靶向Plk1基因的shRNA对肝癌BEL-7402细胞株生物学行为及化疗增敏的影响  

Study of the Influence of siRNA Targeting Plk1 Gene on the Biological Behaviors and Enhancement of Vincristine Chemotherapeutic Sensitivity to 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》2012年第1期2-7,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干扰技术(RNAi),研究表达Polo-like kinase 1(Plk1)的短发夹RNA(shRNA)载体对肝癌细胞株BEL-7402生物学行为及对化疗药物长春新碱敏感性的影响。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,用长春新碱处理细胞后,用MTT法检测四组细胞的生长抑制率,计算IC50值,流式细胞仪检测各组细胞凋亡。结果半定量RT-PCR检测Plk1siRNA-251,Plk1siRNA-1396组Plk1mRNA、Cdc25CmRNA表达较Plk1siRNA-hk转染组与空白对照组明显下降(P<0.01),p53mRNA表达增加。长春新碱处理后,Plk1siRNA-251组和Plk1siRNA-1396组与BEL7402组之间的生长抑制率有显著性差异(P<0.01)。5μg/ml浓度长春新碱作用24 h后,Plk1siRNA-251组和Plk1siRNA-1396组凋亡指数分别为19.09%±3.97%、19.80%±5.47%明显高于Plk1siRNA-hk组和BEL7402组(P<0.01)。结论成功筛选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、Cdc25CmRNA及Plk1蛋白的表达,并使p53mRNA表达增高,并明显抑制细胞增殖,能增加长春新碱的化疗敏感性,从而为进一步研究该基因的功能和作用机制提供了新的手段。Objective To study the influence of shRNA targeting Plk1 on the biological behavior and enhancement of vincristine chemotherapeutic sensitivity of hepatocellular carcinoma cell line BEL-7402.MethodsOne pair of short hairpin RNA targeting Plk1 and a negative control nonsense short hairpin RNA 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.MTT assay was used to detect the cell growth inhibiting rate of transgene tumor cells affected by vincristine and IC50 were calculated.Flow cytometry dyeing was used to demonstrate the apoptosis index of different cells.Results Cell sublines stable transfection was identified by Plk1siRNA-251,Plk1siRNA-1396,Plk1siRNA-hk.Semi-quantitive RT-PCR demonstrated that the expression of Plk1mRNA and Cdc25CmRNA decreased and the expression of p53mRNA significantly elevated in the Plk1siRNA-251,Plk1siRNA-1396 groups,comparing with that in the Plk1siRNA-hk and control group.Observed by MTT assay to hepatocellular carcinoma cells affected by vincristine,the growth inhibiting rate of Plk1siRNA-251 and Plk1siRNA-1396 group cells were significantly higher than that of Plk1siRNA-hk and control group cells（P0.01）.After given 5μg/ml vincristine 24h later,the apoptosis index of Plk1siRNA-251 and Plk1siRNA-1396 group were 19.09%±3.97%,19.80%±5.47%,both increased significantly comparing with the Plk1siRNA-hk group and the control group.Conclusions The transcription of Plk1,Cdc25C mRNA and expression Plk1 protein are inhibited,the transcription of p53 mRNA increase in the stable expressed Plk1 siRNA cell clone.The transfection of targeting Plk1 siRNA vector could inhabit the proliferation of BEL-7402 cells.The siRNAs targeting Plk1 can not only suppress the expression of Plk1 in hepatocellular carcinoma cell,but also enhance the chemotherapy sensitivity,which deserve to be studied further.

关 键 词：肝肿瘤 实验性 RNA干扰 长春新碱 

分 类 号：R735.7[医药卫生—肿瘤]