Rock2对细胞周期检查点Cdc25A的调节作用

目的: 探讨肝癌细胞中Rho相关含卷曲螺旋蛋白激酶2(Rock2)对细胞周期检查点细胞分裂周期蛋白25A(Cdc25A)的调节作用。方法: Western blotting 检测51对肝癌及癌旁组织中Rock2与Cdc25A的蛋白表达情况。构建并筛选shRock2干扰质粒,稳定转染到人肝细胞癌Huh-7和HepG2细胞中,Western blotting检测Cdc25A蛋白表达变化;针对Rock2干扰序列,利用PCR定点突变技术进行碱基突变,构建Rock2突变质粒从而"恢复"Rock2表达,分析Cdc25A蛋白表达变化并用MTT法检测肝癌细胞增殖的改变。Western blotting检测Rock2稳定低表达的肝癌细胞中检查点激酶1(Chk1)和检查点激酶2(Chk2)蛋白的变化,免疫共沉淀及免疫荧光分析Rock2与Cdc25A的相互作用。结果: Rock2与Cdc25A蛋白在肝癌组织中较癌旁组织呈现高表达,而且两者呈正相关。肝癌细胞中稳定低表达Rock2后,Cdc25A蛋白表达明显下降;"恢复"Rock2表达后,Cdc25A蛋白表达增加而且肝癌细胞也出现增殖加快现象。Rock2低表达对Chk1和Chk2蛋白变化无明显影响。免疫共沉淀表明Rock2与Cdc25A直接相互作用,免疫荧光检测表明Rock2与Cdc25A存在共定位。结论: Rock2对细胞周期检查点Cdc25A起直接的正向调控作用,而且不依赖于Chk1/Chk2,可能为肝癌基因表达调控研究提供新的靶基因。

Regulatory effects of Rock2 on cell cycle checkpoint Cdc25A

AIM: To investigate the regulatory effects of Rho-associated coiled-coil-containing protein kinase 2(Rock2) on the cell cycle checkpoint cell division cycle 25A(Cdc25A).METHODS: The protein expression levels of Rock2 and Cdc25A in 51 pairs of hepatocellular carcinoma and the adjacent tissues were detected by Western blotting.shRock2 plasmids were constructed,selected and stably transfected into hepatocellular carcinoma Huh-7 and HepG2 cells.The protein expression of Cdc25A in the cells was determined by Western blotting.Based on the Rock2 interfering sequences,we designed the primers and changed the 4 indicated bases via site-specific mutagenesis.The Rock2-mutant plasmid was verified by sequencing and was transfected into stable Rock2-knockdown cells.The protein expression of Cdc25A was detected by Western blotting,and the cell proliferation was measured by MTT assay.The protein levels of checkpoint kinase(Chk)1/Chk2 were also detected in stable Rock2-knockdown cells.The interaction between Rock2 and Cdc25A was measured by co-immunoprecipitation,and the co-localization of Rock2 and Cdc25A was detected by confocal laser scanning microscopy.RESULTS: Rock2 and Cdc25A were apparently up-regulated in hepatocellular carcinoma,with a significantly positive correlation.The protein expression of Cdc25A was significantly down-regulated in stable Rock2-knockdown cells.The expression of Chk1 and Chk2 was not changed following knockdown of Rock2.The co-immunoprecipitation results verified that Rock2 bound to Cdc25A.The results of confocal laser scanning microscopy showed that Rock2 and Cdc25A were co-localized in hepatocellular carcinoma cells.CONCLUSION: Rock2 positively regulates the cell cycle checkpoint Cdc25A,which is independent of Chk1/Chk2 and this may provide a new target gene for treatment of hepatocellular carcinoma.