miR-145靶向抑制MMP-9表达对大鼠脑动脉血管平滑肌细胞增殖和迁移的影响

摘要

目的 观察miR-145对大鼠脑动脉血管平滑肌细胞(VSMC)增殖和迁移的影响,并探讨其作用机制.方法 利用组织贴块法分离大鼠脑基底动脉VSMC,将细胞分为3组,对照组转染空载体;miR-145组转染miR-145;miR-145+MMP-9组转染miR-145后再转染MMP-9.采用qPCR方法检测各组MMP-9 mRNA表达,Western blotting法检测MMP-9蛋白表达.向三组细胞中加入50 ng/mL TNF-α刺激细胞增殖,采用CCK8法检测细胞增殖情况,Transwell小室检测细胞迁移能力.根据TargetScan和miRanda数据库预测,野生型MMP-9的3′-UTR区域含有miR-145靶向结合片段,采用双荧光素酶报告基因检测验证miR-145和MMP-9的靶向关系.结果 miR-145组MMP-9 mRNA和蛋白表达均低于对照组(P均<0.05),miR-145+MMP-9组MMP-9 mRNA和蛋白表达均高于miR-145组(P均<0.01).加入TNF-α处理后,miR-145组的细胞增殖OD值低于对照组,miR-145+MMP-9组的细胞增殖OD值高于miR-145组(P均<0.01);miR-145组细胞迁移数低于对照组,miR-145+MMP-9组细胞迁移数高于miR-145组(P均<0.01).双荧光素酶报告检测结果显示,miR-145能够抑制野生型MMP-9的荧光素酶活性(P<0.01),但不能抑制突变型MMP-9的荧光素酶活性.结论 miR-145能够通过下调MMP-9抑制TNF-α诱导的大鼠脑动脉VSMC增殖和迁移.%Objective To observe the effects of microRNA-145 on proliferation and migration of rat arterial vascular smooth muscle cells (VSMC) and its mechanism.Methods The rat brain basilar arterial VSMC was isolated by tissue patch assay.Then the cells were divided into three groups: the control group (transfection with empty carrier), miR-145 group (transfection with miR-145 mimics), and miR-145+MMP-9 group (transfection with miR-145 mimics and MMP-9).The mRNA and protein expression of MMP-9 was detected by qPCR and Western blotting, respectively.We added 50 ng/mL TNF-α to stimulate the cell proliferation, which was then examined by CCK8 assay.Transwell chamber was used to detect the cell migration ability.TargetScan and miRNAda identified 3′-UTR of MMP-9 mRNA had the target sequence of miR-145 in VSMCs.Luciferase reporter gene was used to validate the correlation between miR-145 and MMP-9.Results The MMP-9 mRNA and protein expression of the miR-145 group was significantly lower than that of the control group (both P<0.05), while the MMP-9 mRNA and protein expression in miR-145+MMP-9 group was higher than that of the miR-145 group (both P<0.05).After TNF-α treatment, OD value of the miR-145 group was lower than that of the control group, and OD value in miR-145+MMP-9 group was higher than that of miR-145 group (both P<0.05).Meanwhile, the miR-145 group had a significantly smaller number of migration cells than the control group, while the migration cells in miR-145+MMP-9 group were more than those of the miR-145 group (both P<0.01).Luciferase reporter revealed that miR-145 inhibited the relative luciferase activity of wild type MMP-9, but did not inhibit the mutant MMP-9.Conclusion MiR-145 inhibits the proliferation and migration of rat basilar arterial VSMC through down-regulating MMP-9.