Abstract 3045: Identification of specific microRNA signatures in VHL-functional and VHL-silenced renal cell carcinoma cell lines

Abstract

Abstract Objective& Background: Renal cell carcinoma is the most common renal malignancy in adults. Up to 70% of clear cell renal cancers have VHL silencing either because of gene mutation or methylation. MicroRNAs are a class of small non-coding RNAs that control gene expression by targeting mRNAs and trigger either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases including cancer. This study set out to investigate the influences of VHL status and the ensuing differences in miRNA profiles and biological/phenotypic effects in renal carcinoma cell lines. Methods: Normal cell line, HREC, and renal cancer cell lines that are VHL-ve cell lines, A498, A704, 786.O, and 786.O. Vec, VHL+ve cell line,786.O.VHL, VHL-WT cell line, ACHN and Caki.1 and VHL-CH3 cell line, KV6 were used for the VHL-specific genome wide miRNA profiling. miRNAs were extracted, in triplicate, from the above cell lines at 48 hrs time point by using miRVana miRNA isolation kit (Ambion). The extracted miRNAs were subjected to analysis by MEGAPlex RT primers and MEGAPlex Pre-Amp primers, followed by loading them to TaqMan Human miRNA microarrays v2.0 (Human Array A/B (Applied Biosystems). Resulting miRNAs were analysed further by using the RealTimeStatMiner v3.0 software (Integromics) followed by GeneGO pathways analysis. Results: We show that approximately 577 miRNAs were altered in human kidney cancer. 270 miRNAs and 180 miRNAs were upregulated and down-regulated in cancer cell lines when compared against normal HREC cell line, respectively. Upon analysis of differentially expressed miRNAs in RCC cell lines based on VHL status, we found that 7 miRNAs were differentially upregulated in VHL wild type RCC cells. In VHL mutant cell lines, 5 miRNAs and 6 miRNAs were differentially up and down regulated, respectively, while in RCC cell lines with wild type, but silenced (methylated) VHL, 7 and 10 miRNAs were differentially up and down regulated, respectively. Target prediction and microRNA enrichment analysis suggested that the differentially expressed miRNAs are involved in regulation of HIF, angiogenesis, invasion & metastasis, extracellular matrix degradation, EMT phenomenon, metabolism and apoptosis pathways. Conclusion: This MicroRNome analysis reveals a specific spectrum of hypoxia, angiogenesis, tumor metabolism, metastasis, cell cycle and apoptotic specific miRNAs. In vitro experiments to characterize the biological significance differential miRNA expression profiles on different steps of tumorigenesis and tumor progression are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3045.