Abstract 3961: Sec23A is a regulative target of miR-200c and miR-375 in prostate cancer and influences cell growth in vitro

Abstract

Abstract Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs and to analyze the physiological consequences of miRNA deregulation using cell culture models. Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries and microarray analyses. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. The impact of identified target genes on cell growth was examined by a standardized wound healing assay. Results: Two of the miRNAs consistently deregulated in primary prostate cancer samples were miR-200c and miR-375 which were upregulated more than 1.5-fold in deep sequencing and 2.9-fold and 3.9-fold, respectively, in microarray experiments (p<0.001, ANOVA). Independent target prediction databases (including miRanda, Targetscan and PicTar) indicated that the 3’ untranslated region (3’UTR) of the Sec23a gene is a regulative target for both miR-200c and miR-375. This prediction could be confirmed using a luciferase reporter gene construct containing the Sec23a 3’UTR. Co-expression of the reporter gene construct with either miR-200c or miR-375 resulted in a reduction of reporter gene activity while mutation of the miRNA binding sites caused a loss of responsiveness to the respective miRNA. Furthermore we could show that transient expression of miR-200c and miR-375 reduced the amount of Sec23a protein in the HEK293 cell line. A transient knock-down of Sec23a in DU145 prostate cancer cells applying siRNA technique led to acceleration of wound healing and transient overexpression of Sec23a to a deceleration over a time period of 30 hours. In Western blot analyses the majority of primary tissue samples displayed a reduced amount of Sec23a protein. Conclusion: Our findings demonstrate that the 3’-UTR of Sec23a is one regulative target of miR-200c and miR-375, which are commonly deregulated in prostate cancer. Sec23a is reduced in the majority of tumor samples analyzed and has a vital impact on cell growth as determined by wound healing experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3961. doi:10.1158/1538-7445.AM2011-3961