Abstract 5284: A microRNA downregulated in human cholangiocarcinoma induces G2/M arrest through multiple targets.

Abstract

Abstract Background: Cholangiocarcinomas (CCA) are cancers with dismal prognosis, thought to arise from epithelial cells lining the biliary tree. An improved understanding of the pathogenesis of CCA, as well as novel diagnostic and therapeutic approaches are direly needed. MicroRNAs (miRs) are short, single-stranded sequences of RNA, that were recently demonstrated to play a major role in the regulation of virtually all cellular processes. Several previous studies identified miRs that are dysregulated in CCA as well as distinct roles played by miRs in CCA genesis or progression. Methods: We performed microRNA-arrays on 5 normal biliary duct epithelias and 5 CCAs that had been obtained at surgery. After data analysis, we selected mir-494 for all subsequent analysis among top 5 miRs. To confirm the initial miR array data, quantitative real time RT-PCR analysis was performed using 12 human CCA as well as 5 normal cholangiocyte specimens. And then, we transfected 2 CCA cell lines with miR-494 and a non-specific mimic, respectively, and performed mRNA arrays to identify in an unbiased fashion the genes whose expression is downregulated by miR-494. Furthermore, we performed fluorescence activated cell sorting, as well as differential interference contrast microscopy. Results: miR-494 is significantly downregulated in human CCA specimens. Cells transfected with miR-494 showed a significant decrease in growth in the cell proliferation assay. As for the results of mRNA array, Ingenuity Pathway Analysis also indicated that miR-494 appears to coordinately affect several genes involved in the Mitotic Roles of Polo-like Kinase and Cell cycle: G2/M Checkpoint Regulation canonical pathways. We verified that miR-494 modulates the protein level of six genes involved in the G2/M transition. Next, we identified direct binding of miR-494 to the open reading frame (ORF), and downregulation of PTTG1 and TOP2A. Conclusions: our findings suggest that miR-494 has a global regulatory role in cell cycle progression. Therefore, it appears that the simultaneous effects of a single miR species on multiple targets along the same canonical pathway is advantageous for the usage of miRs as therapeutics. Citation Format: Sumitaka Yamanaka, Nathaniel R. Campbell, Scot C. Kuo, Esteban Mezey, Anirban Maitra, Florin Selaru. A microRNA downregulated in human cholangiocarcinoma induces G2/M arrest through multiple targets. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5284. doi:10.1158/1538-7445.AM2013-5284