8/3/2019 embo serkan2

http://slidepdf.com/reader/full/embo-serkan2 1/1

 

microRNAs in Breast Cancer

Selcuklu SD1, Tuna S1, Yakicier MC2, Erson AE1

1Department of Biology, Middle East Technical University, Ankara, Turkey2Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey

hsa-mir-21

0

0,5

1

1,5

2

2,5

3

3,5

4

O V AR Y T ES TE S B R EA S T M C F- 7 M D A- M B-

231

   F  o   l   d

   C   h  a  n  g  e

  METU

http://www.genscript.com/images/MIRNAF.jpg

Table1: Example of common genomic instability

regions in breast cancer. Known genes in these regions

and methods used to identify these alterations are

shown.

Table2: Examples of microRNAs on common

genomic instability regions in breast cancer

5. Results

Up to date, 530 microRNAs are discovered in human,most of them are conserved among related species andmore than 1000 are predicted by computational methods.Various studies showed that microRNAs are regulatingmany cellular pathways in mammalian cells such asdevelopment, cell differentiation, proliferation, apoptosisand tumorigenesis depending on their target. It’s beenestimated that up to 30% of human genes might bemicroRNA targets

2. Introduction

Calin et al, 2004

3. Research Objectives

The aim of this study is to identify significant microRNAs located in common genomic loss orgain regions in breast cancer cells and further investigate the microRNAs at transcript levelwhether there is difference in the expression levels since microRNAs may contribute to breasttumorigenesis and its maintenance

4. Materials & Methods1) Literature Research of genomic instability regions

- 38 mirs were selected as candidates 

2) Semi-quantitative duplex PCR 

- PCR optimizations with GAPDH (housekeeping control) and microRNA primers and Semi-

quantitative duplex PCR with 22 breast cancer cell line DNAs

3) Densitometry Analysis of Semi-quantitative duplex PCR results

4) Semi-quantitative duplex RT-PCR 

- Primer design for precursor structure of candidate miRNAs (pre-miR)- Semi-quantitative duplex RT-PCR with 2 breast cancer cell lines (MCF-7, MDA-MB-231) and

3 normal tissues (breast,ovary, testes)

5) Bioinformatical analysis of microRNAs

- Transciption Start Site (TSS) determination by Eponine TSS finder, Promoter search by

Promo3 and Dragon Estrogen Responsive Element (ERE) finder, microRNA expression

profile in different tissues by microRNA.org, microRNA target predictions by Targetscan,

PicTar, Miranda, miR-target mRNA binding interaction by DINAMelt Server

Figure1: Semi-quantitative duplex PCR with breast cancer cell lines:

Hsa-mir-21 is amplified in MCF7 and MDA-MB-231 breast cancer cell lines

Figure2:  Fold numbers changes for the 36 microRNA genes

mapping to selected genomic instability regions in breast

cancers. Densitometric analysis of microRNA and GAPDH PCR

products of cancer cell lines was compared to that of normal

DNAs.

Figure3: semi-quantitative duplex RT-PCR for hsa-mir-103-2 and hsa-mir-21:

hsa-mir-103-2 transcript is downregulated in MCF7 and MDA-MB-231 breast cancer cell lines

compared to normal breast tissue (RNA from Ambion) whereas hsa-mir-21 transcript is

upregulated in these cell lines.

6.Conclusion & Future Directions 

38 microRNAs were mapped to common genomic alteration regions in breast cancer cells. Semi-quantitative

PCR analysis of DNA regions of these 38 microRNAs in 20 breast cancer cell lines and 2 immortalized breast

cell lines followed fold change calculation. The results showed that 21 of 34 (61%) selected microRNAs are

either lost or amplified in at least 3 breast cancer cell lines. Interestingly, most of the regions shown to

harbour losses had amplifications for these microRNAs and also amplification regions showed loss of some

miRNAs. These results may also help to re-define the borders of these altered regions and indicate the

significant genomic alterations of specific miRNAs in cancer cells.

Expression levels of candidate microRNAs by semi-quantitative RT-PCR results indicate the deregulation of 

candidate microRNAs. Based on these data, candidate microRNAs will be analized and functional analysis of 

significant microRNAs will be done to identify their targets to reveal their involvement in breast

tumorigenesis.

hsa-mir-103-2

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

O VA RY T ES TE S B RE AS T M CF -7 M DA -M B-

231

   F  o   l   d

   C   h  a  n  g  e

hsa-mir-21

0

0,5

1

1,5

2

2,5

3

3,5

4

O VA RY T ES TE S B RE AS T M CF -7 M DA -M B -

231

   F  o   l   d

   C   h  a  n  g  e

1. AbstractGenomic instability is a common event in breast cancers. Various chromosomal and segmental loss or amplification regions have been detected in primary breast tumors and breast cancer cell lines. Search for potent

tumor suppressors or oncogenes in these genomic instability regions continues. MicroRNAs (miRNAs) are ~18-24 nt long non-coding RNAs that regulate protein expression by binding to target sequences in the 3 ’  UTR

regions of mRNAs. A large number of microRNAs are localized to genomic instability regions in cancer cells. Therefore, we hypothesized that microRNAs on common genomic instability regions in breast cancer cells may

contribute to breast tumorigenesis. We investigated more than 30 microRNA genes localized to the common genomic instability regions in breast cancers. It is found that 47% of the investigated microRNA genes show

significant fold number changes compared to internal control gene GAPDH in 3 or more of the total number of 22 breast cancer cell lines. These results may point out these microRNAs as potential targets of the genomic

instability regions as oncogene and tumor suppressor candidates. We focused on chromosomal regions 3p21, 3q23, 8p11-12, 11q23-24 and 17q23, as we detected the most significant amplification/loss data. Our initial

candidates on these chromosomal bands, that are commonly amplified/lost in breast cancer cells, are being investigated in terms of their expression levels by RT-PCR. Moreover, bioinformatical analysis are also being

carried on for promoter search, for transcription start site determination, microRNA target prediction, microRNA-target mRNA binding and also kinetics of these interactions. Based on our expression and bioinformaticalanalyses, further functional assays will be performed to better understand their roles in breast tumorigenesis pathways.

Key References

1. http://www.genscript.com/images/MIRNAF.jpg2. Calin, George Adrian et al. (2004) Proc. Natl. Acad. Sci. USA 101, 2999-30043. Kytola, S., J. Rummukainen, et al. (2000) Genes Chromosomes Cancer 28 ( 3): 308-17.4. Zhao, X., C. Li, et al. (2004). Cancer Res 64(9): 3060-71.

5. Lenora W.M. Loo, et al. (2004) Cancer Research 64, 8541-8549

This project is funded by TUBA (Turkish Academy of Sceiences) Grant no: TUBA-GEBIP 2006

Acknowledgements: Breast cancer cell line DNAs were a generous gift from Assoc. Prof. Dr. Elizabeth M.

Petty, Department of Human Genetics, University of Michigan, USA