Functional characterization of MicroRNA-125b expression in MCF7 breast cancer cell line

Thesis Type: Postgraduate

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

Approval Date: 2010




microRNA dependent gene expression regulation has roles in diverse processes such as differentiation, proliferation and apoptosis. Therefore, deregulated miRNA expression has functional importance for various diseases, including cancer. miR-125b is among the commonly downregulated miRNAs in breast cancer cells . Therefore we aimed to characterize the effects of miR-125b expression in MCF7 breast cancer cell line (BCCL) to better understand its roles in tumorigenesis. Here, we investigated mir-125 family members‟ expression levels in eleven BCCL and MCF10A, by semi-quantitative duplex RT-PCR. pre-miR-125b-1 levels were found to be low or absent in 7 of 11 BCCL. Among these, MCF7 cells were stably transfected with mir-125b-1 (MCF7-125b-1). MCF7-125b-1 cells demonstrated decreased proliferation and migration detected by MTT, in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Putative miR-125b target, ARID3B, was bioinformatically analyzed for miR-125b binding sites. 3‟UTR of ARID3B was cloned downstream of the luciferase gene in pMIR, a reporter vector. ~60% decrease in luciferase activity suggested the interaction between miR-125b and ARID3B 3‟UTR. To further confirm this, a miR-125b binding site was deleted by site directed mutagenesis. Deletion of this predicted site in the ARID3B 3‟UTR resulted with ~30% recovery in luciferase activity. Our results further showed the tumor suppressor functions of miR-125b in MCF7 cells. Revealing the phenotypic effects of miR-125b expression and its mRNA targets may help us shed light on why miR-125b may act as a tumor suppressor in breast cancer cells.