Abstract 4374: Genistein suppresses prostate cancer regulating long non-coding RNA
Authors: Chiyomaru Takeshi , Yamamura Soichiro , Fukuhara Shinichiro , Kinoshita Takashi , Majid Shahana , Saini Sharanjot , Chang Inik , Tanaka Yuichiro , Enokida Hideki , Seki Naohiko , Nakagawa Masayuki 
Published: May 2015

Abstract
Introduction: Genistein has been shown to inhibit various cancers but its mechanism of action is still unknown. Non-coding RNAs (ncRNAs) are involved in many cellular processes and show abnormal expression patterns in cancer tissues. In this study, we tested the hypothesis that genistein can inhibit prostate cancer (PCa) through modulation of ncRNA including long ncRNAs (lncRNAs) as well as coding RNA using various in vitro models. We also tested the functional significance of lncRNAs in PCa cell lines.
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 SurePrint G3 Human GE 8 x 60K microarray platform also includes probes for lncRNAs. To identify the biological processes or pathways potentially regulated by genistein, we performed GeneCodis analysis. To identify networks among their target genes, we analyzed and characterized those genes in KEGG pathway categories. The expression levels of lncRNAs were analyzed by TaqMan quantitative real-time PCR and normalized to GAPDH. For gain-of-function assay, cell proliferation (MTS assay), migration (wound healing assay), invasion (matrigel invasion assay), apoptosis and cell cycle (flow cytometry analysis) were evaluated in human PCa cell lines, PC3 and DU145.
Results: Using mRNA microarray and GeneCodis software analysis, several pathways affected by genistein were identified, such as ‘Pathways in cancer’, ‘Cell cycle’, and‘Regulation of actin cytoskeleton’ and ‘Jak-STAT signaling pathway’ (P&lt;0.05). Using microarray analysis and real-time RT-PCR we observed that HOTAIR was significantly down-regulated with genistein treatment and ZNFX1-AS1 and GAS5 expression was significantly up-regulated with genistein treatment. The expression of HOTAIR was significantly higher in PCa cell lines compared with normal prostate cell (RWPE-1). The expression of GAS5 and ZNFX1-AS1was significantly lower in PCa cell lines compared with RWPE-1. Knockdown of HOTAIR in PCa cells significantly inhibited cell proliferation in vitro and in vivo. Knockdown of HOTAIR induced apoptosis and cell cycle arrest.
Conclusions: Our results show that genistein regulates oncogenic HOTAIR and tumor suppressor GAS5 and ZNFX1-AS1 expression. These findings enhance understanding of how genistein regulates with lncRNA in PCa.
Citation Format: Takeshi Chiyomaru, Soichiro Yamamura, Shinichiro Fukuhara, Takashi Kinoshita, Shahana Majid, Sharanjot Saini, Inik Chang, Yuichiro Tanaka, Hideki Enokida, Naohiko Seki, Masayuki Nakagawa. Genistein suppresses prostate cancer regulating long non-coding RNA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4374. doi:10.1158/1538-7445.AM2014-4374