下调LncRNA-p21抑制p21及p53通路促进血管平滑肌细胞增殖与凋亡促进动脉粥样硬化进展

Coronary artery disease (CAD) is a prevailing cause of death worldwide, atherosclerosis (AS) is the leading cause of CAD, and its pathogenesis has not been fully studied. As the potential therapeutic targets, many long non-coding RNAs (lncRNAs) are indicated to associate with CAD and AS, but the relative molecular mechanisms still need further research. In pre-experiments, we found that serum samples from CAD patients had lower expression of lincRNA-p21 than control samples by lncRNA chip analysis, and the transfection of lincRNA-p21 siRNA could promote vascular smooth muscle cell (HUMC) proliferation and inhibit cell apoptosis. Further experiments showed that transfection of lincRNA-p21 siRNA could decrease the expressions of p21 and p53 downstream target genes included Noxa, Bax and Puma. Combined with references, we hypothesized that “lincRNA-p21 could regulate p21 and p53 pathway, affect proliferation and apoptosis of HUMC, and participate in the process of AS”. Based on the results of pre-experiments, through cell transfection, co-immunoprecipitation and other experimental methods, we will further study the relative molecular mechanisms that lincRNA-p21 regulates HUMC, which provides new molecular targets for the treatment of CAD.

冠状动脉粥样硬化性心脏病（CAD）是当前人类死亡的主要原因之一，而动脉粥样硬化（AS）是导致CAD的主要原因，其发病机制尚未完全阐明。已有多种长链非编码RNA（lncRNA）被发现与CAD及AS存在相关性，在预实验中，本课题组通过lncRNA芯片分析，发现CAD患者外周血样本中lincRNA-p21显著下调，转染lincRNA-p21 siRNA可以促进血管平滑肌细胞的增殖同时抑制细胞凋亡。转染lincRNA-p21 siRNA后，p21及p53通路相关蛋白Noxa、Bax、Puma的表达显著下调。结合文献报道，我们提出如下假说“lincRNA-p21可以调控p21及p53通路，影响血管平滑肌细胞增殖与凋亡，参与AS过程”。在预实验的基础上，本项目拟通过在细胞模型中通过细胞转染、免疫沉淀技术等实验方法，深入探讨其在AS进程中的作用及相关分子机制，为CAD的治疗寻找新的分子靶点。

目的：冠心病（CHD）是最常见的心血管疾病之一，患病率呈持续上升趋势，其防治已成为全球的重大公共卫生问题。动脉粥样硬化（AS）则是引起CHD发生的最主要原因，也是重要的病理过程。近年来的研究已表明，长链非编码RNA（lncRNA）可参与AS发生和发展。本课题拟采用lncRNA表达谱芯片检测CHD患者和非CHD健康人外周血中lncRNA表达量，筛选出与AS相关的lncRNA，进一步探讨其在血管平滑肌细胞（VSMC）凋亡和增值中的作用，为AS机制的深入研究奠定基础。.方法：1.使用lncRNA表达谱芯片检测、比较冠心病与非冠心病健康人外周血中lncRNA表达量的差异。2.采用病例-对照研究分析lincRNA-p21与临床病理特征的关系。3.通过细胞转染和动物模型、MTT实验、多种染色法实验和流式细胞仪探讨和分析lincRNA-p21对VSMC增殖和凋亡的影响。4.应用GO富集分析、KEGG通路数据库、mRNA表达谱芯片和在线预测软件catRAPID分析、筛选与AS相关的通路。5.应用荧光素酶报告基因检测法、Westen blot实验分析、探讨lincRNA-p21与JAK2/STAT3通路之间的关系。.结果：1.冠心病患者和非冠心病健康人外周血中，lincRNA-p21、lncRNA-TUG1、lncRAN-HIF1A-AS1和lncRAN-ANRIL的表达量存在显著差异，其中，lincRNA-p21的差异最为显著。2.AS患者外周血中lincRNA-p21表达量显著低于对照者外周血中的表达量；且患有高血压和糖尿病的患者中，lincRNA-p21表达量显著降低。3.LincRNA-p21可以抑制VSMC增殖、促进凋亡。4.LincRNA-p21通过调控p21和p53影响VSMC增殖和凋亡。5.LincRNA-p21可与STAT3直接结合，参与JAK2/STAT3信号通路的激活，调节VSMC增殖和凋亡。.结论：1.LincRNA-p21通过调节p21、p53参与VSMC增殖和凋亡的调节。2.LincRNA-p21通过调控靶基因STAT3的表达，激活JAK2/STAT3信号通路，参与VSMC增殖和凋亡的调节，促进AS的进展。