Abstract 4161: Genistein inhibits prostate cancer through activation of tumor suppressor microRNA-574-3p.

Abstract

Abstract Introduction: Genistein has been shown to inhibit various cancers but its mechanism of action is still unknown. In this study, we tested the hypothesis that genistein can inhibit prostate cancer (PCa) through modulation of microRNA (miRNAs) using various in vitro models. We also tested the functional significance of miRNAs in PCa cell lines and clinical significance of miRNAs in different stages and grades of PCa. Methods: Subconfluent cells (60-70% confluent) were treated with genistein (25 μM). Genistein administered everyday along with a change of medium, and the cells were grown for 96 h. The miRNA microarray analysis was carried out and analyzed by Phalanx Biotech using the human v3 miRNA OneArray platform that is designed to contain 100% of miRBase Sequence Database Release 17.0. The expression levels of miR-574-3p were analyzed by TaqMan quantitative real-time PCR and normalized to RNU48. The 48 clinical specimens were obtained from patients in San Francisco Veterans Affairs Medical Center from 1998 to 2004. For gain-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), invasion (matrigel invasion assay) and apoptosis (flow cytometry analysis) were evaluated in human PCa cell lines, PC3 and DU145. Predicted target genes and their target miRNA binding site seed regions were investigated using TargetScan. To identify the biological processes or pathways potentially regulated by miR-574-3p, we performed GeneCodis analysis. To identify networks among the miRNAs and their target genes, we analyzed and characterized those genes in KEGG pathway categories. Results: Using miRNA microarray analysis and real-time RT-PCR we observed that miR-574-3p was significantly up-regulated in PCa cells treated with genistein compared with vehicle control. The expression of miR-574-3p was significantly lower in PCa cell lines and clinical PCa tissues compared with normal prostate cells (RWPE-1) and adjacent normal tissues. Low expression level of miR-574-3p was correlated with advanced tumor stage and higher Gleason score in PCa specimens. Re-expression of miR-574-3p in PCa cells significantly inhibited cell proliferation, migration and invasion. miR-574-3p restoration induced apoptosis through reducing Bcl-xL and activating caspase-9 and caspase-3. Using GeneCodis software analysis, several pathways affected by miR-574-3p were identified, such as ‘Pathways in cancer’, ‘Jak-STAT signaling pathway’, and ‘Wnt signaling pathway’. Luciferase reporter assays demonstrated that miR-574-3p directly binds to the 3’ UTR of several target genes (such as RAC1, EGFR and EP300) that are components of ‘Pathways in cancer’. Conclusions: Our results show that genistein up-regulates tumor suppressor miR-574-3p expression targeting several cell signaling pathways. These findings enhance understanding of how genistein regulates with miRNA in PCa. Citation Format: Takeshi Chiyomaru, Soichiro Yamamura, Shinichiro Fukuhara, Hideo Hidaka, Shahana Majid, Sharanjot Saini, Sumit Arora, Guoren Deng, Varahram Shahryari, Inik Chang, Yuichiro Tanaka, Z. Laura Tabatabai, Hideki Enokida, Naohiko Seki, Masayuki Nakagawa, Rajvir Dahiya. Genistein inhibits prostate cancer through activation of tumor suppressor microRNA-574-3p. [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 4161. doi:10.1158/1538-7445.AM2013-4161