microRNA-214在干细胞向平滑肌细胞分化和血管重构中的表观遗传机制

This research project will establish stable expression of miR-214 precursor or antisense mouse ES cell lines by lentiviral vectors on the basis of preliminary studies. First of all, the biological functions of miR-214 in stem cell orientation differentiation to VSMCs will be deeply explored from both positive and negative aspects. Then we will construct 3'UTR plasmid which contains miR-214 potential target genes to do the dual luciferase reporter gene assay. Meanwhile, the change of biological effects of miR-214 due to up or down regulation of target gene will be observed so as to explore the interaction between miR-214 and its potential target molecules. Thus, molecular networks of miR-214 and its target genes will be constructed. Finally, high expression of miR-214 in vascular injury parts will be achieved by local infusion miR-214 mimic or GFP controlled lentiviral vectors on the basis of mouse femoral artery injury model in order to verify the effects of miR-214 on regulatory its target genes, stem/ progenitor cell orientation differentiation to VSMCs and neointimal formation after vascular injury. Implementation of this project will provide in-depth understanding of the molecular mechanisms of stem cell orientation differentiation to VSMCs.

本课题将在前期研究基础上，建立依靠慢病毒载体稳定表达miR-214前体或反义小分子的小鼠ES细胞株。从正负两个效应方面深入探讨miR-214在干细胞向VSMCs定向分化过程中的细胞生物学功能；然后构建含miR-214可能靶基因的3’UTR端质粒，进行萤火素酶双荧光报告基因检测，同时观察miR-214介导的生物学效应是否会由于靶基因的上调/下调而改变，从而探讨miR-214与其潜在靶分子的相互作用关系并初步构建miR-214靶基因交互作用的分子网络。最后，我们还将在小鼠股动脉导丝损伤模型的基础上通过局部输注含有miR-214模拟物或GFP对照的慢病毒载体以实现血管损伤局部miR-214的高表达，从在体水平验证miR-214对其相应靶基因的调控作用、对干/祖细胞定向分化VSMCs的作用及其对血管损伤后新生内膜形成的影响。本项目的实施，将为深入认识干细胞定向分化VSMCs的分子机制提供新的理论依

以动脉粥样硬化为典型病理改变的血管损伤性疾病是人类健康的“第一杀手”，其机制尚未阐明。目前研究认为血管壁干/祖细胞向血管平滑肌细胞（VSMCs）分化及成熟VSMCs的表型转化均参与血管损伤后修复及病理性重塑过程。本项目发现microRNA-214(miR-214)在小鼠胚胎干细胞（ESCs）向VSMCs 分化过程中起促进作用，这种作用是通过抑制其靶基因QKI介导的；同时发现miR-214能够通过作用NCKAP1抑制成熟VSMCs的增殖和迁移，进而抑制血管新生内膜的形成，抑制血管损伤后病理性重塑；此外，本研究还发现miR-22能够调控VSMCs表型转换抑制内膜新生。本研究揭示了miR-214参与动脉粥样硬化、血管损伤内膜增生、移植血管重构的作用和病理机制，加深对血管疾病病理机制的理解，为血管疾病的防治从干细胞和再生医学角度提供新的思路和理论支持。