缺氧状态下miR-145对兔平滑肌细胞生物学行为的影响

Effect of miR-145 on Biological Behavior of Rabbit Smooth Muscle Cells in Hypoxic Condition

目的:探讨缺氧条件下miR-145对血管平滑肌细胞(VSMC)增殖、迁移及凋亡的影响及可能作用机制。方法:采用组织贴壁方法培养获得兔原代VSMC。用脂质体转染法将miR-145mimics转入VSMC细胞。实验设置为常氧条件下(21%O_2,5%CO_2,74%N2)空白对照组、miR-NC组、miR-145mimic组、miR-145inhibitor组;缺氧条件下(3%O_2,5%CO_2,92%N_2)空白对照组,miR-NC组,miR-145mimic组,miR-145inhibitor组。应用real time PCR检测miR-145在各组细胞表达水平。采用EdU染色检测VSMC增殖能力,Transwell实验检测VSMC细胞迁移能力,流式细胞仪检测细胞凋亡。采用targetScan,miRanda和PicTar靶基因预测软件和DAVID数据库对miR-145进行生物信息学分析,预测可能的靶基因。结果:在常氧与缺氧条件下,与miR-NC组相比,转染miR-145mimic组增殖率和迁移率明显减少,细胞凋亡率增高(P<0.05);转染miR-145inhibitor组增殖率和迁移率明显增加,细胞凋亡率下降(P<0.05)。同时与常氧条件相比,缺氧组增殖率和迁移率明显增加以及细胞凋亡率下降(P<0.05)。生物信息学分析提示miR-145靶基因的通路信号富集主要为促分裂素原活化蛋白激酶1(MAPK1),细胞周期蛋白依赖性激酶6(CDK6),钙/钙调素依赖蛋白激酶2D(Ca2+/CAMK2D)信号通路等。结论:缺氧条件下,在VSMC细胞中miR-145低表达、miR-145过表达可以有效的抑制VSMC细胞增殖、迁移及促进该细胞凋亡。

Objective:To explore the effect of miR-145 on proliferation,migration and apoptosis of vascular smooth muscle cell(VSMC)under hypoxic conditions and its possible mechanism.Methods:Primary rabbit VSMCs were collected by tissue anchored method.MiR-145 mimics was transfected stably into cultured VSMC cells in vitro by using cationic liposome.Then,cells were dividedinto following groups:blank group,miR-NC group,miR-145 mimic group,and miR-145 inhibitor group under normoxic condition(21% O_2,5% CO_2,74% N2)and blank group,miR-NC group,miR-145 mimic group,and miR-145 inhibitor group under hypoxic condition(3% O_2,5% CO_2,92% N2).MiR-145 expression level in each group was detected by real-time quantitative PCR.VSMC proliferation,migration and apoptosis were evaluated by using EdU staining,transwell assay and flow cytometry,respectively.Bioinformatic analysis of miR-145 to predict the possible target genes was carried out by mean of targetScan,miRBase,MiRanda target gene prediction software and DAVID database.Results:Under normoxic and hypoxic conditions,in comparison with the miR-NC group,the proliferation and migration rate of the transfected miR-145 mimic group were significantly reduced,while apoptosis rate increased(P<0.05);at the same time,proliferation and migration rate of the transfected miR-145 inhibitor group were significantly increased and apoptosis rate decreased(P<0.05).Furthemore,compared with normoxic conditions,the rate of proliferation and migration of hypoxic group was significantly increased and apoptosis rate decreased(P<0.05).Bioinformatics analysis revealed that miR-145 target gene pathways were mainly mitogen-activated protein kinase 1(MAPK1),cyclin-dependent kinase 6(CDK6),and calcium/calmodulin-dependent protein kinase 2D(Ca2+/CAMK2D),etc.Conclusion:Under hypoxic conditions,miR-145 was low-expressed in VSMC cells,while overexpression of miR-145 could effectively inhibit the proliferation and migration of VSMC and promote the apoptosis.