miR-29c-5p在冠心病患者外周血白细胞中的表达及其对内皮细胞损伤的调控机制

Expression of miR-29c-5p in the peripheral leukocytes of patients with coronary heart disease and its mechanism underlying the regulation of endothelial injury

摘要:

目的]分析微小RNA-29c-5p(miR-29c-5p)在冠心病外周血白细胞中的表达,并探索其在高脂条件下调控内皮细胞氧化损伤和凋亡的机制。 [方法]在冠心病患者外周血白细胞中检测miR-29c-5p的表达水平；利用高脂诱导建立EA.hy926内皮细胞损伤模型,在高脂损伤模型中分别过表达和沉默miR-29c-5p并检测凋亡相关蛋白的表达和活性氧簇(ROS)水平及脂质过氧化程度。利用生物信息学方法预测miR-29c-5p的潜在靶基因,通过双荧光素酶报告基因实验验证其与靶基因的调控关系。 [结果]与健康对照组人群相比,miR-29c-5p在冠心病组的表达水平降低,并对冠心病有诊断意义(AUC:0.712,95%CI:0.632～0.792,P＜0.01)。在高脂损伤模型中,细胞活力下降,细胞凋亡增加(P＜0.01),同时miR-29c-5p的表达水平升高(P＜0.01)。沉默miR-29c-5p表达细胞损伤被逆转,细胞活力增加(P＜0.01),高脂诱导的细胞凋亡减少,ROS及脂质过氧化水平降低(均P＜0.05)；而过表达miR-29c-5p,细胞活力降低(P＜0.01),高脂诱导的细胞凋亡增加,ROS及脂质过氧化水平升高(均P＜0.05)。生物信息学分析预测和双荧光素酶报告基因实验证实miR-29c-5p与组蛋白去乙酰化酶2(HDAC2)基因的3′UTR区相结合,下调HDAC2的mRNA和蛋白表达水平(均P＜0.05)。 [结论]miR-29c-5p在冠心病外周血白细胞中表达下调,对冠心病有诊断意义；抑制miR-29c-5p的表达可通过上调HDAC2减轻高脂诱导的内皮细胞氧化损伤及凋亡。

Abstract:

Aim To analyze the expression of miR-29c-5p in peripheral blood leukocytes of patients with coronary heart disease (CHD), and to explore its mechanism in regulating oxidative damage and apoptosis of endothelial cells induced by high-lipid conditions. Methods The expression levels of miR-29c-5p were detected in peripheral blood leukocytes of patients with CHD. Endothelial cell injury in EA.hy926 cells was induced by exposure to high concentrations of the lipid. miR-29c-5p was overexpressed and silenced respectively in the high-fat-induced model, and the expression of apoptosis-related proteins, the level of reactive oxygen species (ROS), and the degree of lipid peroxidation were detected. Bioinformatics methods were used to predict the potential target genes of miR-29c-5p, and the dual luciferase reporter assay was performed to verify its regulatory relationship with the target genes. Results Compared with the control group, the expression level of miR-29c-5p was decreased in the CHD group and had diagnostic significance for CHD with an AUC value of 0.712 (95%CI:0.632～0.792, P＜0.01). In the high-fat-induced model, cell viability was decreased and cell apoptosis was increased (P＜0.01), together with an elevated miR-29c-5p expression level (P＜0.01).Silencing miR-29c-5p expression alleviated cell damage, increased cell viability (P＜0.01), reduced hight-fat-induced apoptosis, lowered ROS and lipid peroxidation levels (all P＜0.05); while overexpressing miR-29c-5p decreased cell viability (P＜0.01), increased high-fat-induced cell apoptosis and raised ROS and lipid peroxidation levels (all P＜0.05). Moreover, bioinformatics analysis prediction and dual luciferase reporter assay confirmed that miR-29c-5p bound to the 3′UTR region of the histonedeacetylase 2 (HDAC2) gene, thereby downregulating the mRNA and protein expression levels of HDAC2 (all P＜0.05). Conclusion The expression of miR-29c-5p is decreased in peripheral blood leukocytes of patients with CHD, which may serve as a diagnostic biomarker for this disease. Inhibition of miR-29c-5p expression in endothelial cells may alleviate oxidative injury and apoptosis induced by high-fat exposure via upregulating HDAC2.