miR-762通过线粒体调控心肌细胞坏死的机理研究

Cardiovascular disease is presently the leading cause of death in the world. Cardiomyocyte death plays an important role in heart disease. There are three types of cell death: apoptosis, autophagy, and necrosis. Apoptosis and autophagy were well studied in the past, mostly because they are programmed cell death. On the contrary, necrosis was considered to be a random, uncontrolled and accidental cell death that occurred in response to physicochemical insults. But recent studies have shown that necrosis is also regulated by signaling pathway. So necrosis can be classified as uncontrolled and controlled, controlled is also named programmed necrosis. In our previous study, we use high concentrations of H2O2 induced cardiomyocyte necrosis, microRNAs chips analysis were performed in mitochondria fraction. The results show that the levels of miR-762 was significantly increased in mitochondria fraction. This phenomenon prompted miR-762 may be closely related to mitochondria-mediated programmed necrosis pathway. Therefore, this study will focus on the function of miR-762 in programmed necrosis. At the cellular level, we will study the induced cardiomyocyte necrosis through overexpression or knockdown of miR-762. Further, we will explore whether the function of miR-762 is by targeting the mitochondrial genome encodes genes Cytb. In addition, we will construct miR-762 transgenic mice or knockdown endogenous miR-762 by Antagomir to overexpress or knockdown miR-762 in animal level, and further study the role of miR-762 in cardiac infarction induced by ischemia-reperfusion. These studies are expected to illustrate the role of miR-762 in the course of cardiomyocyte programmed necrosis, and miR-762 may serve as prevention or therapeutic target for heart disease.

心血管系统疾病是当今威胁人类健康的重大疾病，心肌细胞死亡在心脏疾病中扮演着重要的角色。过去认为细胞坏死是无序被动的过程，而最近的研究显示坏死也是受多种信号通路调控的主动过程。前期我们通过线粒体microRNA芯片检测发现高浓度H2O2诱导心肌细胞坏死时线粒体内miR-762的水平显著升高。这提示miR-762可能与线粒体介导的程序性坏死密切相关，因此我们将使用过表达（过表达腺病毒）或敲低（Antagomir）的方法，在细胞水平系统研究miR-762在心肌细胞坏死过程中的调控作用，以及研究其是否通过靶向线粒体编码基因Cytb发挥作用。此外，我们还将制作转基因小鼠和用Antagomir的方法在动物水平过表达或敲低miR-762，研究其在缺血再灌注诱导的心肌梗死中的作用。通过本研究可以阐明miR-762在心肌细胞坏死过程中的作用，将为心脏疾病的预防和治疗提供新的途径，具有重要的理论和实践意义

甲状腺功能亢进是一种常见的内分泌疾病，近年来其发病率有增高的趋势。心脏并发症是甲状腺功能亢进的常见并发症，严重时可发展为心衰，是威胁人类健康的原因。迄今为止，自噬是否在甲亢性心肌肥大中发挥作用以及miR-762在其中的作用都是未阐明的，本研究将探究甲亢性心肌肥大的分子机理。我们用腹腔注射甲状腺素（T4）的方法制作甲状腺功能亢进小鼠模型。7周后，进行形态学检测；RT-qPCR检测miR-762、ANP、β-MHC和Beclin-1的表达水平；蛋白印迹检测LC3、Beclin-1的表达水平；透视电镜和免疫荧光染色观察自噬体和自噬溶酶体数量。结果显示，与对照组相比，T4组心肌细胞表面积明显增加，结构紊乱并伴胶原纤维明显增多，ANP、β-MHC明显上调，LC3 II / LC3I、Beclin-1和自噬体数量明显增多，但miR-762明显下降。在新生鼠分离的心肌细胞中，用甲状腺素（T3）处理细胞，同时过表达或敲低miR-762,观察细胞肥大状况和心肌细胞自噬的改变。实验结果与动物实验基本一致。此外，与单纯T3处理相比，T3+miR-762 mimic组的心肌肥大与自噬活性明显缓解，相反，T3+miR-762 inhibitor组的心肌肥大与自噬活性明显加剧。综上所述，miR-762通过抑制其下游靶基因Beclin1，从而抑制心肌细胞自噬，进而调节甲状腺功能亢进引发的心肌肥大。