慢病毒载体介导的RNAi技术构建稳定抑制β-catenin的人鼻咽癌细胞株  

Construction of a stable human nasopharyngeal carcinoma cell line with down-regulated expression of β-catenin

作　　者：姜睿[1] 张弓[1] 马磊[1] 李锡清[1] 肖广惠[1] 姚开泰[1] 

机构地区：[1]南方医科大学肿瘤研究所,广东广州510515

出　　处：《热带医学杂志》2011年第5期520-523,549,F0003,共6页Journal of Tropical Medicine

基　　金：973计划项目(2010CB529400)

摘　　要：目的建立稳定抑制β-catenin基因表达的人鼻咽癌6-10B细胞株,为探讨Wnt/β-catenin信号在鼻咽癌发生中的作用提供细胞模型。方法于β-catenin CTNNB1基因编码区选择3个siRNA靶点合成3对shRNA干扰序列,分别与pLKO.1载体连接,构建pLKO.1-sh-β-catenin质粒(干扰组1)、pLVTHM-sh-β-catenin(干扰组2)和pLKO.1-sh-β-catenin-Neg质粒(阴性对照组);将重组质粒与慢病毒包装质粒共转染293T细胞,收集含慢病毒颗粒的上清液感染人鼻咽癌6-10B细胞,感染pLKO.1-sh-β-catenin和pLKO.1-sh-β-catenin-Neg质粒的细胞经嘌呤霉素筛选并扩大培养后得到稳定克隆株。Western blot检测干扰组β-catenin抑制效率及下游基因c-myc蛋白的表达;四甲基偶氮唑盐(MTT)比色法检测比较细胞增殖状况,Transwell细胞迁移实验检测细胞迁移率。结果 Western blot检测结果显示pLKO.1-sh-β-catenin(干扰组1)干扰效率最佳,β-catenin及c-myc蛋白表达减少;MTT检测显示干扰β-catenin后细胞吸光度下降,细胞增殖受到抑制;穿过Transwell小室底膜的细胞数:干扰组1为(23.5±4.6)个,明显低于阴性对照组(50.3±5.3,P<0.01)及空白对照组(54.3±5.6,P<0.01)。结论成功构建了β-catenin shRNA慢病毒表达载体,建立了稳定抑制β-catenin基因表达的人鼻咽癌6-10B细胞株,为进一步研究以β-catenin为靶点的鼻咽癌基因治疗奠定基础。Objective To establish a nasopharyngeal carcinoma（NPC） cell line in which β-catenin expression is stably suppressed,and to investigate the role of Wnt/β-catenin pathway in nasopharyngeal tumorigenesis.Methods Three siRNA interference sequences targeting β-catenin gene CTNNB1 were designed.Double-stranded shRNA hairpins were synthetized and separately cloned into pLKO.1 and pLVTHM vector so as to produce three plasimds of pLKO.1-sh-β-catenin（Experimental group1）,pLVTHM-sh-β-catenin（Experimental group2） and pLKO.1-sh-β-catenin-Neg（negative control group）.The plasimds and the lentiviral packaging plasmids were co-transfected into the packaging cells 293T,then the supernatant with lentivirus was collected to infect 6-10B.After puromycin selection and culture expansion,stable cell clones were obtained.Intracellular β-catenin and c-myc protein expression was detected by Western blot.The effect of down-regulated expressed β-catenin by RNAi on cell proliferation was quantified by methylthiazoletetrazolium（MTT） assay.Results The total intracellular β-catenin and c-myc proteins were decreased in pLKO.1-sh-β-catenin transfected group.Results from the MTT assay reveaed that the proliferation of β-catenin knockdown cells was significantly decreased（P0.05）.The number of cells passing through the membrane of Transwell chamber in the pLKO.1-sh-β-catenin group（23.5±4.6） was lower than the negative control（50.3±5.3,P0.01） and the blank control group（54.3±5.6,P0.01）.Conclusion Lentiviral shRNA expression vectors targeting β-catenin gene CTNNB1 were successfully constructed and the vextor could effectively downregulate the expression of β-catenin in 6-10B cells.The Wnt/β-catenin signal transduction pathway was also inhibited.This established NPC cell line may be used as a cell model to further study the β-catenin gene-targeted therapy on nasopharyngeal carcinoma.

关 键 词：WNT/Β-CATENIN 慢病毒载体 RNAI 鼻咽癌 

分 类 号：R739.63[医药卫生—肿瘤]