miRNA126调控AS发生发展过程中动脉内膜通透性的变化及其分子机制

The vascular endothelium is a semipermeable barrier between blood and vascular tissue. A major function of the endothelial cell is to serve as a barrier to fluid and solute flux across the blood vessel wall. Breakdown of this barrier leads to increased permeability and the development of atherosclerosis. It's well believed that abnormal protein expression and signal pathway changing play important roles in the development and formation of atherosclerosis. Among them, the expression of endothelial cell tight junction proteins (such as occludin, ZO1) is critical for intimal permeability changes. Mitogen-activated protein kinase (MAPK) pathway activation triggers a series of downstream genes (occludin, ZO1, etc.) expression, which results in the occurrence of atherosclerosis. Recent studies demonstrate that miRNA126 expression is down-regulated in the development and formation of atherosclerosis. Further target gene prediction also indicates that miRNA126 may targeted vascular endothelial growth factor (VEGF), which is an endothelial cell specific mitogen and chemotactic agent. Thus, these promising data strongly suggest that the effects of miRNA126 on the development and formation of atherosclerosis would mediate through VEGF-VEGFR-MAPK signal transduction pathway. The overall goal of this project is to elucidate the effect of miRNA126 on MAPK signaling transduction pathways and arterial intima permeability associated protein expression and activity in the development and formation of atherosclerosis in vivo and in vitro. This project will provide solid evidence and basis for development new drug target for atherosclerosis therapy and prevention through VEGF-VEGFR-MAPK signal transduction pathway.

血管内皮细胞是血管组织和血液之间的第一道屏障，其通透性改变是动脉粥样硬化(AS)发生发展过程中重要的一环。异常蛋白分子的表达和信号通路改变是AS形成的重要机制和研究热点。在AS形成过程中内皮细胞紧密连接蛋白(occludin、ZO1等)表达对内膜通透性变化起至关重要的作用。MAPK通路激活可以引发一系列的下游基因(occludin、ZO1等)表达变化，导致AS的发生。近期发现miRNA126在AS过程中异常低表达，预测提示VEGF为其潜在靶标之一,提示其可能通过VEGF-VEGFR-MAPK通路调控AS过程。本项目旨在整体动物模型（AS发生发展及形成）和体外细胞两个层面考察miRNA126对MAPK信号通路异常改变以及对动脉内膜通透性相关蛋白表达及活性的影响，力图从新的动态的角度在VEGF-VEGFR-MAPK信号通路上寻找新的抗AS作用药物靶点，为AS的预防提供实验依据。

血管内皮细胞是血管组织和血液之间的第一道屏障，其通透性改变是动脉粥样硬化(AS)发生发展过程中重要的一环。异常蛋白分子的表达和信号通路改变是AS形成的重要机制和研究热点。本项目检测动脉粥样硬化（AS）apoE-/-小鼠中miR126的表达，发现在AS中miR126出现明显的低表达。在正常小鼠中miR126 mimic能显著增加miR126表达，而antagomir明显降低miR126表达。研究发现在AS模型apoE-/-小鼠中caspase3活性、细胞运动及骨架蛋白表达明显升高，大分子荧光染料分析表明AS模型apoE-/-小鼠中动脉内膜通透性模型增加，miR126 antagomir进一步增加动脉内膜通透性，而miR126 mimic能减弱AS引起的动脉内膜通透性。同时发现MAPK信号通路在AS的形成过程中具有重要的功能，表明在AS形成过程中内皮细胞内膜通透性变化及MAPK信号通路起至关重要的作用。在此基础上研究miR126对palmitate诱导的HUVEC的细胞凋亡、caspase3 活性、ROS产生、炎性细胞因子TNF-α表达、NADPH 氧化酶及SirT3、细胞运动及骨架蛋白的表达的影响，并探讨MAPK通路在其中可能的作用机制，检测miR126的相应靶标靶基因，结果提示palmitate能够诱导HUVEC细胞发生凋亡，而miR126则可减轻palmitate诱导的细胞凋亡，改善palmitate诱导的HUVEC细胞活性氧产生及TNF-α表达，调节palmitate对HUVEC 中NADPH 氧化酶、SirT3蛋白表达的影响。同时发现miR126可改善palmitate诱导的HUVEC的运动，下调相关蛋白MLCK表达及MLC、MYPT1的磷酸化水平。进一步研究发现miR126直接作用的靶基因，miR126对palmitate诱导的HUVEC 的调控作用与MAPK 信号通路有关。从而明确miR126对HUVEC的保护作用，揭示miR126发挥作用的可能分子机制，项目在整体动物模型和体外细胞两个层面考察miRNA126对相关信号通路异常改变以及对动脉内膜通透性相关蛋白表达及活性的影响，力图从新的角度在MAPK信号通路上寻找新的抗AS作用药物靶点，为AS的预防提供实验依据。