miR-21降解PTEN影响香烟提取物诱导的PASMCs的增殖与迁移

miR-21 Lowers the Level of PTEN to Influence the Proliferation and Migration Induced by Cigarette Smoke Extract(CSE) in Pulmonary Artery Smooth Muscle Cells

目的探讨微小RNA-21(miR-21)的表达对香烟提取物诱导的肺动脉平滑肌细胞中的PTEN/PI_3K/AKT通路及增殖与迁移的影响。方法分离雄性SD大鼠的肺细小动脉平滑肌细胞(PASMC),10%CSE处理PASMCs 24h后分别在PASMCs中转染miR-NC、miR-21、anti-miR-NC、anti-miR-21,通过real-time-PCR检测转染效率。Western blot法检测4组PASMCs增殖及凋亡蛋白PCNA、Bcl-2、caspase-3以及PTEN和相关信号通路中AKT/P-AKT的表达量。通过MTT实验比较4组细胞增殖的变化。用细胞划痕和Transwell实验检测4组细胞迁移能力的变化。结果转染miR-21组的miR-21表达量与转染miR-NC组的比较表达量上升(P=0.000),而anti-miR-21组的比anti-miR-NC的miR-21表达量下降(P=0.000)。在10%CSE处理后,过表达miR-21后抑制了PTEN的表达从而上调了AKT/P-AKT,进而使PCNA和Bcl-2的表达上升,caspase-3的表达被抑制,相反敲降miR-21后PTEN表达上升,AKT/P-AKT活性下降,PCNA以及Bcl-2的表达被抑制,caspase-3表达上升。在10%CSE作用下过表达miR-21后PASMCs的增殖能力显著增强(P<0.01),而敲降miR-21后PASMCs的增殖能力减弱(P<0.01)。在10%CSE作用下过表达miR-21后PASMCs的迁移能力增强,而敲降miR-21后PASMCs的迁移能力减弱。结论 miR-21通过负向调控PTEN从而活化PI3K/AKT信号通路促进了香烟提取物诱导的PASMCs的增殖迁移。

Objective To investigate the role of miR-21 in PTEN/PI 3K/AKT pathway and proliferation,migration induced by cigarette smoke extract(CSE)in pulmonary artery smooth muscle cells(PASMCs).Methods The PASMCs in male SD rats were cultured in vitro and stimulated with 10%CSE for 24h,and then the miR-NC,miR-21,anti-miR-NC,anti-miR21 were transfected into the PASMCs respectively.The expressions of miR-21 mRNA were detected by real-time fluorescence RT-PCR.The expression of PCNA,Bcl-2,caspase-3 and PTEN,AKT/P-AKT were detected by Western blot.The proliferation of PASMCs was examined by MTT assay.(4)The migration of PASMCs was measured by Scratch and Transwell cell test.Results The miR-21 mRNA in transfect miR-21 group were elevated significantly than those in control group(P=0.000)but the miR-21 mRNA level in transfect anti-miR21 group were significantly lower than those in control group(P=0.000).Under 10%CSE stimulates miR-21 overexpression could up-regulation P-AKT/AKT,Bcl-2,PCNA and down-regulation of PTEN,caspase-3 while miR-21 knock down down-regulation P-AKT/AKT,Bcl-2,PCNA and up-regulation of PTEN and caspase-3.miR-21 overexpression promoted PASMCs proliferation(P<0.01),while miR-21 knock down attenued proliferation(P<0.01).Compared with the control,the migration abilitiy of PASMCs in miR-21 overexpression group significantly increased while in miR-21 knock down group significantly decreased.Conclusion miR-21 lowers the level of PTEN then promotes the activiation of PI 3K/AKT pathway,thereby accelerates the proliferation and migration induced by CSE of PASMCs.