牛痘相关激酶1在小鼠肺动脉平滑肌细胞增殖和迁移中的作用及机制

Role and mechanism of vaccinia-related kinase 1 in proliferation and migration of mouse pulmonary arterial smooth muscle cells

目的:探讨牛痘相关激酶1(vaccinia-related kinase 1, VRK1)在小鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)增殖及迁移中的作用及机制。方法:培养小鼠肺动脉平滑肌细胞系,细胞传至第3代至第5代细胞进行后续实验。在低氧条件下检测不同低氧时间干预后PASMC中VRK1蛋白水平的变化。为探究VRK1在低氧所致PASMCs增殖和迁移中的作用,通过携带Vrk1特异短发夹RNA(Vrk1 short hairpin RNA,sh Vrk1)或相关阴性对照RNA(shCon)的慢病毒转染PASMCs,将转染后的PASMCs分别进行常氧、低氧处理,Western blot检测VRK1和β-catenin蛋白水平变化,采用Ki-67细胞免疫荧光染色法检测PASMCs的增殖活性,划痕实验检测PASMCs的迁移能力。加入40μmol/L SKL2001恢复β-catenin活性后再次检测上述指标。结果:与常氧组相比,低氧处理24 h和36 h后PASMCs中VRK1蛋白表达水平升高(P<0.01)。常氧培养条件下,sh Vrk1转染细胞中VRK1及β-catenin蛋白表达水平降低(P<0.01)。相较于常氧培养的shCon转染细胞,低氧条件下shCon转染细胞中Ki-67阳性细胞率升高,划痕愈合速度提高(P<0.01)。低氧培养条件下,sh Vrk1转染后细胞的Ki-67阳性细胞率降低,划痕愈合速度降低(P<0.01)。低氧培养条件下,sh Vrk1+SKL2001组β-catenin蛋白表达水平升高,Ki-67阳性细胞率及划痕愈合速度较sh Vrk1+DMSO组均升高(P<0.01)。结论:沉默Vrk1通过降低β-catenin的表达抑制低氧所致小鼠PASMCs体外的增殖和迁移。

AIM:To investigate the role and mechanism of vaccinia-related kinase 1(VRK1) in the proliferation and migration of mouse pulmonary arterial smooth muscle cells(PASMCs). METHODS:The mouse PASMCs were cultured in hypoxic condition of 3% O2, 5% CO2and 92% N2. The protein expression level of VRK1 after hypoxia exposure was assessed by Western blot. To explore the role of VRK1 in proliferation and migration of mouse PASMCs, lentivirus carrying short hairpin of Vrk1(sh Vrk1) was used to silence of Vrk1. Transfected PASMCs were cultured in normoxia and hypoxia condition. The protein expression levels of VRK1 and β-catenin were determined by Western blot. The proliferation of PASMCs was assessed by Ki-67 immunofluorescence staining. The migration was analyzed by Scratch assay.SKL2001(40 μmol/L) was used to restore the activity of β-catenin, and then the proliferation and migration were estimated by methods as the above mentioned. RESULTS:Compared with shCon(normoxia) group, the protein level of VRK1 was increased after 24 and 36 h of exposure to hypoxia(P<0. 01). Under normoxia condition, the protein levels of VRK1 and β-catenin were obviously reduced in sh Vrk1 transfected cells. Compared with shCon(normoxia) group, the ratio of Ki-67-positive cells and the rate of wound healing were both increased in shCon(hypoxia) group(P<0. 01). Under hypoxia condition, the ratio of Ki-67-positive cells and the rate of wound healing were obviously reduced after sh Vrk1 transfected(P<0. 01). Compared with sh Vrk1+DMSO(hypoxia) group, the ratio of Ki-67-positive cells,the rate of wound healing and the protein level of β-catenin were all increased in sh Vrk1+SKL2001(hypoxia) group(P<0. 01). CONCLUSION:Silencing Vrk1 attenuates proliferation and migration of mouse PASMCs by reducing the activity of β-catenin.