基于miR-30c-2-3p调控内质网应激相关IRE1-XBP1信号通路研究缺血预处理和心肌缺血-再灌注损伤的机制

The RE1-XBP1 signaling pathway is an originating link of the intracellular endoplasmic reticulum stress, and decides the fate of cells. Our previous experiments confirm that different cardiomyocyte outcomes induced by hypoxic preconditioning and hypoxia/reoxygenation injury are closely related to the degree of IRE1-XBP1 signaling pathway openness, which is negatively correlated with endogenous miR-30c-2-3p expression level. By the two-level studies from in vitro and in vivo trials, aims of this project to assess effects of ischemia-reperfusion injury and ischemic preconditioning on the myocardial expressions of miR-30c-2, XBP1 mRNA and XBP1 protein by the real-time quantitative polymerase chain reaction and Western blotting techniques. Combined with the classic indicators of myocardial apoptosis and infarct size, we evaluate the roles of endoplasmic reticulum stress related-IRE1-XBP1 signaling pathway in both myocardial ischemia-reperfusion injury and ischemic preconditioning, and modulation of endogenerous miR-30c-2 on the endoplasmic reticulum stress related-IRE1-XBP1 signaling pathway. At the same time, by in vitro and in vivo transfection of exogenerous miR-30c-2 inhibitor to change myocardial expression of endogenerous miR-30c-2, we measure cardioprotective effect produced by activating endoplasmic reticulum stress related-IRE1-XBP1 signaling pathway. Therefore, this project is expected to achieve a breakthrough in the mechanisms of both myocardial ischemia-reperfusion injury and cardioprotective effect of ischemic preconditioning.

IRE1-XBP1信号通路是内质网应激的始发环节，并决定细胞的命运。我们的前期实验证实，缺氧预处理和缺氧/复氧损伤所致的迥异心肌细胞结局均与IRE1-XBP1信号通路的开放程度密切相关，并且该信号通路的开放程度与内源性miR-30c-2-3p表达量呈明显负相关。本项目从离体和在体实验两个层面，通过检测缺血-再灌注损伤(IRI)和缺血预处理(IPC)对心肌miR-30c-2、XBP1 mRNA和XBP1表达的影响，结合细胞凋亡和心肌梗死面积等指标，评价内质网应激相关IRE1-XBP1信号通路在心肌IRI和IPC中的地位以及内源性miR-30c-2-3p对其的调控作用。同时，通过在体和离体转染外源性抑制剂改变miR-30c-2-3p表达，以确定内质网应激相关IRE1-XBP1信号通路激活所产生的心肌保护效应。因此，本项目有望在心肌IRI发生机制和IPC心肌保护机制方面取得突破性进展。

本项目是从离体和在体实验两个层面，系统评价了缺血-再灌注损伤(IRI)和缺血预处理对心肌miR-30c-2、XBP1 mRNA和XBP1表达的影响，旨在揭示miR-30c-2-3p调控内质网应激相关IRE1-XBP1信号通路在缺血预处理心肌保护和心肌缺血-再灌注损伤发生机制中的作用。通过系列的实验工作，我们完成了本项目研究设计的全部内容，并达到了预期的目标。.离体细胞实验包括两个部分：①XBP1和miRNA-30c-2-3P在心肌细胞缺氧预处理保护作用中的地位及miRNA-30c-2-3p对XBP1的靶向调控关系；②miRNA-30c-2-3p调控XBP1对心肌细胞缺氧/复氧损伤保护作用及其机制的实验研究。主要结果如下：①在心肌细胞内miRNA-30c-2-3p和XBP1具有靶向调控关系。低表达miRNA-30c-2-3p进而增加XBP1表达是缺血预处理产生心肌保护作用的重要内在机制之一。②通过抑制内源性miRNA-30c-2-3p可增加XBP1表达，对离体心肌细胞缺氧/复氧损伤产生明显的保护作用，降低细胞凋亡发生率。③BiP表达可受到miRNA-30c-2-3p对XBP1调控作用的影响，BiP可能是XBP1产生心肌保护效应的下游因子。.在体实验包括两个部分：①缺血-再灌注损伤、缺血预处理和miR-30c-2-3p抑制剂转染预处理对心肌转归影响的实验研究；②缺血-再灌注损伤、缺血预处理和miR-30c-2-3p抑制剂转染预处理对心肌内质网应激相关IRE1-XBP1信号通路和细胞凋亡影响的实验研究。主要结果如下：①心肌细胞miR-30c-2-3p表达对缺血-再灌注中心肌内质网应激相关IRE1-XBP1信号通路状态具有重要调控作用。②通过抑制miR-30c-2-3p表达而调控内质网应激相关IRE1-XBP1信号通路的激活状态是缺血预处理发挥心肌保护作用的主要机制之一。