microRNA-34a在小鼠胚胎干细胞定向分化血管平滑肌细胞中的作用及机制研究

Smooth muscle cell (SMC) differentiation is a critical process during vascular development and SMC proliferative related vascular disease such as atherosclerosis, postangioplasty restenosis and transplantation arteriopathy. Therefore, better understanding of the molecular mechanisms of vascular SMC differentiation is essential for improving the treatment or prevention of cardiovascular disorders. microRNAs (miRNAs) have been recently suggested to play important roles not only in the regulation of multiple biological cellular functions, but also in the cardiovascular diseases. By applying our well-established SMC differentiation model, we have recently found that miR-34a is significantly up-regulated during SMC differentiation from mouse embryonic stem (ES) cells. Data from our miR-34a gain-of-function and lose-of-function studies clearly demonstrated that miR-34 plays a crucial role in SMC differentiation, and that miR-34a mediated SMC differentiation through modulation of SMC transcription factors (SRF, myocardin and MEF2c). In this proposal, we will further investigate the crucial role of miR-34a in SMC differentiation from mouse ES cells, and elucidate its molecular mechanism. We will also investigate the functions of miR-34a in vessel injury-induced neointima formation and the underlying mechanisms. Results of the study would help to better understand the biological mechanisms underlying SMC differentiation and injury-induced neointima formation, and could provide a new strategy for therapeutic intervention in atherosclerosis and postangioplasty restenosis.

胚胎干细胞（ESCs）是一类具有自我更新及多向分化潜能的细胞群，可在体外诱导为血管平滑肌细胞（VSMCs），但其定向分化机制尚未明确。课题组前期研究发现miR-34a在小鼠ESCs向VSMCs分化过程中起关键作用。本研究拟1）建立稳定表达miR-34a前体或反义小分子的小鼠ES株，深入探讨miR-34a促ESCs定向分化VSMCs的作用机制；2）构建含miR-34a可能靶基因SIRT1、CCND1和KLF4的3'UTR质粒，进行报告基因检测，同时观察miR-34a介导的生物学效应是否会由于靶基因的上调/下调而改变，探讨miR-34a与潜在靶分子的相互作用关系；3）建立小鼠血管损伤模型，从在体水平验证miR-34a对其相应靶基因的调控作用及其对血管损伤后新生内膜形成的影响。研究将为深入认识ESCs定向分化VSMCs的分子机制提供新的理论依据, 并为今后有效地防治血管疾病提供新的潜在药物靶点。

血管平滑肌细胞通过干/祖细胞分化及本身表型转化参与血管损伤修复及病理性重塑。课题组前期研究发现miRNA-34a在小鼠胚胎干细胞（ESCs）向血管平滑肌细胞（VSMCs）分化过程中起关键作用，但其具体定向分化机制尚未明确；对于miR-34a是否调控成熟血管平滑肌细胞功能和血管病理重塑仍然知之甚少。本研究希望阐述microRNA-34a在ESCs定向分化VSMCs和VSMCs表型转化后增殖迁移功能以及血管损伤后新生内膜形成的作用及机制。研究方法：建立ESCs向 VSMCs分化模型，并分离小鼠原代血管平滑肌细胞体外培养，利用体外平滑肌细胞表型转化模型，观察miR-34a表达变化，并过表达或抑制miR-34a，研究miR-34a对血管平滑肌细胞分化、增殖、迁移能力的影响。其次，利用生物信息学分析、分子克隆等方法鉴定miR-34a的功能性靶基因。最后，建立小鼠股动脉导丝损伤内膜病理性增生模型，在损伤血管局部过表达 miR-34a，在体水平验证miR-34a对其靶基因的调控作用，研究miR-34a对血管损伤后内膜新生的作用。结果：我们发现miR-34a通过促进小鼠ESCs向VSMCs分化，SIRT1作为miR-34a的靶基因调控小鼠ESCs定向分化VSMCs的分子机制，直接调控干细胞向平滑肌细胞分化关键转录因子（SRF、MEF2c、Myocd）表达。原代血管平滑肌细胞经历血清饥饿，FBS、PDGF-BB、ox-LDL刺激后表型由“收缩型”向“增殖迁移型”转化，在此过程中miR-34a表达显著下降。功能研究发现过表达miR-34a抑制平滑肌细胞增殖、迁移能力，反之抑制miR-34a表达可增加平滑肌细胞增殖、迁移能力。利用生物信息学分析、分子克隆等方法鉴定了miR-34a的功能性靶基因Notch1。成功建立小鼠股动脉导丝损伤内膜增生模型，发现miR-34a在血管损伤后内膜增生过程中显著下降，损伤血管局部高表达miR-34a可明显减轻血管损伤后内膜新生。结论：本研究成功揭示了miR-34a在血管平滑肌细胞中的新功能，miR-34a促进小鼠ESCs向VSMCs分化，抑制血管平滑肌细胞表型转化后的增殖及迁移能力，为今后有效地防治血管疾病提供新的潜在药物靶点，同时为干细胞组织工程人造血管的研发提供实验依据。