Role of MicroRNAs in Ischemic Tolerance

描述（由申请人提供）：缺血耐受是一种亚致死性缺血损伤（预处理）诱导内源性保护机制的现象，该机制可减轻后续更严重缺血损伤的影响。我们使用大鼠和小鼠体内和体外的缺血耐受模型来描述这些内源性系统中神经保护的多种效应物。最近，我们使用DNA微阵列检测小鼠大脑中基因表达的变化，这些小鼠大脑受到短暂缺血、损伤性缺血或耐受性大脑（预处理，然后用损伤性缺血进行挑战）诱导的预处理。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着下调。因此，缺血耐受的特征是基因表达受到抑制。阐明这种集中的转录抑制的分子机制是我们的目标。真核基因表达受microRNA调控，microRNA是一类新发现的小的非蛋白质编码RNA，可调控哺乳动物细胞中mRNA的翻译和染色质的活性。基于缺血耐受的特征，即基因表达的抑制，我们假设缺血耐受导致受调节的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，不同的microRNA子集在预处理，缺血和耐受小鼠大脑中受到调节。因此，为了充分研究microRNA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微RNA在预处理、缺血和耐受小鼠脑中的表达谱，2）确认在预处理、缺血和耐受小鼠脑中调节的微RNA的变化，并检查其时间表达，和3）检查调节的microRNA对靶蛋白表达和细胞存活的影响。这些研究是第一批检查响应于模拟缺血的miRNA的调节表达的研究之一，并且考虑到目前开发的短干扰RNA作为许多疾病的新型治疗剂，包括黄斑变性、哮喘、糖尿病、癌症、亨廷顿病和丙型肝炎感染，这些研究可为开发用于治疗或预防中风和脑缺血的类似策略提供理论基础。

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.