miR-31在心肌保护中的作用及机制

Ischemia preconditioning is an important and intrinsic mechanism for protection of myocardial ischemia and reperfusion injury. It has been demonstrated miRNA is involved in the regulation of myocardial injury and protection.We wish to find efficient target from miRNA of preconditioning myocardium to treat myocardial ischemia and reperfusion injury. In previous study, we performed miRNA micorarray analysis on myocardial samples both after ischemia preconditioning and several time points within 71.5 hours after preconditioning with control tissue from sham-operated mice. The data showed a distinct miRNA expression signature and indicated that miR-31 expression persistly reduced. This miR-31 expression pattern was confirmed by quantitative PCR. We also performed quantitative PCR to detect miR-31 expression on myocardial samples after ischemia and reperfusion injury, then we found miR-31 expression increased. In pre-experiments, we found that inhibition of miR-31 presented a notably cardioprotective effect in mice. The potential targets of miR-31 analyzed by the bioinformatic program TargetScan indicated many genes related to cardiac injury and protection. We plan to confirm the cardioprotective effect by inhibiton of miR-31 in the cardiac myocytes; confirm the target genes of miR-31 by applying experiments, such as reporter gene system; clarify which genes are regulated to realize its cardioprotective effect by experiments, such as recvovery experiment. We will find the miR-31 from the preconditioning myocardium of mice, define its role in mice and cardiac myocytes, and clarify its molecular mechanism. We will supply the new target for drug research and development to treat ischemia and reperfusion injury after this project.

缺血预适应(IPre)是保护缺血再灌注损伤（I/R）心肌的重要内源性机制，已证实miRNA参与心肌缺血损伤及保护的调控，我们从Ipre心肌miRNA中寻找治疗心肌I/R的高效靶点。前期我们利用miRNA microarray方法研究小鼠心肌IPre及之后71.5h内多个时间点miRNA表达谱，发现该过程miR-31表达持续降低，qPCR确定；而qPCR发现小鼠心肌I/R之后miR-31表达增高。预实验中抑制小鼠miR-31具有显著心肌保护作用；预测程序发现miR-31调控众多与心肌受损、保护相关的基因。我们拟再从细胞水平明确抑制miR-31的心肌保护作用；应用报告基因等实验确定miR-31的调控靶基因；应用回复实验等阐明其通过调控哪些靶基因来实现心肌保护作用。本项目从IPre心肌发现miR-31，从整体及细胞水平上明确其作用，从分子水平上阐明其机制，为防治I/R药物研发提供新靶点。

心肌缺血再灌注损伤是心肌梗塞后和体外循环心脏手术心肌细胞必须经历的病理生理过程。不同于大多数研究者从蛋白质组学入手，我们在miRNA领域进行探索，期望找到心肌保护心的特异高效靶点。结合前期研究结果和已发表文献，我们发现miR-31在心肌缺血再灌注损伤时表达异常增高，而在缺血预适应阶段表达降低。因此，我们以miR-31作为研究靶点，明确其在心肌缺血再灌注损伤过程中的作用及机制。我们采用离体心肌细胞经历缺氧再给养和小鼠在体心肌经历缺血再灌注损伤实验模型。通过anti-miR-31转染心肌细胞，发现下调miR-31后能减轻心肌细胞损伤（检测细胞活性、LDH、MDA和SOD等）；通过在体转染anti-miR-31下调miR-31,发现下调miR-31明显减小心肌缺血再灌注损伤后（心梗模型）的心梗面积。从而明确了下调miR-31具有心肌保护效应。应用TARGETSCAN预测，我们发现蛋白激酶Cε（PKCε）可能是miR-31靶基因。通过miR-31 mimics上调miR-31后，抑制了PKCε蛋白表达；而通过anti-miR-31下调miR-31后，则增加了PKCε蛋白表达。采用Luciferase实验，我们证明了miR-31能与PKCε 3’-UTR结合。综合起来，我们证明了PKCε是miR-31的靶蛋白。我们进一步证明，PKCε是否是miR-31的功能靶蛋白。用siRNA抑制PKCε后，破坏了miR-31下调所产生的心肌细胞保护效应。从而证明了下调miR-31所产生的心肌保护效应是通过PKCε实验的。我们进一步验证PKCε的下游分子，发现anti-miR-31下调miR-31后，NF-κB明显激活。而用siRNA抑制PKCε后，NF-κB激活受到抑制，这证明了NF-κB是PKCε的下游分子。而用Adv-dnIκBα抑制NF-κB激活后，则破坏了miR-31下调所产生的心肌保护效应。综而言之，NF-κB是PKCε的下游分子，并介导了下调miR-31所产生的心肌保护效应。本课题是从缺血再灌注损伤和缺血预适应心肌找到了miR-31这个靶点，发现抑制miR-31具有心肌保护效应，并明确miR-31通过调控PKCε/NF-κB途径来实现其保护作用，该研究为心肌缺血再灌注损伤防治药物的开发提供了新的靶点。