论文部分内容阅读
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。
AIM: To investigate the effect of miR-145 on the proliferation, migration and apoptosis of vascular smooth muscle cells (VSMCs) under hypoxia and its possible mechanism. Methods: Rabbit primary VSMCs were obtained by tissue adherent method. MiR-145mimics were transfected into VSMC cells by lipofection. The experiment was set as blank control group, miR-NC group, miR-145mimic group and miR-145inhibitor group under normoxia conditions (21% O2, 5% CO2, 74% N2) CO_2, 92% N_2) blank control group, miR-NC group, miR-145mimic group and miR-145inhibitor group. Real-time PCR was used to detect the expression of miR-145 in each group of cells. The proliferation of VSMCs was detected by EdU staining. The migration of VSMCs was detected by Transwell assay and the apoptosis was detected by flow cytometry. Bioinformatics analysis of miR-145 was performed using targetScan, miRanda and PicTar target gene prediction software and DAVID database to predict possible target genes. Results: Compared with miR-NC group, the proliferation and migration rates of miR-145mimic group were significantly decreased and the apoptosis rate was increased (P <0.05) under the conditions of normoxia and hypoxia. The miR-145inhibitor group Proliferation rate and migration rate increased significantly, the apoptosis rate decreased (P <0.05). At the same time, compared with normoxic condition, the proliferation and migration rates of hypoxia group were significantly increased and the apoptosis rate was decreased (P <0.05). Bioinformatics analysis suggested that the signal pathways of miR-145 target genes were mainly MAPK1, CDK6, and calcium / calmodulin-dependent protein kinase 2D Ca2 + / CAMK2D) signaling pathway. CONCLUSION: Under hypoxia, the expression of miR-145 is low in VSMC cells. Overexpression of miR-145 can effectively inhibit VSMC proliferation, migration and apoptosis.