Genistein, microRNAs and kidney cancer

金雀异黄素、microRNA 和肾癌

• 批准号: 8598022
• 负责人: RAJVIR DAHIYA
• 金额: --
• 依托单位: VETERANS AFFAIRS MED CTR SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8598022
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Address; Apoptosis; Benign; Binding; Biological Assay; Cancer cell line; Cell Cycle; Cell Proliferation; Chromophobe Renal Cell Carcinoma; Clear Cell; CpG Islands; DNA Sequence; Data; Diet; Disease; Drug toxicity; Drug usage; Epigenetic Process; Flow Cytometry; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genistein; Genitourinary system; Goals; Growth; Health; Histone Acetylation; Histones; Human; In Vitro; Incidence; Kidney; Kidney Neoplasms; Lead; Literature; Luciferases; Malignant - descriptor; Malignant Neoplasms; Mediating; Messenger RNA; Methods; Methylation; MicroRNAs; Mission; Molecular; Monitor; Nude Mice; Oncogenes; Oxyphilic Adenoma; Pathologic Processes; Pathway interactions; Promoter Regions; Proteins; Publishing; Regulation; Renal Cell Carcinoma; Renal carcinoma; Repression; Research; Role; Series; Techniques; Testing; Time; Toxic effect; Translations; Tumor Suppressor Genes; Tumor Suppressor Proteins; Untranslated Regions; Veterans; Work; Xenograft procedure; base; cancer cell; cell growth; chromatin remodeling; clinically relevant; design; histone acetyltransferase; in vivo; in vivo Model; migration; mouse model; novel; novel strategies; research study; sodium bisulfite; tumor progression

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.

描述（由申请人提供）： 背景：肾细胞癌（RCC）是退伍军人中最常见的恶性肿瘤之一。肾细胞癌治疗取得进展的主要障碍是治疗药物的高毒性。本提案将通过体外和体内模型利用饮食方法来管理肾细胞癌，从而有助于解决这个问题。该项目的基本原理是，最近的研究表明饮食对体内和体外模型的 miRNA 调节有显着影响。然而，肾癌方面缺乏此类研究。研究目标：该项目的主要目标是利用体外和体内模型研究金雀异黄素是否可以通过激活肿瘤抑制基因 microRNA (miRNA) 来抑制肾癌生长。我们假设金雀异黄素可以激活一组肿瘤抑制 miRNA，从而通过两种不同的途径调节肾癌的进展。首先，金雀异黄素诱导的肿瘤抑制 miRNA 可以通过结合 mRNA 的 3' 非翻译区 (3'UTR) 来抑制癌基因的表达。第二个金雀异黄素诱导的肿瘤抑制 miRNA 可以通过与基因的 5' 上游区域结合来激活肿瘤抑制基因的转录，或通过与 mRNA 的 5' 非翻译区 (5'UTR) 结合来激活翻译。项目设计和方法：具体目标#1。旨在研究金雀异黄素是否通过激活抑癌 miRNA 介导的肾癌抑制作用。在此特定目标下，我们将用金​​雀异黄素处理肾癌细胞系，然后分析金雀异黄素是否通过激活抑癌miRNA来介导抑制肾癌细胞。为了研究这些肿瘤抑制 miRNA 的功能意义，我们将通过金雀异黄素处理激活这些 miRNA，然后分析这些 miRNA 的靶基因。将使用荧光素酶测定来分析 miRNA 介导的癌基因抑制或抑癌基因激活。分别构建 3'UTR 或 5'UTR 序列构建体。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细胞生长的变化。检测包括细胞增殖、流式细胞术、迁移、克隆存活、体外侵袭和细胞凋亡检测。具体目标#2：研究金雀异黄素介导肾癌细胞中肿瘤抑制 miRNA 激活的分子机制。我们将研究肾癌细胞中金雀异黄素介导的抑癌 microRNA 激活的表观遗传机制。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直接 DNA 测序分析 miRNA 推定启动子区域中 CpG 岛的低甲基化。我们将研究这些肿瘤抑制 miRNA 的激活机制是否是通过组蛋白乙酰化和染色质重塑的调节。将分析 miRNA 转染的肾癌细胞中组蛋白乙酰转移酶 (HAT) 和组蛋白脱乙酰酶 (HDAC) 的表达和活性。具体目标#3：研究金雀异黄素介导的肿瘤抑制 miRNA 的激活是否可以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 miRNA 激活是否可以抑制裸鼠肾肿瘤的生长和增殖。将肾癌细胞皮下（异种移植）或原位（肾脏）注射到用金雀异黄素治疗的裸鼠中，并与对照组相比测试其生长和进展。临床相关性：该项目的主要目标是研究金雀异黄酮通过激活抑癌 miRNA 抑制 RCC 的作用。这些信息可以带来治疗和管理肾细胞癌的新策略。由于本提案的目标是通过激活肿瘤抑制 miRNA 来研究饮食介导的 RCC 抑制作用，因此我们相信所提出的工作与退伍军人健康和 VA 使命高度相关。