慢病毒介导的靶向干扰MEX3A基因膀胱癌稳定细胞株的建立

摘要

目的 构建针对MEX3A基因的shRNA慢病毒载体,建立稳定干扰MEX3A基因表达的膀胱癌细胞株.方法 采用实时PCR检测膀胱癌细胞株中MEX3A基因的表达;应用GV115质粒构建重组靶向MEX3A基因的shRNA慢病毒载体,鉴定及测序合格后,与包装载体共转染293T细胞产生慢病毒颗粒,病毒滴度测定后转染膀胱癌细胞,经抗生素筛选建立稳定表达siRNA的细胞株,实时PCR检测敲减效率.结果 MEX3A基因在膀胱癌5637和T24细胞中均有表达,且5637的表达量高于T24;鉴定及测序结果显示成功构建MEX3A-shRNA慢病毒载体,包装后病毒滴度较高,实时PCR结果表明慢病毒转染5637细胞后能稳定抑制MEX3A基因的表达.结论 应用慢病毒介导的RNA干扰技术可成功建立稳定抑制MEX3A基因表达的细胞株.%Objective To construct an shRNA lentiviral vector targeting the MEX3A gene and establish a bladder cancer cell line with stable MEX3A gene knockdown.Methods Real-time PCR was performed to detect the MEX3A gene expression.The recombinant lentiviral vector targeting the MEX3A gene was constructed using the GV115 plasmid.After identification and sequencing,the vectors were co-transfected with the packaging vector into 293T cells to produce lentiviral particles,which were then transduced into bladder cancer cells after viral titer determination.The cell line stably expressing the siRNA was established by antibiotic selection,and real-time PCR was carried out to detect the efficiency of the knockdown.Results Both bladder cancer cell lines,5637 and T24,expressed the MEX3A gene,and its expression was higher in 5637 than in T24.The identification and sequencing results showed that the MEX3A-shRNA lentiviral vector was successfully constructed,and the virus titer was observed to be higher after packaging.The results of the real-time PCR showed that MEX3A gene expression was stably inhibited in 5637 cells after lentiviral transduction.Conclusion Lentivirus-mediated RNAi technology could successfully establish a cell line with stable MEX3A gene knockdown.