27nt-miRNA对血管平滑肌细胞SM22α表达的调节及其对细胞活力、迁移和表型改变的影响

摘要

目的:探讨27nt-微小RNA (27nt-miRNA) 对血管平滑肌细胞 (VSMCs) 中平滑肌22α蛋白 (SM22α表达的调节及其对细胞活力、迁移和表型改变的影响.方法:构建27nt-miRNA高表达、反义序列 (anti-27nt-miRNA) 以及阴性对照的表达质粒, 经慢病毒包装后分别转染大鼠原代VSMCs, 加入血小板源性生长因子BB (PDGF-BB) 诱导VSMCs表型转换.MTT实验检测细胞活力, 划痕实验检测细胞迁移能力, RT-PCR、Western blot和免疫细胞化学染色法检测细胞中SM22α的mRNA和蛋白表达情况.结果:与正常组相比, PDGF-BB组的细胞活力上升 (P <0.05) 、迁移能力上升 (P <0.05) , SM22α的mRNA及蛋白表达量下降 (P <0.05) ;与阴性对照慢病毒组相比, 27nt-miRNA高表达组的细胞活力下降 (P <0.05) , 迁移能力下降 (P <0.05) , SM22α的mRNA及蛋白表达量明显升高 (P <0.05) ;而anti-27nt-miRNA组细胞的细胞活力上升 (P <0.05) , 迁移能力上升 (P <0.05) , SM22α的mRNA及蛋白表达量下降 (P <0.05) .结论:27nt-miRNA促进SM22α表达, 同时抑制VSMCs的活力及迁移, 并有可能抑制VSMCs从收缩型转变为合成型.%AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs).METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs.Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion.The cell viability was measured by MTT assay.The migration ability was detected by scratch assay.The mRNA and protein expression of SM22αwas determined by RT-PCR, immunocytochemical staining and Western blot.RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05) , the migration ability was increased (P<0.05) and the expression of SM22αat mRNA and protein level was decreased (P<0.05).Compared with negative control lentiviral group, the cell viability in 27ntmiRNA over-expression group was decreased (P<0.05) , the migration ability was decreased (P<0.05) , and the mRNA and protein expression of SM22αwas increased (P<0.05).While in anti-27nt-miRNA group, the cell viability was increased (P<0.05) , the migration ability was increased (P<0.05) , and the mRNA and protein expression of SM22αwas decreased (P<0.05).CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.