microRNAs在MLCK调控动脉粥样硬化血管重构中的作用及分子机制

Myosin light chain kinase ( MLCK ) is a key enzyme regulating smooth muscle contraction, and microRNA ( miRNA ) in the pathogenesis of atherosclerosis ( AS ) in vascular remodeling play a very important role. However, MLCK and miRNA how to control the effect and mechanism of AS vascular remodeling is not clear. Our previous use of MLCK gene knockout of vascular smooth muscle cells ( VSMC ), the difference between miRNAs and screened by miRNA chip, the target gene bioinformatics analysis to find miRNA related to vascular remodeling and corresponding. Cytological experiments confirmed that miR-34b-3p, miR-199a-5p, miR-504 in MLCK knockout VSMC expression increased significantly. This project aims to in vivo animal ( MLCK knockout mice ) and in vitro cytology ( MLCK knockout VSMC ), further verify the effect of three kinds of miRNAs in the regulation of MLCK AS in vascular remodeling; clear target gene of three miRNAs; to investigate the molecular mechanism of miRNAs and target gene in MLCK regulation of AS in vascular reconstruction. To provide new ideas for elucidating the molecular mechanisms of AS, and to provide theoretical and experimental basis for prevention and control of drug targets.

肌球蛋白轻链激酶(MLCK)作为调节平滑肌收缩的关键酶，与microRNA (miRNA)在动脉粥样硬化(AS)血管重构中均起着重要的作用。然而MLCK与miRNA如何调控AS血管重构的作用及机制尚不清楚。我们前期利用MLCK基因敲除血管平滑肌细胞（VSMC），通过miRNA芯片筛选出差异miRNAs，经生物信息学分析后找到与血管重构相关的miRNA及对应的靶基因。细胞学实验证实miR-34b-3p、miR-199a-5p、miR-504在MLCK基因敲除VSMC表达明显增高。本项目拟通过体内动物（MLCK基因敲除小鼠）和体外细胞学（MLCK基因敲除VSMC），进一步验证三种miRNAs在MLCK调控AS血管重构中的作用；明确三种miRNAs的靶基因；探讨miRNAs及靶基因在MLCK调控AS血管重构中的分子机制。为揭示AS的发病机制提供新的思路，并为寻找防治药物的靶点提供理论和实验依据。

MLCK和miR-92a分别是动脉粥样硬化的重要调节因子，但它们在动脉粥样硬化形成中的关系仍然是一个热门研究课题。我们研究了miR-92a在MLCK信号通路中对PDGF-BB调控的VSMCs增殖和迁移的作用。在小鼠动脉粥样硬化斑块形成过程中，MLCK和miR-92a的表达均增加，而且这种增加呈现一致的趋势。在ML-7（MLCK抑制剂）治疗小鼠中，血管壁的脂质沉积量减少，miR-92a的表达量降低。miRNA阵列分析显示在MLCK基因缺失的血管平滑肌细胞（VSMC）中miR-92水平降低。我们推测MLCK可能参与了miR-92a的调控。因此，我们通过使用分离的动脉平滑肌细胞和3种不同血管平滑肌细胞系，重点研究MLCK信号通路与miR-92a的关系。我们发现MLCK和miR-92a均参与调节VSMCs的迁移和增殖。pre-miR-92a部分恢复了MLCK缺失对VSMC增殖和迁移下调的影响。ML-7增加小鼠动脉平滑肌细胞中miR-92a的靶基因KLF4的表达，siKLF4增加VSMCs的增殖和迁移。令人鼓舞的是，抑制MLCK增强了KLF4的表达。MLCK上调miR-92a抑制KLF4的表达，促进PDGF-BB介导的VSMC增殖和迁移，这种新的信号通路可能是改善AS血管功能的治疗药物。