Student: Tea BecirevicSupervisor: Assist. Prof. Dr. Daria Ler

Analysis of MACC1 and c-Met expression in cytobrush-collected healthy and HPV infected cervical epithelial cells

International University of Sarajevo Faculty of Engineering and Natural Sciences

Genetics and Bioengineering department

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

INTRODUCTION

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 parts 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)

Breast

Lung, bronchial, tracheal

Cervical

Ovarian

Gastric

Uterine

Rectal

Colon

Brain, nervous system

Pancreatic

Others (without melanoma cancer)

613

198

190

133

122

118

118

117

83

77

681

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 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

Cervical Cancer precursors

How cervical cancer develops?

Infection with Human Papilloma Virus (HPV) is the central cause for the devel-opment of invasive cervical cancer and its precursor lesions!

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)

HPV – sexually transmitted disease (STD)

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

Papanicolaou (PAP) test – cytology based test used to identify abnormal cervical cells and early can-cers. 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 can-cer

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.

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 sig-naling 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

MATERIALS AND METHODS

95 cervical specimens tested for HPV infection at Institute for Biomedical Diagnostics and Research “NALAZ” (February 2014 – March 2015)

70 sample – High Risk HPV 15 samples – Low Risk HPV

(42) HPV16

(23) HPV52

(11) HPV18

(10) HPV57/71

(4) HPV40/61

(1) HPV54/55

Controls:

I. Cervical brush sample obtained from healthy donor not tested for HPV infectionII. Blood sample obtained from healthy donor

Samples:

1. RNA extraction

2. Spectrophotometry

3. RNA purification

4. Agarose gel electrophoresis

5. cDNA synthesis

6. Real-Time Polymerase Chain Reac-

tion (PCR)

1. RNA Extrac-tion

Two different methods used!

1. Commercially available kit for RNA extraction: The GeneJET™ RNA Purifica-tion Kit (Thermo Fisher Scientific, USA)

2. TRIzol® Reagent (Invitrogen, USA) extraction

Method based on Phenol-Chloroform RNA extraction

2. Spectrophotometry – to measure the RNA con-centration

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 Sci-entific, 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 in-terest by applying specific set of primers designed for that specific sequence

RESULTS

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 ran-domly chosen samples

RNA extraction: TRIzol® Reagent following the manufacturer’s protocol (Invit-rogen, 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 Sci-entific)

2% gel electrophoresis of RNA ex-traction and purification.

RNA purification: DNase I, RNase-free (supplied with MnCl2) (Thermo Fisher Sci-entific)

A B

2% gel electrophoresis of RNA extraction using:

A. TRIzol® Reagent

B. The GeneJET™ RNA Purification Kit

HD – Healthy Donor cervical cytobrush sample

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

Amplification plot for MACC1 and c-Met using Real-Time qPCR SYBR green method in sam-ples 40, 43, 44 and 84.

Negative controls : • Reverse Transcriptase – (RT-) • No Template Control (NTC)

Positive control: • GAPDH

5 cDNA samples have been used in Real-Time qPCR analysi!s

Ubiquitin gene – used as endogenous control

DISCUSSION

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 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.

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….

CONCLUSION

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 stud-ies!!

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 Atten-tion!