mitoKATP介导抗心肌缺血-再灌注损伤的microRNA调控机制

It has been reported that microRNAs (miRNAs) play important roles in regulating the pathophysiologic processes of angiocardiopathy. Myocardium derived miRNAs significantly affect different diseases such as cardiomyogenesis, cardiac trauma and heart failure. Our previous studies showed that mitochondrial adenosine triphosphate-sensitive potassium channel (mitoKATP) efficiently reduced myocardial ischemia-reperfusion injury (MIRI), but the detailed anti-MIRI mechanisms of mitoKATP remains to be fully elucidated. The present study is to ① use langendorff ischemic/reperfusion model of adult rat heart to filter out specifically expressed miRNAs in four different mitoKATP states (normal, ischemic, open and blocked) after specific pattern analysis of miRNA and mRNA expression in rat hearts; ② To identify the anti-MIRI miRNAs by constructing their regulatory networks at the help of bioinformatics; ③ Gain- and loss-of-function experiments in vitro, ex vivo and in vivo will be designed to identify their effects on MIRI. In the hope of providing theoretical bases for clinical researches as well as affording new avenues for the prevention and treatment of MIRI, this project focuses on whether the anti-MIRI effect of mitoKATP is contributed by miRNAs and how do miRNAs specificly affect MIRI.

microRNA（miRNA）在心血管疾病的发生发展中发挥着重要的调控作用。心肌miRNA参与调控心脏损伤、心力衰竭等病理生理过程。前期研究发现线粒体ATP敏感性钾通道（mitoKATP）开放可有效减轻心肌缺血-再灌注损伤（MIRI），但具体机制不明。本项目拟：① 采用大鼠心脏缺血-再灌注模型，对mitoKATP正常、缺血、mitoKATP开放与阻断四种状态心肌细胞miRNA和基因表达模式进行分析，筛选并鉴定mitoKATP不同状态下的特征miRNA；② 辅以生物信息学等方法分析特征miRNA介导的调控网络及关键节点；③ 通过功能获得/缺失型实验，从心肌细胞、离体心脏和在体动物水平，验证miRNA在mitoKATP抗 MIRI中的作用。本课题重点关注mitoKATP介导的抗MIRI与miRNA调控的关系，同时明确相关特征miRNA的作用效果，为临床MIRI的防治研究提供新的理论依据和策略。

本项目在国家自然科学基金委员会的资助下，借助microRNA测序技术高通量筛选了缺血-再灌注大鼠和二氮嗪后处理大鼠心脏差异表达的microRNA；选取表达量高，且在各组间表达具有显著性差异的18个miRNA，利用qRT-PCR验证了包括rno-miR10a等9个miRNA在各组间具有显著性差异；通过Gene Ontology、KEGG Pathway等数据库分析上述9个miRNA及可能调控的mRNA的调控通路及网络，最终确定深入观察miR-10a的心肌保护作用及可能的分子机制：①利用gain-/loss-of-function实验，在细胞、离体心脏及在体心脏的缺血-再灌注模型均证实miR-10a可加重心肌缺血-再灌注损伤，而其抑制剂具有心肌保护作用；②pull-down实验和luciferase reporter gene实验均证实IRE1是miR-10a的靶基因；③miR-10a可在缺氧-复氧心肌细胞模型和活体动物心脏中通过IRE1调节内质网应激反应，从而保护缺血-再灌注损伤的心肌。该研究为mitoKATP开放剂二氮嗪对缺血-再灌注心肌的保护作用提供了分子机制方面的实验依据，并且提示miR-10a可作为潜在的心肌保护分子。