靶向Polo-like kinase 1基因的shRNA对肝癌BEL-7402细胞株生物学行为的影响  

Study of the Influence of siRNA Targeting Plk1 Gene on the Biological Behaviors of Hepatocellular Carcinoma Cell Line BEL-7402

作　　者：郑核 钟德玝[2] 何自力[2] 苗雄鹰[2] 文宇[2] 刘威[2] 陈广[2] 

机构地区：[1]湖南省邵阳市中心医院,邵阳422000 [2]中南大学湘雅二医院,长沙410011

出　　处：《中国现代手术学杂志》2011年第6期401-406,共6页Chinese Journal of Modern Operative Surgery

基　　金：邵阳市科技计划项目(CO968)资助课题

摘　　要：目的利用RNA(RNAi)干扰技术,研究表达Polo-like kinase 1(Plk1)的短发夹RNA(shR-NA)载体对肝癌细胞株BEL-7402生物学行为的影响。方法根据靶基因的不同区域构建针对Plk1mRNA的两组干扰质粒shRNA-Plk1:Plk1siRNA-251,Plk1siRNA-1396以及阴性对照质粒Plk1siRNA-hk,利用电穿孔法转染入肝癌细胞株BEL-7402,获得稳定表达的克隆后,RT-PCR检测肝癌细胞株BEL-7402的Plk1mRNA的变化,Western blot检测Plk1蛋白的表达,MTT法检测细胞增殖,流式细胞仪检测细胞周期变化。结果利用电穿孔将载体转入肝癌细胞株BEL-7402,利用G418筛选,获得稳定表达的克隆。RT-PCR检测Plk1siRNA-251,Plk1siRNA-1396组Plk1mRNA表达较Plk1siRNA-hk转染组与空白对照组明显下降(P<0.01)。Western blot检测转染BEL-7402细胞shRNA载体后的Plk1蛋白的表达分别为0.032±0.007、0.040±0.008、0.272±0.041、0.278±0.053。MTT检测发现转染shRNA载体后BEL-7402细胞增殖明显减慢。细胞周期检测证实转染Plk1siRNA可以引起BEL-7402细胞G0/G1期减少,G2/M期增高,而对S期影响不大。结论成功构建出两条能特异而高效抑制Plk1基因表达的shRNA,可以显著抑制Plk1 mRNA的转录及Plk1蛋白的表达,并明显抑制细胞增殖,从而为肝癌的基因治疗提供新的切入点。Objective To study the influence of short hairpin RNA（shRNA） targeting Plk1 on the biological behavior of hepatocellular carcinoma cell line BEL-7402.Methods One pair of shRNA targeting Plk1 and a negative control nonsense shRNA were designed and synthesized and inserted into plasmid pGenesil-2 to generate siRNA eukaryotic expression vector.The recombinant eukaryotic plasmids were transmitted into BEL-7402 cells by electroporation.After gained stable expressed clones,the biological behaviors of the Plk1 siRNA transfected cells were observed by the experiments such as RT-PCR、Western blot、MTT and flow cytometry.Results The stable expressed Plk1 siRNA cell clone were selected by G418 after electroporation transfection into BEL-7402 successfully.Cell sublines stable transfection was identified by Plk1siRNA-251,Plk1siRNA-1396 and Plk1siRNA-hk.RT-PCR demonstrated that the expression of Plk1 mRNA decreased in Plk1siRNA-251,Plk1siRNA-1396 groups significantly compared with that in Plk1siRNA-hk and control groups.Western blot demonstrated that the expression ratio of Plk1 protein were 0.032±0.007、0.040±0.008、0.272±0.041、0.278±0.053 in Plk1siRNA-251,Plk1siRNA-1396,Plk1siRNA-hk and control group separately.Compared with the other two groups,Plk1siRNA-251 and Plk1siRNA-1396 groups grew more slowly and had a higher proportion of cells in G2/M phase whereas fewer cells in G0/G1 phase.Conclusion The expression of Plk1 mRNA was inhibited in stable expressed Plk1 siRNA cell clone.The transfection of targeting Plk1 siRNA vector could inhabit the proliferation of BEL-7402 cells.

关 键 词：肝肿瘤 实验性 RNA干扰 PLK1 

分 类 号：R735.7[医药卫生—肿瘤]