TFEB的SUMO化修饰调控外泌体miR-296介导慢性肾衰竭血管平滑肌细胞表型转化及钙化的机制研究

血管平滑肌细胞(VSMCs)表型转化是慢性肾衰竭血管钙化的关键环节。外泌体miRNA在VSMCs表型转化中尤为重要。我们前期研究发现TFEB的SUMO化修饰调控巨噬细胞外泌体miR-296的分泌；miR-296在慢性肾衰竭血管钙化组织中高表达，生物信息学预测其靶基因是调控VSMCs表型转化的重要分子SRF。因此，我们推测TFEB的SUMO化修饰通过外泌体miR-296/SRF途径参与慢性肾衰竭血管钙化。本项目拟通过：①构建TFEB的SUMO化位点突变的巨噬细胞系，通过外泌体提取、外泌体与细胞共培养以及细胞非接触共培养等技术，阐明TFEB的SUMO化修饰调控外泌体miR-296参与VSMCs表型转化及钙化的机制；②应用在体小鼠模型，揭示TFEB的SUMO化修饰在慢性肾衰竭血管钙化中的作用，证实干预外泌体miR-296可延缓慢性肾衰竭血管钙化的进展，为慢性肾衰竭心血管疾病的防治提供新靶点。

Phenotypic transition of vascular smooth muscle cells (VSMCs) is a crucial event during vascular calcification of chronic renal failure. Exosome miRNA was reported to act an important role in VSMCs phenotypic transition. Our previous studies have found that TFEB SUMOylation can regulate the secretion of exosomes containing miR-296 in macrophage. The expression of miR-296 was dramatically upregulated in the calcified aortas of chronic renal failure; further computational analysis indicated that the target gene of miR-296 is SRF, which is a regulator of VSMCs phenotypic transition. Based on the above findings, we hypothesize that TFEB SUMOylation may regulate phenotypic transition and calcification of VSMCs through exosome miR-296/SRF pathway. We will address our hypothesis by performing the following studies: ①Construct macrophage cell lines that stably expressing TFEB with mutation of SUMOylation site using lentivirus-mediated gene delivery. Investigate the role of TFEB SUMOylation in regulating exosome miR-296 on VSMCs phenotypic transition and calcification based on new technologies, such as exosomes isolation, co-culture of exosomes and cells, and non-contact in vitro co-culture assay; ②Reveal the role of TFEB SUMOylation in vascular calcification of chronic renal failure on the animal models. Confirm the possibility of the retarding effect of exosome miR-296-based therapeutic strategies on progression of vascular calcification in chronic renal failure. The outcome of our study may provide new targets for treatment of cardiovascular complications in chronic renal failure.