Bmi-1基因沉默对鼻咽癌细胞CNE2生物学行为的影响  

Effects of silencing Bmi-1 on biological behavior of nasopharyngeal carcinoma cell line CNE2

作　　者：王巧 罗清 李亚军[2,3] 邹彦[2] 

机构地区：[1]贵州省人民医院,贵阳550002 [2]遵义医学院附属肿瘤医院 [3]遵义医学院第三附属医院

出　　处：《山东医药》2018年第5期1-4,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81560444);贵州省科技厅社会发展攻关课题(黔科合SY字[2015]3046);贵州省遵义市汇川区科技局课题(20120724)

摘　　要：目的探讨慢病毒靶向沉默Bmi-1基因对鼻咽癌细胞CNE2生物学行为的影响。方法设计3条Bmi-1shRNA干扰系列,构建慢病毒重组载体PLVx-shRNA2-Bmi-1,用重组载体转染293T细胞,收集上层病毒液浓缩、滴定。将CNE2细胞随机分为5组,对照组不进行感染,shRNA1、2、3组分别感染表达shRNA1、2、3的慢病毒,空载体组感染空载的慢病毒。感染48 h,用RT-PCR及Western blotting法分别检测Bmi-1基因及蛋白的相对表达量,筛选沉默Bmi-1效率最佳的干扰序列。用平板克隆形成实验检测Bmi-1基因沉默后CNE2细胞增殖能力,用流式细胞术检测细胞凋亡率和周期分布。结果插入的序列与设计的3条shRNA序列碱基排列一致,说明目的片段均已正确插入,PLVx-shRNA_2-Bmi-1慢病毒表达载体构建成功。与其他两组比较,Bmi-1-shRNA1、2、3组Bmi-1 mRNA及蛋白相对表达量低(P均<0.05),Bmi-1-shRNA3组最低(P均<0.05),沉默效率最佳。与空载体组比较,Bmi-1-shRNA3组增殖能力弱(P均<0.05),细胞凋亡率高(P均<0.05),Bmi-1-shRNA3组G1期细胞比例高、S期细胞比例低(P均<0.05)。结论沉默Bmi-1基因可抑制鼻咽癌CNE2细胞增殖、促进其凋亡,并改变细胞周期。Objective To investigate the effects of silencing Bmi-1 gene on the biological behavior of nasopharyngeal carcinoma cell line CNE2. Methods Three primers of shRNA targeting the Bmi-1 were designed,and the recombinant plasmid PLVx-shRNA2-Bmi-1 was constructed. Virus particles were collected and the viral titer was determined after the recombinant plasmid was transfected into 293 T cells. CNE2 cells were randomly divided into five groups,control group were named for untransfected lentivirus. CNE2 cells were transfected with shRNA1,2,and 3,respectively,which were named as the shRNA1 group,shRNA2 group,and shRNA3 group; cells transfected with the empty lentivirus were named as the empty vector group,and the control group was not transfected. The protein and mRNA expression levels of Bmi-1 were detected by using real-time PCR and Western blotting at 48 h after transfection and we screened out the best interference sequence with highest inhibition efficiency. Colony formation assay was used to detect ability of cell proliferation,and flow cytometry to detect the cell cycle distribution and apoptosis after silencing Bmi-1 gene. Results The sequences were inserted into the vectors which were consistent with the three designed primers,demonstrating that the recombinant lentivirus vectors containing PLVx-shRNA2-Bmi-1 were successfully constructed. The Bmi-1 shRNA 1,2,and 3 groups had lower expression of Bmi-1 mRNA and protein than that of the other two groups（ both P 0. 05）,and the Bmi-1 shRNA 3 group had the lowest expression of Bmi-1 mRNA and protein,and had the highest inhibition efficiency（ both P 0. 05）. Compared with the empty vector group,the proliferation increased and the apoptosis rate increased in the Bmi-1 shRNA 3 group（ both P 0. 05）. There were higher percentage of cells in the G1 phase and lower percentage of cells in the S phase（ both P 0. 05）. Conclusion Silencing Bmi-1 gene can inhibit the proliferation of nasopharyngeal carcinoma CNE2 cells,promote its apoptosis,and change the cell cycle.

关 键 词：鼻咽癌 BMI-1基因 CNE2细胞 细胞增殖 细胞凋亡 细胞周期 

分 类 号：R739.6[医药卫生—肿瘤]