(1.宁夏医科大学总医院 心内科,宁夏银川市 750003;2.宁夏医科大学总医院 心脏中心大血管外科,宁夏银川市 750003)
1.Department of Cardiology,Ningxia 750003, China ;2.Department of Heart Center Macrovascular Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia 750003, China)
目的]探讨长链非编码RNA(lncRNA)HOX转录反义RNA(HOTAIR)对冠心病(CHD)的影响及其作用机制。[方法]通过实时定量PCR检测CHD患者和健康志愿者外周血血样和内皮祖细胞(EPC)中lncRNA HOTAIR、微小RNA-126(miR-126)和Polo样蛋白激酶4(PLK4)的表达水平；噻唑蓝法检测EPC细胞活力；流式细胞仪检测细胞凋亡率；Western blot检测自噬相关蛋白LC3-Ⅱ和Beclin1的表达。激光共聚焦显微镜观察细胞自噬情况。miRcode软件和双荧光素酶报告基因实验分析lncRNA HOTAIR和miR-126的相互关系。TargetScan软件和双荧光素酶报告基因实验分析miR-126和PLK4的相互关系。Western blot检测lncRNA HOTAIR通过miR-126对EPC细胞哺乳动物雷帕霉素靶蛋白(mTOR)通路的影响。[结果]lncRNA HOTAIR和PLK4在CHD血样和EPC细胞中的表达明显上调(P＜0.01),miR-126表达明显下调(P＜0.01)。下调lncRNA HOTAIR或PLK4促进EPC细胞自噬,抑制CHD进展。上调lncRNA HOTAIR通过miR-126抑制了EPC细胞自噬,促进细胞凋亡。lncRNA HOTAIR通过miR-126激活了EPC内mTOR通路。[结论]HOTAIR/miR-126/PLK4轴介导EPC细胞自噬,通过mTOR信号通路影响CHD。
Aim To investigate the effect of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) on coronary heart disease (CHD) and its mechanism. Methods The expression levels of lncRNA HOTAIR, microRNA-126 (miR-126) and Polo-like kinase 4 (PLK4) in peripheral blood samples and endothelial progenitor cells (EPC) of CHD patients and healthy volunteers were detected by quantitative real-time PCR. EPC cell viability was detected by methyl thiazolyl tetrazolium assay. Apoptosis rate was detected by flow cytometry. The expressions of autophagy-related proteins LC3-Ⅱ and Beclin1 were detected by Western blot. Cell autophagy was observed by confocal microscopy. The relationship between lncRNA HOTAIR and miR-126 was analyzed by miRcode software and dual-luciferase reporter gene assay. The relationship between miR-126 and PLK4 was analyzed by TargetScan software and dual-luciferase reporter gene assay. The effect of lncRNA HOTAIR on mammalian target of rapamycin (mTOR) pathway through miR-126 in EPC was detected by Western blot. Results The expressions of lncRNA HOTAIR and PLK4 were significantly up-regulated in CHD blood samples and EPC (P＜0.01), and the expression of miR-126 was significantly down-regulated (P＜0.01). Down-regulation of lncRNA HOTAIR or PLK4 promoted autophagy in EPC, and inhibited CHD progression. Up-regulation of lncRNA HOTAIR inhibited EPC autophagy and promoted apoptosis through miR-126.The lncRNA HOTAIR activated the mTOR pathway in EPC via miR-126. Conclusion HOTAIR/miR-126/PLK4 axis mediates autophagy in EPC and affects CHD via mTOR signaling pathway.