microRNA-124通过Sp1控制人血管平滑肌细胞表型转换影响颅内动脉瘤发展的机制

The mechanism by which microRNA-124 influences cerebral aneurysm development by controlling the phenotypic switch of human vascular smooth muscle cells via Sp1

目的探究微小RNA-124(miR-124)通过特异性β1糖蛋白(Sp1)控制人血管平滑肌细胞(HVSMC)表型转换影响颅内动脉瘤(IA)发展的机制。方法HVSMC进行原代培养,HVSMC在5%胎牛血清的生长培养基SmGM-2(Lonza)中于37℃在加湿的5%CO_(2)培养箱中培养。根据研究方案将HVSMC细胞分为对照组、miR-124模拟转染组和miR-124-pCMV-Sp1Mut共转染组。通过定量RT-PCR分析HVSMC在血小板源生长因子(PDGF-BB)刺激后miR-124表达。通过BrdU检测和刮擦试验检测HVSMC的增殖迁移。通过荧光素酶报告基因测定检测miR-124与Sp1的靶向关系。通过蛋白印迹分析HVSMC标记基因和Sp1的蛋白表达。结果与0 h相比,PDGF-BB(20 ng/mL)诱导后第6、12、24 h HVSMC中miR-124表达降低,差异有统计学意义(P<0.05)。miR-124模拟转染组细胞增殖和细胞迁移能力较对照组增强,差异均有统计学意义(P<0.05)。miR-124模拟转染组与Sp1-WT3'-UTR融合的荧光素酶活性较对照组降低,差异有统计学意义(P<0.05),而miR-124模拟转染组与Sp1-MUT的3'-UTR融合的荧光素酶活性与对照组相比,差异无统计学意义(P>0.05)。miR-124模拟转染组α-SMA、SM22a、calponin和MYH11蛋白表达较对照组升高,miR-124-pCMV-Sp1Mut共转染组α-SMA、SM22a、calponin和MYH11蛋白表达较miR-124模拟转染组降低,差异均有统计学意义(P<0.05)。miR-124模拟转染组Sp1蛋白表达较对照组降低,miR-124-pCMV-Sp1Mut共转染组Sp1蛋白表达较miR-124模拟转染组升高,差异均有统计学意义(P<0.05)。结论miR-124通过Sp1调节HVSMC的表型并显著抑制IA发展,miR-124对Sp1表达的调节可能为miR-124的功能脑血管疾病的分子机制提供新的见解。

Objective To explore the mechanism by which microRNA-124(miR-124)controls the phenotype switch of human vascular smooth muscle cells(HVSMC)through Sp1 and affects the development of cerebral aneurysm.Methods The HVSMC were cultured in 5%fetal bovine serum growth medium SMGM-2(Lonza)in a humidified 5%CO_(2) incubator at 37℃.According to the study protocol,HVSMC cells were divided into control group,miR-124 simulated transfection group and miR-124-PCMV-SP1MUT co-transfection group.The expression of miR-124 in HVSMC after PDGF-BB stimulation was analyzed by quantitative RT-PCR.Value added migration of HVSMC was detected by BrdU and scratch test.The targeting relationship between miR-124 and Sp1 was detected by luciferase reporter gene assay.The expression of HVSMC marker gene and Sp1 protein was analyzed by Western blotting.Results Compared with 0 h,miR-124 expression was decreased in HVSMC at 6 h,12 h and 24 h after pdgf-bb(20 ng/mL)induction,the difference was statistically significant(P<0.05).The ability of cell proliferation and cell migration in the miR-124 mock transfection group was enhanced compared with the control group,the differences were statistically significant(P<0.05).The luciferase activity of the miR-124 simulated transfection group was lower than that of the control group,the difference was statistically significant(P<0.05);however,there was no significant difference in the luciferase activity of the miR-124 mock transfection group and the 3'-UTR fusion of Sp1-MUT compared with the control group(P>0.05).The protein expressions ofα-SMA,SM22a,Calponin and MYH11 in miR-124 simulated transfection group were higher than those in control group,the differences were statistically significant(P<0.05);the protein expressions ofα-SMA,SM22a,Calponin and MYH11 in the miR-124-PCMV-SP1MUT co-transfection group were lower than those in the miR-124 simulated transfection group,the differences were statistically significant(P<0.05).Compared with the control group,the expression of Sp1 protein in the miR-124 simulated transfection group was decreased,and the expression of Sp1 protein in the miR-124-PCMV-SP1MUT co-transfection group was increased compared with the miR-124 simulated transfection group,the differences were statistically significant(P<0.05).Conclusion MiR-124 regulates the phenotype of HVSMC through Sp1 and significantly inhibits IA development,and the regulation of Sp1 expression by miR-124 may provide new insights into the molecular mechanism of miR-124 function in cerebrovascular disease.