International University of Sarajevo
Faculty of Engineering and Natural Sciences
Genetics and Bioengineering department
Analysis of MACC1 and c-Met expression in cytobrush-collected healthy
and HPV infected cervical epithelial cells
Student: Tea Becirevic
Supervisor: Assist. Prof. Dr. Daria Ler
15 October 2015
Presentation Outline
1. INTRODUCTION



Cervical cancer precursors, epidemiology and screening
Human Papilloma Virus (HPV)
MACC1 and HGF/c-Met Pathway
2.
AIM OF THE STUDY
3.
MATERIALS AND METHODS
4.
RESULTS
5.
DISCUSSION
6.
CONCLUSION
Cervical cancer?
 Cancer is a disease in which cells in the body
grow out of control.
 Cancer is always named for the part of the body
where it starts, even if it spreads to other body p
arts later.
 When cancer starts in the cervix, it is called
cervical cancer.
 The cervix is the lower, narrow end of the uterus.
Cervical Cancer epidemiology
Public Health Institute of the Federation of Bosnia and Herzegovina (FB&H)
Others (without melanoma cancer)
681
Pancreatic
77
Brain, nervous system
83
Colon
117
Rectal
118
Uterine
118
Gastric
122
Ovarian
133
Cervical
190
Lung, bronchial, tracheal
198
Breast
613
Number of all woman (2450) in Federation B&H suffering from ten most common malignancies in 2010
(Public Health Institute of Federation B&H, 2010)
Cervical Cancer precursors
Cervical cancer develops from well defined precursor lesions know as:
Cervical Intraepithelial Neoplasia (CIN)
or
Squamous Intraepithelial Lesions (SIL)
There are 3 stages of CIN:
 CIN 1 (Mild dysplasia) – 1/3 epithelial thickness
 CIN 2 (Moderate dysplasia) – 2/3 epithelial thickness
 CIN 3 (Severe dysplasia or Carcinoma in situ) – more then 2/3
How cervical cancer develops?
Infection with Human Papilloma Virus (HPV) is the central cause for the developm
ent of invasive cervical cancer and its precursor lesions!
HPV – sexually transmitted disease (STD)
Virtually all cervical cancer cases (99%) are linked to genital infection with HPV, making it the most
common viral infection of the reproductive track (WHO)
There are more than 40 HPV types that can infect the genital areas of males and females!
 Low-oncogenic risk (LR) (HPV 6 & 11) – anogenital warts and CIN1
 High-oncogenic risk (HR) (HPV 16 & 18) – CIN2/3 and Invasive Cervical Carcinoma (ICC)
Cervical Cancer screening
 Screenings are tests that look for diseases before any symptoms appear!
 Biomarker is a biological molecule found in blood, other body fluids or tissue that is a sign of normal
or abnormal processes and condition of a diseases.
Papanicolaou (PAP) test – cytology based test used to identify abnormal cervical cells and early cancers.
European Guidelines recommend PAP test every 3-5 years starting at age 22 – 30 !!
HPV testing – primary screening marker for cervical cancer!
High specificity – consequent high negative predictive value
Poor sensitivity – consequent low positive predictive value
Only a subset of neoplastic lesions with HPV infection persist and progress to invasive cancer
MACC1 – Metastasis-Associated in Colon Cancer 1
Induces migration, invasion and proliferation of cancer cells
Firstly discovered in: Colon Cancer
Hepatocellular, Nasopharyngeal, Ovarian, Cervical Carcinoma
Key regulator of hepatocyte growth factor (HGF)/c-Met signaling pathway
MACC1 activates c-Met transcription by binding to endogenous c-Met 60bp long promoter sequence
HGF/c-MET
signaling
pathway
Aim of the Study:
Aim of this study was to extract mRNA from cytobrush-collected
healthy and HPV infected cervical epithelial cells and investigate
the expression levels of MACC1 and c-Met transcripts in healthy
and infected samples
Samples:
 95 cervical specimens tested for HPV infection at Institute for Biomedical Diagnostics
and Research “NALAZ” (February 2014 – March 2015)
70 sample – High Risk HPV
(42) HPV16
(23) HPV52
(11) HPV18
15 samples – Low Risk HPV
(10) HPV57/71
(4) HPV40/61
(1) HPV54/55
Controls:
I.
II.
Cervical brush sample obtained from healthy donor not tested for HPV infection
Blood sample obtained from healthy donor
1. RNA extraction
2. Spectrophotometry
3. RNA purification
4. Agarose gel electrophoresis
5. cDNA synthesis
6. Real-Time Polymerase Chain Reaction (PCR)
1. RNA Extraction
Two different methods used!
1.
Commercially available kit for RNA extraction: The GeneJET™ RNA Purification Kit
2.
TRIzol® Reagent (Invitrogen, USA) extraction
(Thermo Fisher Scientific, USA)
 Method based on Phenol-Chloroform RNA extraction
2. Spectrophotometry – to measure the RNA concentration
Wavelengths: 260nm DNA
280nm RNA


OD 1 = ̴ 50 μg/mL of DNA
OD 1 = ̴ 40 μg/mL of RNA
Formula to calculate the concentration of RNA:
(1) c [ng/μl] = A260 x DF x 40
3. RNA purification
DNase I, RNase-free (supplied with MnCl2) (Thermo Fisher Scientific, USA)
 DNase I is an endonuclease that digests single- and double- stranded DNA
4. agarose gel electrophoresis
 Used to separate RNA or DNA fragments
 2% agarose gel used
5. cDNA synthesis and Real-Time PCR
cDNA synthesis: The Thermo Scientific RevertAid First Strand cDNA Synthesis Kit
(Thermo Fisher Scientific, USA)
→ cDNA is a copy synthesized from mRNA
→ The enzyme required for this reaction is reverse transcriptase (usually found in retroviruses)
Real-Time (PCR) – used for amplification and quantification of sequence of interest by
applying specific set of primers designed for that specific sequence
RNA extraction: The GeneJET™ RNA Purification Kit (Thermo Fisher Scientific, USA)
 48 samples used – 32 visualized on the 2% agarose gel electrophoresis
Gel electrophoresis of
isolated nucleic acids
from 17 randomly
chosen samples
RNA extraction: TRIzol® Reagent following the manufacturer’s protocol (Invitrogen, USA)
 20 samples used – 4 visualized on the 2% agarose gel electrophoresis
2% Gel electrophoresis of isolated nucleic
acids from seven (7) randomly chosen
samples
RNA purification: DNase I, RNase-free (supplied with MnCl2) (Thermo Fisher Scientific)
2% gel electrophoresis of RNA extraction
and purification.
RNA purification: DNase I, RNase-free (supplied with MnCl2) (Thermo Fisher Scientific)
HD – Healthy Donor cervical cytobrush sample
A
B
2% gel electrophoresis of RNA extraction using:
A.
TRIzol® Reagent
B.
The GeneJET™ RNA Purification Kit
cDNA Synthesis: The Thermo Scientific RevertAid First Strand cDNA Synthesis Kit
(Thermo Fisher Scientific, USA)
2% gel electrophoresis of cDNA products from five
randomly selected samples GAPDH - control
Real-Time PCR
5 cDNA samples have been used in Real-Time qPCR analysi!s
Ubiquitin gene – used as endogenous control
Amplification plot for MACC1 and
c-Met using Real-Time qPCR SYBR
green method in samples 40, 43, 44
and 84.
Positive control:
• GAPDH
Negative controls :
• Reverse Transcriptase – (RT-)
• No Template Control (NTC)
Frequency of HPV 16 genotype confirmed 
 out of 95 samples = 42 samples HPV 16 !!
Identifying the prevalence of particular HPV types for specific region – good in predicting the HPV
vaccines efficiency
Cervarix® a bivalent HPV 16/18 vaccines from GlaxoSmithKline Biologicals (GSK, UK)
Gardasil® a quadrivalent HPV16/18/6/11 vaccine from MSD Merck (USA)
Study based on gene expression analysis – MACC1 and c-Met target genes
Samples obtained by regular Papa testing – non invasive procedure
Gene expression analysis – RNA extraction necessary !!
RNA – unstable and susceptible to degradation!
•
Presence of 2’OH (2’hydroxyl group) – very reactive in nature
•
RNases are everywhere!!
skin, hands, laboratory equipment, air dust….
RNA extraction challenging process !!
 Requires sterile conditions and RNases-free environment
RNA purification- necessary step for assurance of RNA quality !!
? Longer storage time and conditions affected RNA degradation
 RNA extraction from fresh sample = same results
Samples used in this particular study, obtained using standard cytobrush technique, were not suitable
for gene expression analysis.

The most common HPV genotype HPV 16

RNA extraction from cytobrush-collected cervical epithelial cells used in this study couldn’t
be accomplished with either of the used techniques

The storage time and conditions are not the only factors that have contributed to RNA
degradation in the samples!
→ The very act of sampling could already damage any RNA molecules present in that
area of epidermis.

Process of RNA purification is a necessary step in all gene expression analysis studies!!
Acknowledgments
Mentor Assist. Prof. Dr. Daria Ler
International University of Sarajevo (IUS) and Management Assistant of Research and
Development Centre (RDC) Jasmin Sutkovic
Institute for Biomedical Diagnosis and Research “NALAZ” and Prof. Mirsada Hukic
Members of committee: Assoc. Prof. Dr. Sabina Semiz and Assist. Prof. Dr. Mirza Suljagic
My family and friends
The End…
Thank You for Your Attention!