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

Tuna, Serkan
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.


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Citation Formats
S. Tuna, “Functional characterization of microrna-125b expression in MCF7 breast cancer cell line,” M.S. - Master of Science, Middle East Technical University, 2010.