous in size, typically ranging 40 - 80nm in diameter.
 It is classified into 7 genotypes and more than 60 subtypes.
 Egypt has the highest HCV prevalence in the world and Genotype 4 is
predominant, representing >85% of all HCV cases.
 Potential long‐term outcomes in chronically HCV‐infected people are liver
cirrhosis and HCC, which remain the leading cause
for liver transplantation. HCV is globally
infecting over 150 million individuals.
HCV infection
cycle
HCV
symptoms
 Pain in the right upper abdomen
 Abdominal swelling due to fluid (ascites)
 Clay-colored or pale stools
 Dark urine
 Fatigue
 Fever
 Itching
 Jaundice
 Loss of appetite
 Nausea and vomiting
Liver Cirrhosis (LC)
Liver cirrhosis (LC) represents the final stage of liver
fibrosis, the wound healing response to chronic liver
injury.
The natural course of fibrosis begins with a longlasting
rather
asymptomatic
period,
called
‘compensated’ phase followed by a rapidly
progressive phase, named ‘decompensated’ cirrhosis
characterised by clinical signs of complications of
portal hypertension and/or liver function impairment.
Staging of liver cirrhosis
Child-Pugh scoring system
Criteria
Serum total
A
B
C
<2 mg/dl
2-3
mg/dl
>3 mg/dl
>3.5 g/dl
2.8-3.5 g/dl
<2.8 g/dl
INR
<1.7
1.71-2.20
>2.2
Ascites
No
Mild-Moderate
Sever
No
Grade I - II
Grade III - IV
bilirubin
Serum albumin
Encephalopathy
Hepatocellular carcinoma (HCC)
HCC is the most common primary liver cancer and
the second most common cause of cancer-related
mortality globally and usually develops in patients
with underlying liver cirrhosis.
 Egypt ranks the third and 15th most populous
country in Africa and worldwide, respectively,
representing the sixth most common cancer
worldwide and the fourth common cancer in Egypt.
Diagnostic approach
for HCC
Metastases/Carcinogenesis
Epithelial-mesenchymal transition
(EMT)
 It is a developmental regulatory program, identified as
transformed epithelial cells loss epithelial constraints and then
acquires the abilities to invade, resist apoptosis and metastasis.
 EMT allows a polarized epithelial cell, to undergo multiple
biochemical changes to assume a mesenchymal cell
phenotype.
Classification of EMT
Regulation of Epithelial–
Mesenchymal Transition (EMT)
Cadherins
 Cadherins are family of membranous calciumdependent glycoproteins, which is responsible for
Ca2+-dependent cell-cell adhesions, carries out
functions essential for intercellular adhesion and is
involved in embryo development and the maintenance
of normal tissue.
 According to the distribution of tissue it is divided into
more than 10 subgroups including E (epithelium)-, N
(nerve)-, P (placenta)- and R (retina)-cadherins.
Cadherin types
PTEN
 named is phosphatase and tensin homologue (PTEN)
gene
 Making an enzyme that is found in almost all tissues in
the body
 PTEN protein acts as a phosphatase to dephosphorylate
Located on chromosomal region 10q23
Acts as a negative regulator of the (PI3K/AKT)
phosphatidylinositol 3-kinase/AKT signaling
Cell growth
cell survival
proliferation
Cell migration and angiogensis by phosphorylating
pten
ph
phosphatidylinositol (4,5)trisphosphate (PtdIns
(4,5)P3 or PIP2).
phosphatidylinositol (3,4,5)trisphosphate (PtdIns
(3,4,5)P3 or PIP3).
Clinical significance
Cancer
• mutations and deletions of PTEN occur that
inactivate leading to increased cell proliferation
and reduced cell death







lung cancer
prostate cancer
breast cancer
liver cancer
gastric carcinoma
colorectal cancer
osteosarcoma
Correlation between
PTEN and E- Cad
MicroRNA-21 (miR-21)
 MicroRNA-21 (miR-21), identified from the location on
chromosome 17q23.1, 18–25 nucleotides in length has been
proven to be a p53-inducible miRNA with the capability of
enhancing p21 levels and mediating cell cycle arrest.
MiR-21 Targets
MiRNAs and HCV infection
Mir- 21
 To the best of knowledge, no previous study was
conducted on the relationship between these
triangle
parameters
(miR-21,
PTEN,
and
cadherins) on HCV- related HCC patients.
 Thus, this work was carried out to investigate
their circulating expression level in HCV-infected
patients with different clinical manifestations (from
cirrhosis to HCC).
subjects
and
methods
Study population and clinical investigations
 The current investigation was carried out at the Clinical
Pathology Department, National Liver Institute, Menofia
University, Egypt.
 One hundred forty‐five (145) participants were studied;
 100 HCV‐infected patients
 75 had cirrhosis
 25 had HCC.
 45 age‐matched healthy volunteers attending hospital
blood bank after blood donation considered as controls.
 Cirrhotic patients were classified according to the Child‐Pugh scoring
system
 Class A patients have a total score of ≤6 (mild liver disease),
 Class B has a total score of 7 to 9 (moderate liver disease),
 Class C has a total score >9 (severe liver disease).
 Demographic data included age and gender, routine liver
functions were performed for all participants.
 Screening for hepatitis (HBsAg and HCV antibodies) was
done by using specific kits for Cobas 6000 (Roche
Diagnostics GmbH, Germany) confirmed by reverse
transcription polymerase chain reaction (PCR)
 Abdominal ultrasonography for hepatic scanning and
evaluation of the presence and/or severity of ascites and
abdominal CT scan or MRI imaging were done.
E/N‐cadherin and PTEN level detection
 Serum levels of E‐cadherin
,N‐cadherin and PTEN gene were
measured using an enzyme‐linked
immunosorbent assay (ELISA) kit
(INTRON Bioneovan Co,Ltd
China). ELISA PR 4100
Absorbance Microplate
 Reader was used to process the 450
nm absorbance data into a standard
curve from which E‐cadherin and
N‐cadherin ,PTEN gene
concentrations were derived.
miR‐21 expression by quantitative real‐time
reverse transcription assay
miRNA extraction
 Fresh blood samples
were collected from all
patients and controls,
centrifuged,
aliquoted
and stored at −80°C for
further investigations.
 Total RNA was extracted
from plasma samples
using the miRNeasy Mini
Kit (Cat no. 218073;
QIAGEN) according to
manufacturer
instructions.
miR‐21 expression by qRT-PCR
 Two‐step RT‐PCR for miR‐21 was performed using miScript II RT
Kit (Cat nos. 218073; Qiagen) for the conversion of miR to cDNA
in PCR PERKIN‐ELMER 2400 Thermal cycler
 Amplification and quantification of miR‐21 were done by real‐time
PCR system 7500 (Applied Biosystems) using miScript SYBR
Green PCR kit (Cat no. 218073; Qiagen) according to
manufacturer instructions. Specific primer for miR‐21 was as
follows:Hs‐miR‐21‐/miScript primer assay
 5'- UAGCUUAUCAGACUGAUGUUGA-3'.
 U6 was used as a housekeeping reference gene to calculate the
relative expression level of miR‐21.
 The cycling conditions of the reactions were as follows: 95°C for
15minutes for initial denaturation, followed by 40 cycles at 95°C
for 15 seconds for denaturation, 55°C for 30 seconds for
annealing, and 72°C for 30 seconds for extension, and a final
stage was 95°C for 15 seconds, 60°C for 1 minute and 95°C for
15 seconds.
 Relative expression levels were determined by using the
equation 2(-ΔΔCT) method (ΔCT = CTmiR-21 - CTU6).
Statistical analysis
 All statistical analyses were performed using the Statistical Package for
Social Science version 19 (LEAD Technology Inc).
 Clinical data are presented as means with corresponding standard
deviation.
 Comparisons among different groups were performed by one‐way
analysis of variance.
 Tukey test was used as a posthoc test. The frequency of categorical
data was compared using the χ2 test.
 Correlation between variables was determined using Pearson’s
correlation test. In all tests, the level of significance was P < .05.
Patient characteristics
 The demographic and biochemical characteristics of all
patients in this study are listed in the following tables.
Parameter
Control
Cirrhosis
HCC
N=45
N=75
N=25
P
Correlation
with disease
progression
Age
Sex (M:F)
48.29±6.85
56.02±12.19
56.24±5.75
NS
43:2
58:17
22:3
0.05
r=0.380; 0.001
ALT
17.09±5.79
42.66±49.34
54.28±28.35
0.001
AST
20.93±6.11
53.36±61.48
67.76±40.08
0.001
r=0.452;
ALB
4.3±0.47
3.22±0.96
3.4±0.71
0.001
r= -0.607; 0.001
TP
9.91±14.38
6.58±1.32
12.13±18.37
TBIL
0.62±0.29
3.92±6.09
1.32±0.7
0.001
r=0.271; 0.001
DBIL
0.09±0.11
2.96±5.34
0.51±0.53
0.001
r=0.249; 0.01
INR
1±0.1
1.34±0.29
1.17±0.11
0.001
r=0.467; 0.001
AFP
3.32±1.05
63.72±394.13
1229.13±3501
0.05
r=0.229; 0.01
0.001
NS
Sensitivity: 80%
Specificity: 57%
Sensitivity: 88%
Specificity: 100%
r=-o.455, P<0.01
r=-o.255, P<0.05
 In conclusion, the presented work investigated the
expression patterns of EMT-related genes (miR-21,
PTEN, and N-and E-cadherin) in HCV-associated
HCC patients and their relationships with one
another.
 In this study, upregulated miR-21 was linked to
chronic HCV infection and eventual complications of
carcinogenesis.
 This could indicate that persistent HCV infection
changes the expression of numerous proteins,
including PTEN and N-and E-cadherin, driving
normal hepatocytes to malignancy via miR-21.
 These findings could lead to new targets for
preventing and treating HCC metastasis.
 Further studies are needed using human
malignant tissues to improve the results.
First of all, Thanks to Allah the most gracious and the most
merciful
I would like to express my sincere gratitude to
Prof. Dr. Omaima Khamiss ,
Prof. Dr. Roba Mohamed Talaat
Dr. Yasser Bastawy Mohamed Ali ,
I would like to express my deep gratitude and
deepest thanks to
Classmate of molecular immunology specially
Dr: abeer elmaghraby
& finally thanks my sister to
support me all the time