MiR-142-3p通过Rictor/Akt/eNOS途径调控内皮细胞衰老在动脉粥样硬化发病机制中的作用研究

Vascular endothelial cells (VECs) senescence is thought to be an initial stage of atherosclerosis (AS). Rapamycin-insensitive companion of mTOR（Rictor）plays an essential role in VECs senescence. However, regulation of Rictor expression in VECs is not well documented. Our previous studies showed that the expression of miR-142-3p was markedly up-regulated in ox-LDL induced senescence of VECs. Bioinformatics analysis suggested that Rictor may be a target gene of miR-142-3p. MiR-142-3p could inhibit the expression of Rictor and phosphorylation of Akt. Knockdown of miR-142-3p increased the expression of Rictor and phosphorylation of Akt. We hypothesize that inhibition of miR-142-3p alleviates VECs senescence and atherogensis via enhancing Rictor expression and activating downstream Akt/eNOS pathway. In order to test this hypothesis, we will utilize dual luciferase reporter assay system to confirm that whether Rictor is one of the direct target of miR-142-3p in VECs. We will also analysis whether miR-142-3p participates in regulating the Rictor/Akt/eNOS signal pathway and senescence of VECs. ApoE-/- mice will be used to explore the effect of miR-142-3p on atherosclerosis development. This study will reveal the role of miR-142-3p in modulating VECs senescence and AS and bring new targets for prevention and treatment of AS.

血管内皮细胞（VECs）衰老是动脉粥样硬化（AS）形成的关键始动环节。Rictor基因是调节VECs衰老的关键因子，但其基因表达的调控机制不明。我们前期研究发现：ox-LDL诱导VECs衰老过程中miR-142-3p高表达，软件预测Rictor是miR-142-3p的靶基因。MiR-142-3p水平与Rictor蛋白表达和Akt磷酸化负相关。我们假设抑制miR-142-3p可直接靶向上调Rictor基因，激活下游Akt/eNOS途径，延缓VECs衰老和AS发生。本项目拟采用报告基因分析miR-142-3p与Rictor的靶向结合位点；细胞实验观察miR-142-3p对Rictor/Akt/eNOS途径活化和VECs衰老的调控作用；动物实验探讨miR-142-3p对ApoE-/-小鼠AS形成的影响。研究将揭示miR-142-3p调控VECs衰老和AS进展的作用及机制，并为AS防治提供新靶点。

动脉粥样硬化（AS）是全球范围内的主要致病和致死原因。血管内皮细胞（VECs）衰老是AS形成的关键始动环节。抑制血管内皮细胞衰老，保持内皮细胞活力是预防和治疗AS的重要靶点之一。Rictor基因是调节VECs衰老的关键因子，但其基因表达的调控机制尚未阐明。本研究通过构建氧化低密度脂蛋白（ox-LDL）诱导人主动脉内皮细胞（HAECs）衰老的细胞模型，证实了miR-142-3p在此过程中表达水平显著上调。下调miR-142-3p的表达水平可以抑制ox-LDL诱导的HAECs衰老和凋亡，促进细胞增生并削弱了氧化应激反应。生物信息学软件预测和双荧光素酶报告基因载体系统验证确认在HAECs中Rictor是miR-142-3p直接作用的靶基因之一。抑制miR-142-3p可以通过上调下游靶基因Rictor的表达并磷酸化激活Akt/eNOS信号途径，从而减轻HAECs衰老和凋亡。于在体动物层面，建立ApoE-/-小鼠AS模型，尾静脉注射miR-142-3p antagomir在整体动物水平下调miR-142-3p的表达，可以使AS小鼠体内血脂水平（甘油三脂、总胆固醇和低密度脂蛋白胆固醇）、血清炎性细胞因子（IL-6、IL-1b、TNF-a和MCP-1）水平明显降低，血清NO水平显著上调。同时使AS小鼠主动脉组织ROS水平、动脉壁脂质沉积和AS斑块面积显著降低。进一步机制研究显示在体内条件下抑制miR-142-3p的表达水平可以上调小鼠主动脉中Rictor蛋白表达并激活Akt/eNOS信号通路从而延缓主动脉内皮细胞衰老和减少内皮细胞凋亡。本研究结果有助于阐明AS进程中miRNAs调控VECs衰老和功能障碍的机制，为AS早期诊断与治疗提供新靶点。