miR-499a-5p调控靶基因FOXO4对心肌缺血再灌注损伤线粒体凋亡的作用及其机制研究

Myocardial ischemia-reperfusion injury is the leading cause for perioperative cardiac events. During this process, mitochondrial apoptosis accounts for its main mechanism, and FOXO4 transcription gene takes an important part. Studies found that miR-499a-5p inhibited mitochondrial apoptosis and alleviated myocardial infraction. Our preliminary experiments showed excessive apoptosis with miR-499a-5p downregulation and FOXO4 upregulation in cardiomyocyte exposed to hypoxia-reoxygenation. Therefore, we hypothesize that miR-499a-5p protects the heart against ischemia-reperfusion injury by regulating mitochondrial apoptosis via targeting FOXO4 dependent mechanisms. To test this hypothesis, we evaluate the role of FOXO4 and miR-499a-5p in regulation of mitochondrial apoptosis under the in vivo model of myocardial ischemia-reperfusion injury in rats and in vitro model in primary neonatal rat cardiomyocytes by using various methods including confocal microscopy, qRT-PCR, Western Blot, and RNA interference. Furthermore, we investigate the binding site of miR-499a-5p targeting FOXO4 gene and the underline mechanisms for regulating mitochondrial apoptosis by using various methods including Luciferase gene detection, gene silence, and lentivirus overexpression. We aim to demonstrate that miR-499a-5p provides cardioprotection by inhibition of mitochondrial apoptosis via FOXO4 dependent mechanisms in order to provide new ideas and theoretical basis for perioperative cardioprotection.

心肌缺血再灌注损伤是围术期发生心血管事件的主要原因之一，线粒体凋亡是其重要机制，FOXO4转录调控基因参与了此过程。有研究发现miR-499a-5p抑制线粒体凋亡和减轻心肌梗死。我们的预实验提示：心肌缺氧复氧时，心肌细胞发生凋亡，miR-499a-5p表达降低，FOXO4表达增加。为此，我们推测miR-499a-5p可能通过靶基因FOXO4来调控线粒体凋亡而发挥心肌保护作用的。为验证该假说，我们通过细胞缺氧复氧和在体缺血再灌注模型，采用共聚焦显微镜、qRT-PCR、Western blot、RNA干扰等方法，从分子、细胞和整体分别来明确miR-499a-5p和FOXO4对线粒体凋亡的调控作用，同时进一步通过双荧光素酶靶基因检测、基因沉默、慢病毒过表达等技术，深入研究miR-499a-5p与靶基因FOXO4的结合位点，以及该靶向作用对线粒体凋亡的调控机制，为围手术期心肌保护提供新的理论依据。

心肌缺血再灌注损伤是围术期发生心血管事件的主要原因之一。大量研究表明FOXO4参与各类生理活动的调控，包括：细胞周期，细胞代谢和细胞凋亡。在生理条件下，miR-499大量分布于心脏，其阻断后能增强凋亡，并增加心肌梗死面积。本研究旨在探讨miR-499-5p和FoXO4信号通路参与心肌缺血再灌注损伤的机制以及右美托咪定预处理是否通过调控miR-499-5p和FoXO4信号发挥心肌保护作用。我们的研究结果表明：FoxO4的mRNA和蛋白水平在H9c2细胞H/R损伤处理早期显著增加，shRNA-FoXO4 转染H9c2细胞显著降低H/R损伤过程中Bim和Bax mRNA以及蛋白表达，增加Bcl-2 mRNA和蛋白表达，提高H9c2细胞生存率，降低线粒体膜电位和细胞凋亡率； Bim的过表达可以逆转shRNA-FoXO4对心肌细胞H/R损伤的保护作用。H9c2细胞H/R损伤处理晚期可以通过上调miR-499-5p调控NF-κB信号介导的炎症反应减轻心肌细胞H/R损伤，并且右美托咪定预处理可以通过α2受体调控miR-499-5p和FoXO4的表达减轻小鼠心肌缺血再灌注损伤。