ox-LDL致血管内皮损伤中线粒体miRNA动态变化的意义及其转运机制的研究

Mitochondrial damage is one of the most important initial event in atherosclerosis (AS), which is caused by the injury of endothelial cells induced by ox-LDL. Mitochondrial miRs which are located in mitochondria and mainly encoded by nuclear genome are the direct regulators of mitochondrial function. However, the transport mechanism, functional regulation and abnormalities in pathological conditions of mitochondrial miRs are still poorly understood until now. Our preliminary study indicated that ox-LDL could cause abnormalities of expression and/or distribution of mitochondrial miR in human umbilical vein endothelial cells (HUVEC). These abnormalities were closely related to apoptosis and mitochondrial function. Nevertheless, their dynamic characteristics, their effects on endothelial injury induced by ox-LDL and in particular the mechanism of mitochondrial miR transport has not yet been reported at present. So, we plan to study the overall characteristics and transport mechanisms of mitochondrial miR in endothelial cells. Then, it is planned to reveal the dynamic changes of mitochondrial miR in endothelial cells treated with ox-LDL and the effects of mitochondrial miR on endothelial injury induced by ox-LDL as well as their mechanism of action. Finally, we hope to uncover the dynamic changes, significance and transport mechanism of miRNA in mitochondria in the injury of endothelial cells induced by ox-LDL. The proposal will help us to understand the mechanisms of endothelial injury induced by ox-LDL from the perspective of mitochondrial miR, expand the understanding of the regulation of mitochondrial function and provide a new means for improving the pathological effects of ox-LDL on endothelial cells which eventually lead to atherosclerosis development.

线粒体受损是Ox-LDL损伤内皮细胞引起动脉粥样硬化（AS）的关键始动事件之一，而位于细胞线粒体内的主要由细胞核编码的线粒体miR则是线粒体功能的直接调控者，目前对其转运机制、功能调控和病理条件下的异常等仍知之甚少。我们前期研究表明ox-LDL能使内皮细胞线粒体miR的含量和/或分布显著异常，这些异常与线粒体功能和凋亡密切相关，但它们的动态变化特征、在ox-LDL致内皮损伤中的意义、特别是线粒体miR的转运机制等问题迄今仍未见报道。因此，本申请将利用miR-Seq、蛋白质组、亲和纯化以及报告基因等功能研究技术，揭示内皮细胞线粒体miR的整体特征和转运机制，阐明线粒体miR在ox-LDL作用下的动态变化及其在ox-LDL致内皮损伤中的作用和机制。本项目将有助于从线粒体miR角度理解ox-LDL损伤血管内皮的机制，拓展对线粒体功能调控的认识，为改善ox-LDL损伤内皮和致AS的作用提供新的手段

线粒体受损是Ox-LDL损伤内皮细胞引起动脉粥样硬化（AS）的关键始动事件之一，而位于细胞线粒体内的主要由细胞核编码的线粒体miRNA则是线粒体功能的直接调控者，目前对其转运机制、功能调控和病理条件下的异常等问题仍知之甚少。本项目按照计划任务要求，完成了项目预期的主要研究目标和研究内容，获得了项目计划的研究成果。具体来说，利用二代测序技术揭示了ox-LDL处理HUVEC不同时间点线粒体miRNA的动态变化，筛选出了与ox-LDL病理作用关系密切的关键差异线粒体miRNA miR-381-3p；发现了与线粒体miRNA特异结合的蛋白MSI2，并证实MSI2能结合具有UAG/UAC短序列的miRNA，在这些miRNA的线粒体转运中发挥关键作用；揭示了关键差异线粒体miRNA miR-381-3p能通过靶基因NFIA和LRP6及靶基因的下游分子p53, p21和CCND1, c-Myc组成的调控网络促进ox-LDL诱导的内皮细胞凋亡和增殖抑制。项目成果的科学意义在于首次系统地揭示了在ox-LDL诱导内皮功能障碍过程中miRNA线粒体分布的动态变化。发现很多因为总表达量不变而被忽视的miRNAs也可能通过线粒体-细胞质分布的变化来调节信号网络，在ox-LDL诱导的内皮细胞损伤中发挥重要作用。miR-381-3p就是这些miRNA的典型代表。此外，还首次发现MSI2蛋白能与具有UAG/UAC短序列的miRNA结合，促进它们向线粒体转运。项目的研究结果为揭示miRNA的线粒体转运机制提供了新的线索，拓展和加深了对miRNA调控内皮稳态作用的认识。