Objectives: To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD).
Methods: Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration.
Results: Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05).
Conclusions: Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.
目的: 研究抑制Ras同源基因（Ras homolog gene, Rho）/Rho相关卷曲螺旋形成蛋白激酶（Rho-associated coiled-coil forming protein kinase, ROCK）通路对心肌素（myocardin, MYOCD）参与的气道平滑肌细胞增殖与迁移的影响及分子机制。方法: 体外构建腺病毒载体Ad-ZsGreen-shRNA-hROCK1感染人气道平滑肌细胞，将细胞随机分为4组：ROCK1基因沉默对照（shNC）组、shNC+花生四烯酸（arachidonic acid, AA；Rho/ROCK通路激活剂）组、ROCK1基因沉默（shROCK1）组、shROCK1+AA组（各组n=3）。采用实时荧光定量聚合酶链反应法、Western blot法检测ROCK1、MYOCD mRNA和蛋白表达水平，ELISA法检测球状肌动蛋白和丝状肌动蛋白水平，免疫荧光染色法及细胞划痕实验检测细胞增殖及迁移情况。结果: 与shNC+AA组相比，shROCK1+AA组ROCK1、MYOCD mRNA和蛋白表达水平降低，球状肌动蛋白、丝状肌动蛋白表达降低，细胞增殖、细胞迁移能力降低（P<0.05）。结论: 抑制Rho/ROCK通路致气道平滑肌细胞增殖及迁移能力受抑，其机制可能与MYOCD的下调有关。.
Keywords: Airway smooth muscle cell; Asthma; Myocardin; Proliferation; Rho/ROCK pathway.