内皮祖细胞来源的外泌体通过has-miR-1246/ELF5和has-miR-1290/SP1信号通路介导心脏成纤维细胞向内皮细胞转化的作用机制研究

Myocardial fibrosis is the key pathological feature of heart failure, therefore, studying on the pathogenesis and therapy of myocardial fibrosis is of great significance for improving patients treatments rate. This study investgated endothelial progenitor cells derived exosomes which plays the role of reversing myocardial fibrosis progression by carrying the microRNA regulating fiber cells to differentiate into endothelial cells. Bioinformatic analysis showed that hsa-miR-1246 and hsa-miR-1290 binding sites were found in the 3'UTR of ELF5 and SP1, respectively. We also predict that the transcription factor binding sites of ELF5 and SP1 were found in the promoter position of CD31. Therefore, we hypothesize that hsa-miR-1246 and hsa-miR-1290 are the upstream regulators of ELF5 and SP1, repectively, and ELF5 and SP1 could regulate the transcriptional activity of CD31. Hsa-miR-1246 and ELF5, hsa-miR-1290 and SP1 communicate with CD 31 and comprise a microRNA-based regulation network, it is crucial for mesenchymal-endothelial transition which alleviated deterioration of cardiac function. We would investigate the molecular mechanisms of hsa-miR-1246/ELF5 and hsa-miR-1290/SP1 regulation pathways on cell and animal level. Our project will provide new information for the therapy of endothelial progenitor cells derived exosomes on angiogenesis through reversing mesenchymal-endothelial transition.

心肌纤维化是心力衰竭的关键病理特征，探明心肌纤维化的发病机制对于患者的治疗具有重要意义。本研究探讨内皮祖细胞来源的外泌体能够通过介导microRNA调控成纤维细胞向内皮细胞分化，生物信息学预测hsa-miR-1246和hsa-miR-1290分别与转录因子ELF5和SP1存在结合位点，同时ELF5和SP1与内皮细胞分化标志物CD31的启动子区域皆存在结合位点。故提出假说：EPCs源性外泌体中hsa-miR-1246和hsa-miR-1290可以分别调控其下游靶基因ELF5和SP1的表达，ELF5和SP1可以同时调控CD31的转录与翻译，从而构成hsa-miR-1246/ELF5和hsa-miR-1290/SP1调控通路，发挥逆转心肌纤维化进展的作用。因此，我们将从细胞和动物整体水平阐明这两条通路的调控功能，为内皮祖细胞来源的外泌体促进间质内皮转化和血管新生奠定了新的理论基础。

心肌梗死是导致全世界发病率和死亡率高的主要原因。内皮祖细胞来源的外泌体可有效缓解心肌梗死，然而其中的具体机制尚不清晰。本研究旨在确定内皮祖细胞来源的外泌体miR-1246和miR-1290对心肌梗死造成的损伤的保护作用，并探讨其潜在的分子机制。外泌体从内皮祖细胞中提取，通过qRT-PCR检测基因表达水平，western blot和免疫荧光染色检测蛋白表达水平。通过小管形成实验和PKH67免疫荧光染色法检测人心脏成纤维细胞的血管生成和增殖。荧光素酶报告实验、CHIP和EMSA检测确定了miR-1246/1290与靶基因(EFL5和SP1)之间的相互作用。并在心肌梗死大鼠模型中评估了miR-1246/1290对心肌梗死的保护作用。内皮细胞来源的外泌体显著上调了miR-1246和miR-1290的表达，促进了成纤维细胞-内皮细胞转化，促进血管生成和增殖。过表达miR-1246或miR-1290促进成纤维细胞-内皮细胞转化和血管生成，而敲低miR-1246或miR-1290则显示相反的作用。从机制上讲，内皮祖细胞来源的外泌体的miR-1246和miR-1290通过靶向基因的启动子区域诱导了ELF5和SP1的上调。在成纤维细胞中，过表达ELF5和SP1增强了成纤维细胞-内皮细胞转化和血管生成，而敲除EFL5和SP1则发挥相反的作用。ELF5和SP1均可与CD31的启动子结合，导致CD31在成纤维细胞中上调。此外，动物体内研究表明，高表达miR-1246或miR-1290的内皮祖细胞来源的外泌体可减轻心肌梗死诱导的大鼠心脏损伤，导致ELF5、SP1和CD31表达增加，抑制心肌组织中α-SMA的表达。总之，我们的研究表明，内皮祖细胞来源的外泌体中miR-1246和miR-1290可促进心肌梗死的体内和体外血管生成，促进心肌梗死后心脏损伤和心肌纤维化的改善。