Abstract 3168: Functional analysis and target identification of specific miRNAs involved in development of metastases in renal cell carcinoma

Abstract

Abstract MicroRNAs (miRNAs) are short, non-coding RNA sequences which play an important role in cellular gene expression regulation on the post-transcriptional level. Recent studies have shown that they influence the regulation of tumor development in clear cell renal cell carcinoma (ccRCC), but their impact on metastasis is not investigated so far. We aimed in defining a specific miRNA expression pattern of metastasis in ccRCC. Furthermore, we focused on analysis of cellular functions and identification of target proteins of miRNAs involved in the metastatic process using cell lines. Total RNA from 44 ccRCC samples (including 22 non-metastatic and 22 metastatic tumors) and from 21 metastases was isolated using snap frozen tissue. Microarray analyses were performed for a global miRNA expression profiling. Results were validated by qRT-PCR. For functional analysis and target protein identification a metastatic (786-O) and a non-metastatic (KTCTL-30) ccRCC cell line was transfected with miRNAs differently expressed in metastatic tumors and metastases. Proliferation, migration and invasion assays were performed to investigate the influence of these miRNAs on the metastastic process. For identification of target proteins multiplex-fluorescent 2D gel electrophoresis of the transfected cell lines and database research were carried out. Microarray and qRT-PCR analyses showed that expression of specific miRNAs distinguishes between metastatic and non-metastatic ccRCC. Metastases have high similarity in miRNA expression to metastatic tumors. 9 miRNAs (including miR-451) are differentially expressed in primary metastatic tumors and metastases compared to non-metastatic tumors. Furthermore, a specific metastases miRNA pattern was detected. In addition, miR-30c expression level discriminates non-metastatic, metastatic tumors and metastases. Functional analysis on cell lines showed an significantly decreased migration after overexpression of miR-30c. It was possible to identify specific target candidates of miR-30c and miR-451 differently expressed in transfected cell lines by multiplex-fluorescent 2D gel electrophoresis. A specific miRNA expression pattern characterizes metastatic ccRCC. MiRNA expression in metastases is in accordance with primary metastatic tumors. Specific expression in metastases might be caused by a specific microenvironment at the metastatic site. MiR-30c regulates cellular processes involved in metastasis. MiRNA target identification can provide an insight into signal pathways deregulated in metastasis. Thus, several candidate target proteins of miR-30c and miR-451 were identified which have to be validated in this ongoing study using specific antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3168. doi:1538-7445.AM2012-3168