抑制HSP70-2基因表达诱导肝癌细胞G_2/M期阻滞的机制研究  

Knockdown of HSP70-2 induces G_2/M arrest in hepatocellular carcinoma cells

作　　者：夏丽敏[1] 田德安[1] 张琼[1] 晏维[1] 周珍珍[1] 朱倩[1] 罗敏[1] 张全乐[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院消化内科,湖北武汉430030

出　　处：《胃肠病学和肝病学杂志》2008年第12期966-970,共5页Chinese Journal of Gastroenterology and Hepatology

摘　　要：目的探讨HSP70-2在肝癌组织中的表达,以及抑制HSP70-2表达对肝癌细胞生长和周期影响的详细作用机制。方法免疫组织化学检测HSP70-2在45例肝癌组织和癌旁组织中的表达,Western blot检测HSP70-2在5种肝癌细胞株中的表达。设计合成针对HSP70-2基因的特异性shRNA,构建真核表达载体HSP70-2 shRNA1和HSP70-2 shRNA2。转染肝癌细胞后,MTT法检测细胞增殖,流式细胞仪检测细胞周期,Western blot检测HSP70-2表达及细胞周期相关蛋白p-Cdc2(Tyr15)、Cdc25C、Cdc2和cyclinB1的表达变化。结果免疫组织化学检测显示,45例肝癌组织中有33例(73.3%)HSP70-2蛋白表达阳性,45例癌旁组织中仅4例(8.9%)表达阳性,表达差异有显著性(P<0.05)。HSP70-2蛋白在HepG2、Bel-7402、Huh-7、Hep3B、SMMC-7721肝癌细胞中的表达明显高于其在L02细胞中的表达。HSP70-2 shRNA1和shRNA2均能有效抑制肝癌细胞HepG2和Bel-7402中HSP70-2的表达。与control shRNA组相比,转染HSP70-2 shRNA1和shRNA2组HepG2和Bel-7402细胞生长增殖速度均明显减慢,细胞周期表现为处于G2/M期的细胞比例显著增加。Western blot检测发现,转染HSP70-2 shRNA1和shRNA2后肝癌细胞中磷酸化的p-Cdc2(Tyr15)表达增加,Cdc25C、Cdc2和cyclinB1表达显著减少。结论HSP70-2在肝癌中过表达,抑制HSP70-2表达可以显著抑制肝癌细胞的增殖,诱导细胞周期阻滞,其诱导G2/M期阻滞的机制是通过影响Cdc25C-Cdc2/cyclinB1通路来实现的。Objective To explore the expression of HSP70-2 in hepatocellular carcinoma （HCC） and to determine the effect of short hairpin RNA targeting HSP70-2 on proliferation and cell cycle of HCC cells. Methods The expressions of HSP70-2 in HCC tissues （n = 45） and corresponding adjacent tissues were detected by immunohistochemistry. Western blot analysis was used to detect the expression of HSP70-2 in five HCC cell lines. The recombinant plasmid HSP70-2 shRNA1 and shRNA2 were separately transfected into HCC cells conducted by lipofectamineTM2000. The expression of HSP70-2 was detected by Western blot and the subsequent effects on proliferation of HepG2 and Bel-7402 cells were determined by MTT. Cell cycle was determined by flow cytometery. Western blot was used to analyze the expressions of cell cycle related proteins including p-Cdc2 （Tyr15）, Cdc25C, Cdc2 and cyclinB1. Results The expression of HSP70-2 was elevated in majority HCC patient samples （36/45, 73.3% ） and I4CC cell lines. Two pairs of DNA sequences containing small hairpin structure to HSP70-2 were proved to be successfully cloned into expression vector pSilence-2, 1-U6. HSP70-2 shRNA1 and shRNA2 could efficiently suppress the expression of HSP70-2 at protein levels. After transfected with HSP70-2 shRNA1 and shRNA2 for 48 h, the proliferaiion of HCC cells was significantly suppressed and the proportion of ceils in GJM phase increased. Knockdown of HSP70-2 increased the expression of p- Cdc2 （Tyrl5） and decreased the expression of Cdc25C, Cdc2 and cyclinB1. Conclusion Our data indicate that knockdown of HSP70-2 significantly suppresses the proliferation of HCC cells and induces cell cycle arrest. The GJM arrest by HSP70-2 depletion is associated with Cdc25C-Cdc2/cyclinB1 pathway.

关 键 词：HSP70-2 肝癌 RNA干扰 细胞周期 

分 类 号：R735.7[医药卫生—肿瘤]