MicroRNA靶向调节sPLA2-ⅡA表达在动脉粥样硬化发病机制中的作用

Our former research found that the plasma concentration of sPLA2-ⅡA significantly increased in the patients with acute coronary syndrome(ACS). And sPLA2-ⅡA as a predictive factor for the atherosclerosis(AS) plaque stability predicted the nature of lesions and the prognosis of ACS. Our recent studies showed that the plasma concentration of miR-30c-1-3p obviously decreased in the patients with ACS, and there was a negative correlation between the miR-30c-1-3p lever and the sPLA2-ⅡA lever in the plasma.We discovered that miR-30c-1-3p targeted the 3'UTR sequence of sPLA2-ⅡA mRNA through using the microRNA forecasting software system, the gene sequence of which in human was akin to that in mice. While actually, it is presently lacked of the systematic study on the effects of miR-30c-1-3p regulating sPLA2-ⅡA expression on the pathogenic mechanism of AS. In this study, we will elucidate deeply the effects of miR-30c-1-3p on regulating sPLA2-ⅡA expression in vascular smooth muscle cells（VSMCs）and adjusting VSMCs transforming foam cells by constructing eukaryotic expression vector carrying miR-30c-1-3p gene sequences. And we will further observe the effects of miR-30c-1-3p on the pathophysiology of the AS process in the apolipoprotein E-knock out(ApoE-/-) mice. The project will provide a new interfering target and a scientific laboratorial basis for preventing and curing AS and diseases related to AS.

我们前期研究发现急性冠脉综合征（ACS）患者血浆ⅡA分泌型磷脂酶（sPLA2-ⅡA）水平明显增高，可作为动脉粥样硬化（AS）斑块稳定性的预测因子预测ACS病变性质及预后；近期研究发现ACS患者血浆miR-30c-1-3p水平与sPLA2-ⅡA水平呈显著负相关，运用miRNA靶基因预测软件发现miR-30c-1-3p可靶向作用于sPLA2-ⅡA，且人源miR-30c-1-3p序列与小鼠种系完全一致。目前尚缺乏miR-30c-1-3p是否可靶向调节sPLA2-ⅡA表达的相关性研究。本课题利用携带miR-30c-1-3p的真核表达载体，在血管平滑肌细胞（VSMC）上探讨miR-30c-1-3p对sPLA2-ⅡA表达的调节、对VSMC转化为泡沫细胞的调控作用；并用ApoE-/-小鼠观察miR-30c-1-3p对在体动物AS形成的影响。为临床防治AS及相关性疾病提供新的干预靶点和科学实验依据。

动脉粥样硬化（Atherosclerosis，AS）是众多心脑血管疾病共同的病理基础。sPLA2-IIA在动脉粥样硬化病变处含量明显增多、活性显著增强，同时伴随中性粒细胞的增多,参与AS发生发展的各个阶段。本课题前期研究发现ACS患者血浆sPLA2-IIA水平显著增高，且miR-30c-1-3p水平与sPLA2-IIA水平呈显著负相关，但目前关于miR-30c-1-3p是否可靶向调节sPLA2-IIA表达的相关性研究还未有报道。.本项目首先构建了miR-30c-1-3p表达载体，并选取原代大鼠主动脉血管平滑肌细胞进行研究，以慢病毒转染miR-30c-1-3p到大鼠主动脉血管平滑肌细胞。体外实验结果表明，miR-30c-1-3p可以明显抑制IL-1β，TNF-α以及ox-LDL诱导的sPLA2-IIA表达水平和平滑肌细胞的增殖，同时，miR-30c-1-3p可明显抑制ox-LDL诱导的细胞内胆固醇的水平。.本课题组使用ApoE基因敲除小鼠建立动脉粥样硬化模型，苏丹IV染主动脉组织，观察到miR-30c-1-3p组主动脉脂质斑块面积明显小于对照组。进一步对主动脉窦组织进行冰冻切片，油红染色结果发现，miR-30c-1-3p明显抑制主动脉窦脂质的蓄积。HE染色主动脉根病灶处的坏死区，结果显示：miR-30c-1-3p可明显抑制小鼠动脉粥样硬化主动脉根病灶处的坏死核心面积。收集小鼠血清，结果发现，miR-30c-1-3p组血脂（甘油三酯，低密度脂蛋白和高密度脂蛋白）的水平明显低于对照组，同时，western-blotting结果显示，注射miR-30c-1-3p agomir组的小鼠动脉组织中，sPLA2-IIA的表达明显下调。.综上所述，我们的体外和在体实验结果提示：miR-30c-1-3p可能通过靶向下调sPLA2-IIA的表达，抑制动脉粥样硬化的发展。本研究为临床防治AS及相关性疾病提供新的干预靶点和科学实验依据。