MicroRNAs in the Progression and Metastisis of Prostate Cancer

描述（由申请人提供）：前列腺癌进展中的MicroRNA。该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。该项目的基本原理是，miRNA通过抑制翻译或指导互补mRNA的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miRNA和非编码双链RNA也可以激活多种基因。基于这些新的观察结果，我们假设一组microRNA的下调可以抑制肿瘤抑制基因或激活癌基因，这些miRNA的重新表达可以逆转这些作用，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。具体目标#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系分析miRNAs是否可以调节细胞增殖和进展。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miRNA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定义的单个miRNA，并分别使用实时PCR和Western分析在mRNA和蛋白质水平上评估靶基因的调节。将分别使用3'UTR或5'UTR序列构建体通过荧光素酶测定来分析miRNA介导的癌基因抑制或肿瘤抑制基因激活。通过监测增殖、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。待使用的测定包括细胞增殖、流式细胞术、迁移、克隆形成存活、体外侵袭和基于TUNEL的ELISA凋亡测定。具体目标#2：研究前列腺癌细胞中microRNA失活的分子机制。我们将测试特定的miRNAs通过表观遗传途径失活的假设。人前列腺癌组织将用于分析miRNA的甲基化。将通过亚硫酸氢钠甲基化技术检查miRNA的推定启动子区域中的CpG甲基化，并通过直接DNA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（组蛋白去乙酰化酶和组蛋白乙酰转移酶）是否在控制特定miRNA表达中发挥作用。具体目标#3：研究microRNA是否可以在裸鼠模型中抑制人异种移植前列腺肿瘤的生长和增殖。为了验证我们的体外结果，我们还将使用具有人前列腺癌细胞的小鼠异种移植物的体内模型。 公共卫生相关性：这些实验的成功完成将证明特定microRNA在抑制前列腺癌生长中的功能作用，以及这些基因在前列腺癌中失活的机制。在未来，这些结果可能为前列腺癌的管理提供更好的策略。

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. PUBLIC HEALTH RELEVANCE: Successful completion of these experiments will demonstrate the functional role of specific microRNAs in the suppression of prostate cancer growth and also the mechanism of inactivation of these genes in prostate cancer. In the future, these results may provide better strategies for the management of prostate cancer.