Role of MicroRNAs in Ischemic Tolerance

描述(申请人提供)：缺血耐受是一种现象，在这种现象中，亚致死性缺血损伤，即预适应，诱导内源性保护机制，以减轻随后更严重的缺血性损伤的影响。我们在体内和体外使用了大鼠和小鼠的缺血耐受模型，来描述这些内源性系统中神经保护的多个效应因子。最近，我们使用DNA微阵列检测了小鼠脑内基因表达的变化，包括短暂的脑缺血、损伤性脑缺血或耐受脑(先预适应，再用损伤性脑缺血)。这些实验表明，损伤性缺血上调基因表达，而缺血耐受导致基因表达显著下调。因此，缺血耐受的标志是基因表达受抑。阐明这种集中转录抑制的分子机制(S)是我们的目标。真核基因的表达受microRNAs的调控，microRNAs是新近发现的一类小的非蛋白编码RNA，调节哺乳动物细胞中的mRNA翻译和染色质活性。基于缺血耐受的特征，一种基因表达的抑制，我们假设缺血耐受导致调节的microRNA表达，进而导致受保护的表型。为了支持这一假设，我们的初步数据显示，在预适应、缺血和耐受的小鼠脑中，microRNAs的不同亚群受到调节。因此，为了全面研究microRNAs在缺血耐受中的作用，我们提出了以下具体目标：1)通过微阵列分析建立预适应、缺血和耐受小鼠脑中microRNAs的表达谱；2)确认在预适应、缺血和耐受小鼠脑中调节的microRNAs的变化，并检测其在时间上的表达；以及3)检测调控的microRNAs对靶蛋白表达和细胞存活的影响。这些研究是第一批检测miRNAs对模拟缺血反应的调控表达的研究之一，鉴于目前短干扰RNAs作为一系列疾病的新治疗剂的发展，包括黄斑变性、哮喘、糖尿病、癌症、亨廷顿氏症和丙型肝炎感染，这些研究可能为开发类似的治疗或预防中风和脑缺血的策略提供理论基础。

DESCRIPTION (provided by applicant): Ischemic tolerance is a phenomenon whereby a sub-lethal ischemic injury, preconditioning, induces endogenous protective mechanisms that lessen the impact of a subsequent, more severe ischemic insult. We have used modeled ischemic tolerance in rat and mouse, both in vivo and in vitro, to describe multiple effectors of neuroprotection in these endogenous systems. Recently, we used DNA microarray to examine changes in gene expression in mouse brains subjected to preconditioning induced by a brief duration of ischemia, injurious ischemia, or a tolerant brain (preconditioned, then challenged with injurious ischemia). These experiments revealed that injurious ischemia up-regulated gene expression, while ischemic tolerance resulted in significant down-regulation of gene expression. Thus the signature of ischemic tolerance is that of suppressed gene expression. Elucidating the molecular mechanism(s) that underlie this focused transcriptional suppression is our goal. Eukaryotic gene expression is regulated by microRNAs, a newly identified class of small non-protein coding RNAs that regulate mRNA translation and chromatin activity in mammalian cells. Based on the signature of ischemic tolerance, a suppression of gene expression, we hypothesize that ischemic tolerance leads to regulated microRNA expression that in turn leads to the protected phenotype. In support of this hypothesis, our preliminary data show that distinct subsets of microRNAs are regulated in preconditioned, ischemic, and tolerant mouse brain. Thus to fully examine a role for microRNAs in ischemic tolerance, we propose the following specific aims: 1) establish the expression profile of microRNAs in preconditioned, ischemic, and tolerant mouse brain by microarray analysis, 2) confirm changes in, and examine the temporal expression of, microRNAs regulated in preconditioned, ischemic, and tolerant mouse brain, and 3) examine the effect of regulated microRNAs on target protein expression and cell survival. These studies are among the first to examine the regulated expression of miRNAs in response to modeled ischemia, and given the current development of short, interference RNAs as novel therapeutic agents for a number of diseases including macular degeneration, asthma, diabetes, cancer, Huntington's, and Hepatitis C infection, these studies may provide rationale for the development of similar strategies for the treatment or prevention of stroke and brain ischemia.