摘要
目的探讨miR-142-5p在动脉粥样硬化组织中的表达及对人巨噬细胞凋亡的作用。方法构建动脉粥样硬化大鼠模型,qRT-PCR检测动脉粥样硬化组织中miR-142-5p的表达水平。50μg/L氧化型低密度脂蛋白(ox-LDL)刺激人巨噬细胞、血管平滑肌细胞(VSMC)、内皮细胞24 h后,提取细胞RNA,qRT-PCR检测细胞中miR-142-5p的表达水平。靶基因预测软件预测miR-142-5p的靶基因,双荧光素酶报告基因鉴定靶基因的正确性。细胞转染miR-142-5p mimic、mimic control、miR-142-5p inhibitor、inhibitor control,Western blot和qRT-PCR检测miR-142-5p对靶基因的调控作用,流式细胞仪检测miR-142-5p和靶基因对巨噬细胞凋亡的作用。结果 miR-142-5p在动脉粥样硬化组织中表达上调,与正常组织相比差异显著(P<0.01)。血管平滑肌细胞和内皮细胞经ox-LDL刺激后miR-142-5p的表达水平与刺激前没有明显变化,而巨噬细胞经ox-LDL刺激后miR-142-5p的表达水平较刺激前明显升高(P<0.01)。预测miR-142-5p的靶基因为转化生长因子β2(TGF-β2)。miR-142-5p mimic与TGF-β2共转染后荧光素酶活性最低;miR-142-5p mimic组TGF-β2蛋白和mRNA的表达水平与mimic control组相比明显下降,miR-142-5p inhibitor组TGF-β2蛋白和mRNA的表达水平与inhibitor control组相比明显升高(P<0.01);miR-142-5p mimic组细胞凋亡率明显高于mimic control组,TGF-β2 siRNA组细胞凋亡率明显高于siRNA control组(P<0.01)。结论 miR-142-5p在动脉粥样硬化中过度表达,miR-142-5p通过下调靶基因TGF-β2促进人巨噬细胞凋亡。
Aim To study the expression of miR-142-5p in atherosclerosis tissues and its effect on human macrophage apoptosis. Methods Rat atherosclerosis model was constructed, qRT-PCR was used to detect the expression of miR-142-5p in atherosclerotic tissues. 50 μg/L ox-LDL stimulated human macrophage, vascular smooth muscle cell, endothelial cell for 24 h, qRT-PCR was used to detect the expression of miR-142-5p in the cells. Target gene prediction software was used to predict the target gene of miR-142-5p, dual luciferase reporter gene was used to identify the target gene. Cells were transfected with miR-142-5p mimic, mimic control, miR-142-5p inhibitor, inhibitor control into macrophage, Western blot and qRT-PCR were used to to detect the expression of miR-142-5p on target gene, the effect of miR-142-5p and target gene on the apoptosis of macrophages was detected by flow cytometry. Results MiR-142-5p expression was up-regulated in atherosclerotic tissues compared with normal tissue, and the difference was significant （P〈0.01）.After stimulation with ox-LDL, the levels of miR-142-5p in vascular smooth muscle cells and endothelial cells did not change significantly compared with before stimulation, but the level of miR-142-5p in the macrophages was significantly higher than that before stimulation （P〈0.01）. Predicting the target gene of miR-142-5p was transforming growth factor-β2 （TGF-β2）. The luciferase activity was the lowest after transfection with miR-142-5p mimic and TGF-β2. The expression of TGF-β2 protein and mRNA in miR-142-5p mimic group was significantly decreased compared with mimic control group （P〈0.01）, the expression of TGF-β2 protein and mRNA in miR-142-5p inhibitor group was significantly higher than that in inhibitor control group （P〈0.01）, the apoptosis rate of miR-142-5p mimic group was significantly higher than that of mimic control group （P〈0.01）, the apoptosis rate of TGF-β2 siRNA group was significantly higher than that of siRNA control group （P〈0.01）. Conclusion MiR-142-5p was over expressed in atherosclerosis tissues, and miR-142-5p could promote the apoptosis of human macrophages by regulating the target gene TGF-β2.
出处
《中国动脉硬化杂志》
CAS
北大核心
2017年第5期475-479,共5页
Chinese Journal of Arteriosclerosis
