稳定表达DNA结合蛋白A慢病毒载体的构建及其对大肠癌细胞增殖的影响

Lentivirus-mediated RNA interference suppresses DNA binding protein A expression and inhibits proliferation of colorectal cancer cells

目的 观察慢病毒介导的RNA干扰（RNAi）沉默DNA结合蛋白A（dbpA）基因对大肠癌细胞株SW620增殖的影响,探讨该基因的作用.方法 实验分为3组：慢病毒干扰组（shRNA-dbpA组）、非特异性序列组（CON组）和空白对照组（NC组）.制备dbpA-短发卡RNA（shRNA）慢病毒表达载体,聚合酶链反应（PCR）筛选阳性克隆,测序鉴定.转染SW620细胞48h后,反转录-聚合酶链反应（RT-PCR）法对细胞中dbpA mRNA表达水平进行分析,Western blot检测SW620细胞的dbpA蛋白表达水平;同时采用流式细胞仪检测细胞凋亡和细胞周期变化,噻唑蓝（MTT）比色法检测细胞增殖抑制率,并检测细胞克隆形成能力.结果 PCR和测序证实,成功构建shRNA-dbpA病毒表达载体,shRNA-dbpA显著下调了SW620细胞中dbpA的表达,所构建的重组慢病毒有较高的基因沉默效率,Western blot检测结果表明shRNA-dbpA组的dbpA表达水平下调.流式细胞仪检测各组的凋亡率分别为（26.60±0.38）％、（1 2.54±0.25）％和（4.46±0.19）％,shRNA-dbpA组显著高于其他两组（P＜0.01）,且G0/G1期细胞增多,S期细胞减少,G2/M期细胞增多（P＜0.05）;MTT法检测结果显示各组的增殖第5天吸光度值分别为1.183±0.226、5.295±0.282和10.207±0.383,shRNA-dbpA组显著低于其他两组（P＜0.01）;细胞克隆数分别为（37±3）、（64±5）和（175±10）个,shRNA-dbpA组显著低于于其他两组（P＜0.01）.结论 以dbpA为靶标的RNA干扰能下调大肠癌细胞株SW620中的dbpA表达,显著增加细胞的凋亡,阻滞细胞于G0/G1期,降低了细胞的增殖克隆能力.

Objective To investigate the effects of lentivirus-mediated RNA interference （RNAi） targeting DNA binding protein A （dbpA） on the proliferation of colorectal cancer cell line SW620.Methods The lentiviral vector shRNA-dbpA was constructed and verified by polymerase chain reaction （PCR） and DNA sequencing.SW620 cells were transfected with shRNA-dbpA plasmid,nontargeting shRNA plasmid,or empty plasmid.Forty-eight hours after the transfection,the cells were examined for dbpA expression using Western blotting.Seventy-two hours after transfection,flow cytometry was used to detect the cell apoptosis and cell cycle changes.The cell growth inhibition rate was detected by methyl thiazol tetrazolium （MTT） assay,and then clone formation was detected.Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting dbpA gene was successfully inserted into the lentiviral vector.shRNA-dbpA transfection resulted in obviously reduced expression of dbpA in SW620 cells.After transfection,the apoptosis rate of shRNA-dbpA-transfected cells were increased to （26.60 ±0.38）%,significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid [（12.54 ±0.25）% and （4.46 ±0.19）%,respectively,P 〈0.01] ; the cell number in G0/G1 phase increased while that in G2/M phase decreased in shRNA-dbpA-transfected cells.The growth inhibition test indicate that the OD value of the fifth day in shRNA-dbpA group was 1.183 ± 0.226,significantly lower than that in the other two groups （5.295 ± 0.282 and 10.207 ± 0.383,respectively,P〈0.01）.The colony formation number is （37 ±3）、（64 ±5） and （175 ± 10） respectively,shRNA-dbpA is significantly higher than that in the other two groups（P 〈 0.01）.Couclusion Lentivirus-mediated RNAi targeting dbpA can effectively suppress the expression of dbpA in SW620 cells and decrease the proliferation of the colorectal cells.