受DNA甲基化调控的microRNA-125a-3p抑制动脉平滑肌细胞的增殖和迁移研究

Arteriosclerosis obliterans (ASO) caused by peripheral vascular atherosclerosis incidence up to 12%-29% in the crowd，leading to thousands of patients with limb disability, is a serious threat to human health. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) are the key factors leading to ASO and vascular restenosis after surgical intervention. Previous studies found that the expression of miR-125a-3p was down-regulated in ASO vascular tissues and upregulation of mir-125a-3p could down-regulate the target genes mapk1 and Smad2 to inhibit the proliferation and migration of VSMCs in vitro, however the internal molecular mechanism needs to be clarified. At the same time, it was found that the promoter of MIR-125A was hypermethylation. Accordingly, we put forward the hypothesis: the hypermethylation of MIR-125A promoter leads the down-regulation of mir-125a-3p in ASO; miR-125a-3p could regulate the target genes ERB2, mapk1, Smad2 and Smad4 to influence activity of MAPK / ERK and TGF-beta/SMAD signaling pathway, affect the expression of downstream protein and then control the proliferation and migration of VSMCs, effect the occurrence and development of ASO. This study intends to use a variety of techniques of molecular biology and animal experiment, in order to obtain the reliable evidence that miR-125a-3p could regulate MAPK / ERK and TGF-beta/SMAD signaling pathways activity and interpret the mechanism of miR-125a-3p regulating the proliferation and migration of VSMCs, in order to provide more adequate scientific basis to establish mir-125a-3p as a new target of treating ASO and vascular restenosis after surgical intervention.

动脉硬化闭塞症（ASO）在人群中发病率高达12%-29%，导致成千上万患者肢体残疾，严重威胁人类的健康。血管平滑肌细胞（VSMCs）的过度增殖和迁移是导致ASO和外科治疗后血管再狭窄的关键因素。前期研究发现，miR-125a-3p在ASO血管组织中表达下调，体外上调miR-125a-3p表达可通过下调靶基因MAPK1和SMAD2抑制VSMCs增殖和迁移; 同时我们发现基因MIR-125A启动子区域存在DNA的过甲基化。本研究将进一步采用各种分子生物学技术及动物实验验证受DNA甲基化调控的 miR-125a-3p可以通过调控靶基因ERB2、MAPK1、SMAD2、SMAD4来影响MAPK/ERK 和TGF-beta/SMAD信号通路的活性，进而控制VSMCs的增殖与迁移，影响ASO的发生和发展。本研究拟为miR-125a-3p作为治疗ASO和外科治疗后血管再狭窄的新靶点提供更充分的科学依据。

项目背景：动脉硬化闭塞症（ASO）在人群中发病率高达12%-29%，导致成千上万患者肢体残疾，严重威胁人类的健康。介入治疗是目前重要的治疗手段，但术后血管再狭窄严重制约了患者的预后。血管平滑肌细胞（VSMCs）的过度增殖和迁移是导致ASO和外科介入治疗后血管再狭窄的关键因素。多项研究表明MicroRNA积极参与了其中的各种病理过程。.主要研究内容：前期高通量芯片研究提示了miR-125a-3p在再狭窄血管和正常血管组织中的表达是有差异的，本研究通过独立的RT-PCR确认miR-125a-3p在正常组织和狭窄血管组织中的表达差异；原位杂交研究miR-125a-3p 的在血管组织中的表达分布；体外细胞研究miR-125a-3p在血管平滑肌细胞中的功能；进一步探讨miR-125a-3p实现其功能的作用机制以及miR-123a-3p在动脉狭窄的动物模型中的作用。.重要结果：miR-125a-3p在ASO和再狭窄血管组织中表达下调，其主要分布于血管平滑肌细胞中，体外上调miR-125a-3p表达可通过下调靶基因MAPK1抑制VSMCs增殖和迁移。同时miR-125a-3p在体内能够通过抑制MAPK1来抑制动物模型中的血管再狭窄。.关键数据及科学意义：miR-125a-3p能够抑制MAPK1抑制血管平滑肌细胞的增殖和迁移从而抑制血管的再狭窄，其意义是后期临床转化，为ASO和血管介入术后的血管再狭窄治疗提供潜在的治疗靶点。