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）是我国退伍军人中最常见的恶性肿瘤之一。RCC治疗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个问题，利用饮食方法的管理RCC使用在体外和体内模型。该项目的基本原理是，最近的研究表明，使用体外和体内模型，饮食对miRNAs的调节具有显著影响。然而，这种研究缺乏肾癌。研究目的：本项目的主要目的是研究染料木黄酮是否可以通过激活肿瘤抑制microRNA（miRNAs）来抑制肾癌的生长，使用体外和体内模型。我们假设染料木黄酮可以激活一组肿瘤抑制miRNAs，从而通过两种不同的途径调节肾癌的进展。首先，染料木黄酮诱导的肿瘤抑制miRNAs可以通过与mRNA的3 '非翻译区（3'UTR）结合来抑制癌基因的表达。第二，染料木黄酮诱导的肿瘤抑制miRNAs可以通过结合到基因的5 '上游区域来激活肿瘤抑制基因的转录，或者通过结合到mRNA的5'非翻译区（5'UTR）来激活翻译。项目设计和方法：具体目标#1。探讨染料木黄酮是否通过激活肿瘤抑制基因miRNAs介导对肾癌的抑制作用。在这个特定的目标下，我们将用染料木黄酮处理肾癌细胞系，然后分析染料木黄酮介导的肾癌细胞抑制是否是通过激活肿瘤抑制miRNA。为了研究这些抑癌miRNAs的功能意义，我们将通过染料木黄酮激活这些miRNAs，然后分析这些miRNAs的靶基因，分别使用3'UTR或5'UTR序列构建体通过荧光素酶分析来分析miRNAs介导的癌基因抑制或抑癌基因激活。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。分析包括细胞增殖、流式细胞术、迁移、克隆存活、体外侵袭和凋亡分析。具体目标#2：研究染料木黄酮介导的肾癌细胞中肿瘤抑制miRNA活化的分子机制。我们将研究在肾癌细胞中染料木黄酮介导的肿瘤抑制microRNA激活的表观遗传机制。我们将用染料木黄酮处理肾癌细胞，然后使用亚硫酸氢钠甲基化技术和直接DNA测序分析推定的miRNA启动子区域中CpG岛的低甲基化。我们将研究这些肿瘤抑制基因miRNAs的激活机制是否是通过调节组蛋白乙酰化和染色质重塑来实现的。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：在裸鼠模型中研究染料木黄酮介导的肿瘤抑制miRNAs的激活是否可以抑制肾肿瘤的生长和增殖。我们将研究染料木黄酮介导的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（异种移植物）或原位（肾脏）注射到用染料木黄酮处理的裸鼠中，并与对照相比测试其生长和进展。临床相关性：本课题的主要目的是研究染料木黄酮通过激活肿瘤抑制miRNA在抑制肾细胞癌中的作用。这些信息可以导致新的策略，治疗和管理RCC。由于该提案的目标是通过激活肿瘤抑制基因miRNAs来研究饮食介导的RCC抑制，因此我们认为所提出的工作与退伍军人健康和VA使命高度相关。