MicroRNAs in the Progression and Metastisis of Prostate Cancer

描述(申请人提供)：前列腺癌进展过程中的微RNA。这个项目的主要目标是研究一组microRNAs(MiRNAs)在前列腺癌进展中的作用。这个项目的基本原理是miRNAs通过抑制互补mRNA的翻译或引导序列特异性降解来调节基因表达，我们的初步结果提供了一个新的概念，即miRNAs和非编码双链RNAs也可以激活各种基因。基于这些新的观察结果，我们假设一组microRNAs的下调可以抑制肿瘤抑制基因或激活癌基因，而这些miRNAs的重新表达可以逆转这些效应，从而调节前列腺癌的进展。这些假设将通过追求以下三个具体目标来检验。具体目的1.研究一组miRNAs在人前列腺癌组织中的表达，并利用前列腺癌细胞株分析miRNAs是否能调节细胞的增殖和进展。基于我们的初步数据，我们已经确定了一组在前列腺癌中显著下调的miRNAs。我们将分析这些miRNAs在人类前列腺癌样本中的表达。我们将在前列腺癌细胞系中过表达一组特定的miRNAs，并将分别使用实时荧光聚合酶链式反应和蛋白质分析在mRNA和蛋白质水平上评估靶基因的调节。MiRNAs介导的癌基因抑制或肿瘤抑制基因的激活将分别使用3‘UTR或5’UTR序列构建的荧光素酶分析。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分析细胞生长的变化。检测方法包括细胞增殖、流式细胞术、迁移、克隆存活、体外侵袭和TUNEL-BEL法检测细胞凋亡。特定目的#2：研究前列腺癌细胞中microRNA失活的分子机制。我们将测试特定miRNAs通过表观遗传途径失活的假设。人类前列腺癌组织将被用于分析miRNAs的甲基化。MiRNAs可能启动子区的CpG甲基化将通过亚硫酸氢钠甲基化技术进行检测，并通过DNA直接测序进行确认。我们还将研究组蛋白乙酰化、染色质重塑和相关酶(组蛋白去乙酰酶和组蛋白乙酰转移酶)是否在控制特定miRNAs表达方面发挥作用。具体目的#3：研究microRNAs是否能抑制裸鼠模型中人前列腺癌移植瘤的生长和增殖。为了验证我们的体外结果，我们还将使用小鼠与人前列腺癌细胞异种移植的体内模型。

DESCRIPTION (provided by applicant): MicroRNAs in the progression of prostate cancer. The main goal of this project is to investigate the role of a set of microRNAs (miRNAs) in the progression of prostate cancer. The rationale of this project is that miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA, and our preliminary results have provided a novel concept that miRNAs and non-coding double stranded RNAs can also activate various genes. Based on these novel observations, we hypothesize that down-regulation of a set of microRNAs can inhibit tumor suppressor genes or activate oncogenes and re-expression of these miRNAs can reverse these effects thereby regulating prostate cancer progression. These hypotheses will be tested by pursuing the following three specific aims. Specific Aim # 1. To investigate the expression of a set of miRNAs in human prostate cancer tissues and also analyze whether miRNAs can regulate cell proliferation and progression using prostate cancer cell lines. Based on our preliminary data, we have identified a set of miRNAs that are significantly downregulated in prostate cancer. We will analyze the expression of these miRNAs in human prostate cancer samples. We will over-express a defined set of individual miRNAs in prostate cancer cell lines and the modulation of targeted genes will be evaluated at both the mRNA and protein levels using real-time PCR and Western analysis, respectively. The miRNAs-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. Changes in cell growth will be analyzed by monitoring proliferation, cell cycle distribution, apoptosis and in vitro invasion. Assays to be used include cell proliferation, flow cytometry, migration, clonogenic survival, in vitro invasion and TUNEL-based ELISA apoptosis assays. Specific Aim #2: To investigate the molecular mechanisms of microRNA inactivation in prostate cancer cells. We will test the hypothesis that specific miRNAs are inactivated through epigenetic pathways. Human prostate cancer tissues will be used for analysis of methylation of miRNAs. CpG methylation in putative promoter regions of miRNAs will be examined by sodium bisulfite methylation techniques and confirm by direct DNA sequencing. We will also investigate whether histone acetylation, chromatin remodeling and associated enzymes (histone deacetylases and histone acetyltransferases) play a role in controlling expression of specific miRNAs. Specific Aim #3: Investigate whether microRNAs can inhibit growth and proliferation of human xenograft prostate tumors in a nude mouse model. To validate our in vitro results, we will also use an in vivo model of mouse xenografts with human prostate cancer cells.