Rock2对紫外线诱导肝癌细胞DNA损伤的保护作用及调节机制

摘要

背景与目的:Rock2基因在肝癌等多种恶性肿瘤中呈现高表达,本研究利用紫外线(ultraviolet,UV)诱导DNA损伤,观察Rho相关卷曲螺旋形成蛋白激酶2(Rho associated coiled coilforming protein kinase 2,Rock2)基因对肝癌细胞生存的保护作用,并探讨其可能的作用机制.方法:将特异性抑制Rock2表达的干扰质粒shRock2转染到人肝癌细胞Huh-7和HepG2中,用荧光定量PCR及蛋白质印迹法(Western blot)检测Rock2 mRNA及蛋白的表达.通过UV照射造成肝癌细胞DNA损伤模型,用MTT比色法检测肝癌细胞增殖的变化,Western blot检测细胞分裂周期素25A(cell division cycle 25A,Cdc25A)蛋白表达的变化,并用能量共振转移法检测Cdk2/Cyclin E活性的改变.结果:shRock2转染到肝癌细胞后,其mRNA及蛋白的表达均明显降低;经过UV诱导肝癌细胞DNA损伤时,Rock2低表达组肝癌细胞较Rock2正常组肝癌细胞增殖受到抑制.Western blot检测发现,UV诱导DNA损伤时抑制Rock2表达可导致肝癌细胞中Cdc25A表达进一步下降,并且Cdk2/CyclinE活性也随Cdc25A的降低而降低.结论:UV诱导肝癌细胞DNA损伤时,Rock2通过调节Cdc25A对肝癌细胞的生存起保护作用,可能为肝癌基因表达调控研究提供新的靶基因.%Background and purpose: Rock2 is overexpressed in many malignant tumors such as bepatocellular carcinoma (HCC). This study was aimed to investigate the effects of Rock2 on ultraviolet induced DNA damage of HCC. Methods: Specific inhibition plasmid shRock2 was transfected into Huh-7and HepG2 cells and Rock2 rnRNA and protein was detected by Real-time PCR and Western blot. DNA damage model was induced using ultraviolet exposure. MTT assay was used to detect cell proliferation, the change of Cdc25A expression was measured by Western blot and Cdk2/Cyclin E activity was detected by fluorescence resonance energy transfer kit. Remits: Rock2 mRNA and protein showed significantly decreased alter the transfeclion of shRock2 in Huh-7and HepG2 cells. MTT assay showed that proliferation of shRock2 cells was inhibited compared with siControl cells following exposure to UV induced DNA damage. Western blot aria lysis revealed that when exposed to UV, Cdc25A decreased more obviously in shRoek2 cells compared to siControl cells. After exposure to UV, the decrease in Cdk2/Cyclin E activity was more apparent in shRock2 cells compared to the si Control cells. Conclusion: When UV-induced DNA damage, Rock2 plays a protective effect on the survival of HCC by regulating Cdc25A and this provided a new target for treatment and gene expression regulation of HCC.