Genistein, microRNAs and kidney cancer

描述(由申请人提供)： 背景：肾细胞癌是退伍军人中最常见的恶性肿瘤之一。肾癌治疗取得进展的主要障碍是用于治疗的药物的高毒性。目前的建议将有助于解决这一问题，通过使用体外和体内模型利用饮食方法管理肾细胞癌。这个项目的基本原理是，最近的研究表明，在体外和体内模型中，饮食对miRNAs的调节都有显著影响。然而，在肾癌方面缺乏这样的研究。研究目的：本课题的主要目的是通过体外和体内模型研究金雀异黄素是否通过激活肿瘤抑制因子microRNAs(MiRNAs)来抑制肾癌的生长。我们假设金雀异黄素可以激活一组肿瘤抑制基因miRNAs，从而通过两种不同的途径调节肾癌的进展。首先，金雀异黄素诱导的抑癌基因miRNAs通过与3‘端非翻译区(3’UTR)结合来抑制癌基因的表达。第二，金雀异黄素诱导的抑癌基因miRNAs可以通过与基因5‘上游区结合来激活抑癌基因的转录，或通过与5’端非翻译区(5‘UTR)结合来激活翻译。项目设计与方法：特定目的#1.探讨染料木素是否通过激活抑癌基因miRNAs来抑制肾癌的发生。在这个特定的目标下，我们将金雀异黄素处理肾癌细胞株，然后分析金雀异黄素是否通过激活抑癌基因miRNAs来抑制肾癌细胞。为了研究这些抑癌基因miRNAs的功能意义，我们将通过染料木素处理激活这些miRNAs，然后对这些miRNAs的靶基因进行分析。miRNA介导的癌基因抑制或抑癌基因的激活将分别用3‘UTR和5’UTR序列构建的荧光素酶活性来分析。细胞生长的变化将通过监测细胞增殖、细胞周期分布、细胞凋亡和体外侵袭来评估。检测方法包括细胞增殖、流式细胞仪、迁移、克隆存活、体外侵袭和细胞凋亡检测。具体目的#2：探讨金雀异黄素在肾癌细胞中激活抑癌基因miRNAs的分子机制。我们将探讨金雀异黄素介导的肿瘤抑制基因在肾癌细胞中激活的表观遗传学机制。我们将用金雀异黄素处理肾癌细胞，然后使用亚硫酸氢钠甲基化技术和DNA直接测序分析miRNAs可能启动子区CpG岛的低甲基化。我们将研究这些肿瘤抑制因子miRNAs的激活机制是否通过组蛋白乙酰化和染色质重塑来调节。分析组蛋白乙酰转移酶(HATS)和组蛋白脱乙酰基酶(HDACs)在miRNA转染的肾癌细胞中的表达和活性。具体目的#3：研究金雀异黄素介导的肿瘤抑制基因miRNAs的激活是否能抑制裸鼠模型中肾肿瘤的生长和增殖。我们将研究金雀异黄素介导的miRNAs激活是否能抑制裸鼠肾肿瘤的生长和增殖。肾癌细胞将被注射到用金雀异黄素治疗的裸鼠的皮下(异种移植)或原位(肾脏)，并与对照组相比测试它们的生长和进展。临床意义：该项目的主要目标是研究金雀异黄素通过激活肿瘤抑制因子miRNA来抑制肾癌的作用。这些信息可以为肾癌的治疗和管理带来新的策略。由于本提案的目的是通过激活肿瘤抑制因子miRNAs来研究饮食对肾癌的抑制作用，我们认为拟议的工作与退伍军人健康和退伍军人退伍军人使命高度相关。

DESCRIPTION (provided by applicant): Background: Renal cell carcinoma (RCC) is one of the most common malignancies among our Veterans. The major barrier to progress in the management of RCC is the high toxicity of the drugs used for the treatment. The present proposal will help to address this problem by utilizing dietary methods for the management of RCC using in vitro and in vivo models. The rationale for this project is that recent studies have shown significant effects of diet on modulation of miRNAs using both in vitro and in vivo models. However, such studies are lacking in kidney cancer. Research Objectives: The main goal of this project is to investigate whether genistein can inhibit kidney cancer growth through activation of tumor suppressor microRNAs (miRNAs) using both in vitro and in vivo models. We hypothesize that genistein can activate a set of tumor suppressor miRNAs thereby regulating kidney cancer progression through two different pathways. First, genistein induced tumor suppressor miRNAs can repress oncogene expression by binding to the 3' untranslated region of mRNA (3'UTR). Second genistein induced tumor suppressor miRNAs can activate the transcription of tumor suppressor genes by binding to the 5' upstream region of the gene or activate translation by binding to the 5' untranslated region of mRNA (5'UTR). Project Design and Methods: Specific Aim #1. To investigate whether genistein mediated inhibition of kidney cancer is through activation of tumor suppressor miRNAs. Under this specific aim, we will treat kidney cancer cell lines with genistein and then analyze whether genistein mediated inhibition of kidney cancer cells is through activation of tumor suppressor miRNAs. To investigate the functional significance of these tumor suppressor miRNAs, we will activate these miRNAs by genistein treatment and then target genes of these miRNAs will be analyzed.The miRNA-mediated repression of oncogenes or activation of tumor suppressor genes will be analyzed by luciferase assays using 3'UTR or 5'UTR sequence constructs, respectively. Alterations in cell growth will be assessed by monitoring cell proliferation, cell cycle distribution, apoptosis and in vitro invasion. Assays include cell proliferation, flow cytometry, migration, clonogenic survival, in vitro invasion and apoptosis assays. Specific Aim #2: To investigate the molecular mechanisms of genistein mediated activation of tumor suppressor miRNAs in kidney cancer cells. We will investigate epigenetic mechanisms of genistein mediated activation of tumor suppressor microRNAs in kidney cancer cells. We will treat kidney cancer cells with genistein and then analyze hypomethylation of CpG Islands in putative promoter regions of miRNAs using sodium bisulfite methylation techniques and direct DNA sequencing. We will investigate whether the mechanisms of activation of these tumor suppressor miRNAs are through modulation of histone acetylation and chromatin remodeling. The expression and activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in miRNA transfected kidney cancer cells will be analyzed. Specific Aim #3: Investigate whether genistein mediated activation of tumor suppressor miRNAs can inhibit growth and proliferation of kidney tumors in a nude mouse model. We will investigate whether genistein mediated activation of miRNAs can inhibit growth and proliferation of kidney tumors in nude mice. Kidney cancer cells will be injected sub-cutaneously (xenograft) or orthotopically (kidney) into nude mice treated with genistein and tested for their growth and progression compared to controls. Clinical Relevance: The main goal of this project is to investigate the role of genistein in the inhibition of RCC through activation of tumor suppressor miRNA. This information can lead to novel strategies for treatment and management of RCC. Since the goal of this proposal is to investigate the dietary mediated inhibition of RCC through activation of tumor suppressor miRNAs, we believe that the work proposed is highly relevant to Veterans health and the VA mission.