Investigation of microRNAs on genomic instability regions in breast cancer

Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2007




Genomic instability is commonly seen in breast cancers. To date, various chromosomal or segmental loss or amplification regions have been detected in primary tumors and cell lines. Hence, an intensive search for potent tumor suppressors or oncogenes located in these regions continues. MicroRNAs (miRNAs) are ~18-24 nt long non-coding RNAs that regulate protein expression either by target mRNA cleavage or translational repression. We hypothesized that miRNAs located in genomic instability regions in breast cancer cells may contribute to the initiation or maintenance of breast tumors. Here, we investigated genomic levels of miRNAs on frequent loss or gain regions of breast cancer cells. First, using bioinformatics resources we mapped known miRNAs and candidate miRNAs to reported genomic instability regions. Our extensive searches resulted with more than 30 known miRNAs and 35 candidate miRNAs. To further confirm loss or amplification of miRNA genes on these chromosomal regions in breast cancer cells, we designed specific primers for the known pre-miRNA DNA regions and performed semi-quantitative PCR in 20 breast cancer cell lines, 2 immortalized mammary cell lines, and 2 control samples. Densitometry results suggested that a striking 61 % (22/36) of selected miRNAs showed either loss or amplification in at least 3 different breast cancer cell lines. Interestingly most of these alterations were found to be amplifications even in regions reported to harbor losses in breast tumors. Genomic fold change results of these microRNAs provide a biologically relevant starting point for further expression and functional experiments of microRNAs in breast cancer studies. Genomic fold change analysis followed expression analysis of two significant microRNAs (hsa-miR-21 and hsa-miR-383) was done by qRT-PCR method. Our data provide a wide screen of genomic instability of 36 microRNA genes in 20 breast cancer cells and normal samples detected by semi-quantitative duplex PCR method as well as expression analysis of two microRNAs. To this date, such an extensive data on genomic status of microRNA genes in breast cancer cells did not exist. Therefore, our results are the first comprehensive investigation of many microRNA genes on genomic instability regions in breast cancers and provide further clues to the potential involvement of these microRNAs in breast tumorigenesis MicroRNA genomic instability may affect their expression and therefore their targets’ expressions. Understanding how these microRNAs regulate their targets and contribute to the neoplastic events will also contribute to the field by using this information for future diagnostic and threaupetical applications.