lncRNA-4321/miR-702-5p/Notch1轴向调控自噬减轻心肌缺血再灌注损伤

Despite appreciated as a cellular self-protection mechanism, autophagy functions as a double-edged sword on myocardial ischemia reperfusion injury(IRI). Based on previous publications and our preliminary data, we hypothesize that lncRNA-4321/miR-702-5p/Notch1 axial reduces myocardial IRI through regulation of autophagy. On the premise of clarifying the cardioprotective effect of autophagy, we will elucidate the biological effects of Notch1 on reducing myocardial IRI by enhancing autophagy in series experiments in vitro and in vivo, such as cell viability, infarct size and left heart function. Transgenic mediated compensation experiment will be utilized to demonstrate that miR-702-5p reduces the cardioprotective effect of autophagy by downregulation of Notch1 expression. Dual luciferase reporter gene assay and biotin-avidin pulldown experiments will be applied to investigate the molecular mechanism of lncRNA-4321 in regulating the expression of Notch1 depend on miR-702-5p, transgenic technology will be employed to investigate the physiological function of lncRNA-4321/miR-702-5p/Notch1 axial in reducing myocardial IRI through regulation of autophagy. In short, this study will reveal the molecular mechanism of lncRNA/miRNA in heart protection against myocardial IRI, and will provide a novel strategy for clinical myocardial protection.

自噬作为细胞自我保护机制，在心肌缺血再灌注损伤(IRI)中却发挥“双刃剑”效应。在总结前人与自身工作基础上，我们假设lncRNA-4321/miR-702-5p/Notch1轴向调控自噬，减轻心肌IRI。在多参数确认自噬性心肌保护作用前提下，从细胞活力、心梗面积、左心功能等经典指标，在体内外水平阐明Notch1通过增强自噬，减轻心肌IRI；利用转基因介导的回补实验，探讨miR-702-5p下调Notch1表达，削弱自噬性心肌保护作用；应用双荧光素酶报告基因与biotin-avidin pulldown技术研究lncRNA-4321以miR-702-5p为节点调控Notch1表达的分子机理，再分层论述lncRNA-4321/miR-702-5p/Notch1轴向调控自噬，减轻心肌IRI的生物效应，旨在揭示lncRNA/miRNA防治心肌IRI的分子机制，为临床心肌保护策略提供新的理论依据。

我们依据 “lncRNA-4321/miR-702-5p/Notch1轴向调控自噬，减轻心肌IRI”假设，在确认Notch1信号通路调控自噬性心肌保护作用基础上，构建miR-702-5p、lncRNA-4321腺病毒表达与干扰载体，感染H9c2mRFP-GFP-LC3稳转细胞株，建立缺血再灌注模型，Western blot检测N1ICD、p62、LC3Ⅱ/LC3Ⅰ蛋白表达，确认lncRNA-4321/miR-702-5p/Notch1在自噬中的调节作用，再检测心肌细胞存活率及心肌自噬改变，观察明确lncRNA-4321/miR-702-5p/Notch1轴向调控自噬的心肌保护效应，采用双荧光素酶报告基因技术探讨lncRNA-4321/miR-702-5p/Notch1之间的调控机制，我们实验数据表明增强lncRNA-4321表达，可以抑制miR-702-5p 活性，使Notch1表达上调，通过增强心肌自噬，提高心肌细胞存活率，从而减轻心肌缺血再灌注损伤。该课题已阐明lncRNA-4321/miR-702-5p/Notch1轴向调控自噬，减轻心肌IRI相关分子机制，并可作为新的靶点，为研制新型心肌保护药物奠定的理论与实验基础。