微小RNA-130a调控人巨噬细胞三磷酸腺苷结合盒转运体A1在动脉粥样硬化的作用

Role of human macrophage ABCA1 regulated by miR-130a in atherosclerosis

目的探讨微小RNA-130a(microRNA-130a,miR-130a)靶向调控人巨噬细胞三磷酸腺苷结合盒转运体A1(ABCA1)在动脉粥样硬化的作用。方法培养人单核巨噬细胞(THP-1),分为实验组和对照组。利用lipofectamine2000转染试剂,分别将miR-130amimics或阴性对照转染到THP-1中,通过液体闪烁计数测定荷脂THP-1内胆固醇流出情况,高效液相色谱检测细胞内脂质含量;油红O染色显示细胞内脂滴情况。生物信息学软件预测miR-130a的靶基因,并利用双荧光素酶报告基因系统进行验证。通过Western blot检测靶基因ABCA1蛋白表达变化。结果实验组较对照组显著抑制THP-1内胆固醇流出[(5.24±2.36)%vs(12.58±3.92)%,P<0.05]。实验组THP-1内胆固醇、胆固醇酯、游离胆固醇较对照组明显增高(P<0.05),细胞内脂滴稠密且体积肥大。生物信息学分析显示,miR-130a的靶基因为ABCA1,经体外双荧光素酶报告基因系统检测,证实ABCA1是miR-130a的靶基因。实验组ABCA1蛋白表达水平较对照组明显降低(0.13±0.05 vs 0.38±0.09,P<0.05)。结论 miR-130a具有调控THP-1内胆固醇流出的功能,其机制可能与靶向调控ABCA1有关。

Objective To study the role of human macrophage ABCA1 regulated by miR-130 ain atherosclerosis.Methods Cultured human mononuclear macrophages（THP-1）were divided into experimental group and control group.The miR-130 amimics or negative controls were transinfected into the THP-1 with lipofectamine2000.Intracellular 3 H-cholesterol efflux was assayed with a liquid scintillator,intracellular lipid level was measured by HPLC,and intracellular lipid droplets were detected with oil red O staining.The target gene of miR-130 awas identified by bioinformatics software and verified by dual luciferase reporter assay.The ABCA1 protein expression was detected by Western blot.Results The cholesterol efflux from THP-1was significantly lower in experimental group than in control group（5.24% ±2.36% vs12.58% ±3.92%,P〈0.05）.The intracellular cholesterol,lipid and free cholesterol levels were significantly higher in experimental group than in control group（P〈0.05）.The intracellular droplets were denseer and their size was larger in experimental group than in control group（P〈0.05）.Bioinformatics analysis showed that the target gene of miR-130 awas ABCA1which was confirmed by dual luciferase reporter assay.In vitro cell experiment revealed that the ABCA1 protein expression level was significantly lower in experimental group than in control group（P〈0.05）.Conclusion MiR-130 a regulates the cholesterol efflux from THP-1by targeting the ABCA1.