MicroRNA-1在小鼠动脉粥样硬化斑块中的表达及通过TGF-β2调控巨噬细胞凋亡功能的研究

Expression of microRNA-1 in atherosclerotic plaque and its regulating effects on macrophage apoptosis via TGF-β2 in mice

目的探讨微小核糖核酸(microRNA)-1在动脉粥样硬化斑块中的表达及通过转化生长因子(TGF)-β2调控巨噬细胞凋亡功能的分子机制。方法实验选用5周龄的雄性Apo E-/-小鼠50只,按实验要求分为对照组(25只)与动脉粥样硬化组(实验组,25只)。实验组小鼠采用高脂喂食以建立动脉粥样硬化模型,对照组小鼠采用正常饲料喂养。造模成功后,实验组及对照组分别取动脉粥样硬化斑块组织及正常动脉组织,采用实时荧光定量PCR(qRT-PCR)检测microRNA-1的表达。培养小鼠内皮细胞、平滑肌细胞及巨噬细胞,并用50 ng/ml氧化修饰低密度脂蛋白(ox-LDL)刺激24 h,应用qRT-PCR技术比较miRNA-1在不同细胞中的表达变化。通过数据库及生物信息学软件预测microRNA-1的靶基因,并利用双荧光素酶报告基因系统进行验证。实验组与对照组分别通过转染microRNA-1 mimics和microRNA-1 mimics的阴性对照至巨噬细胞株后观察其对细胞凋亡功能的影响。结果MicroRNA-1在实验组动脉粥样硬化斑块中的表达比在对照组正常动脉组织中的表达明显增加(P<0.05)。MicroRNA-1高表达于小鼠血管巨噬细胞,而低表达于血管平滑肌细胞及内皮细胞(P<0.05),经ox-LDL刺激后,其在巨噬细胞中的表达量较刺激前明显增加(P<0.05),内皮细胞及平滑肌细胞的表达量无明显差异(P均>0.05)。生物信息学分析发现microRNA-1的靶基因为TGF-β2,并经体外双荧光素酶报告基因系统检测证实。体外细胞功能实验发现microRNA-1能增加巨噬细胞的凋亡,TGF-β2具有抑制其凋亡的功能。结论 MicroRNA-1在动脉粥样硬化斑块中表达上调,其可能通过介导靶基因TGF-β2调控巨噬细胞的凋亡进而参与动脉粥样硬化的发生过程。

Objective To investigate the expression of micro ribonucleic acid （microRNA）-1 in atherosclerotic plaque and the molecular mechanism regulating macrophage apoptosis via transforming growth factor-β2 （TGF-β2） in mice. Meth- ods Fifty ApoE-/- male mice of 5 weeks old were randomly divided into control group （ n = 25 ） and atherosclerosis group （ experimental group, n = 25 ）. High fat feeding was given in experimental group to establish atherosclerosis experimental an- imal model, and normal feeding was given in control group. The atherosclerotic plaque tissue and normal artery tissue were respectively taken in experimental group and control group after modeling. The real-time fluorescence quantitative polymer- ase chain reaction （qRT-PCR） was used to detect the expressions of microRNA-I in tissues. After vascular smooth muscle cells, endothelial ceils and macrophages were cuhured in vitro and stimulated by 50 ng/ml of oxidized low density lipopro- tein （ox-LDL） for 24 h,the expressions of microRNA-1 in different cells were detected by qRT-PCR. The target gene of mi- croRNA-1 was predicted by database and bioinformatics software and verified by Dual-Luciferase Reporter Assay System. After being respectively transfected with microRNA-1 mimics and its negative control in macrophage line of experimental group and control group, the influence of microRNA-1 on cell apoptosis function of macrophage was observed. Results Mi- croRNA-I expression in atherosclerotic plaque （experimental group） increased significantly compared with normal artery tissue in control group （P 〈 0.05 ）. MicroRNA-1 expression in macrophage was significantly higher than that in vascular smooth muscle cells and endothelial cells （all P 〈 0.05 ）. After being stimulated by ox-LDL, microRNA-1 expression level in macrophages increased significantly compared with before stimulating（ P 〈 0.05 ） ,while its expression levels in endothe- lial cells and smooth muscle cells remained unchanged （ all P 〉 0.05 ）. Bioinformatics analysis showed that the target genes of microRNA-1 might be TGF-β2, whieh was confirmed by Dual-Luciferase Reporter Assay System in vitro. The cell func- tion test in vitro showed that microRNA-1 can increase apoptosis of macrophage,while TGF-β2 can inhibited the apoptosis of macrophage. Conclusion The expression of microRNA-1 is up-regulated in atherosclerotic plaque. MicroRNA-1 might regulate the apoptosis of macrophages via mediating target gene TGF-β2 and participates in the occurring process of athero- sclerosis fhrther.