小干扰RNA抑制成纤维细胞激活蛋白3的表达对肺癌干细胞增殖和侵袭的影响  

Effect of siRNA-Mediated Down-Regulation of Fibroblast Activation Protein 3 on the Proliferation and Invasion of Human Lung Cancer Stem Cells

作　　者：张立凡[1] 刘特 

机构地区：[1]复旦大学附属华东医院胸外科,上海200040 [2]上海中医老年医学研究所,上海200031

出　　处：《中国临床医学》2013年第1期5-7,共3页Chinese Journal of Clinical Medicine

基　　金：上海市科委医学引导基金项目(编号:10411967100)

摘　　要：目的:探讨小干扰RNA(siRNA)抑制成纤维细胞激活蛋白3(fibroblast activation protein 3,FAP3)基因表达对肺癌干细胞增殖和侵袭的影响。方法:采用流式细胞术从肺癌细胞株A549中分选出CD44+/CD133+的肺癌的肿瘤干细胞(cancerstem cells,CSCs)。将肺癌CSCs分为3组:空白对照组(不转染任何质粒)、siRNA-FAP组(转染靶向沉默FAP3表达的siRNA质粒)、阴性对照组(转染包含随机序列的siRNA质粒)。采用MTT比色法检测靶向FAP3表达的siRNA(siRNA-FAP3)转染12、24、48和72h后对细胞增殖的影响;采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)法和蛋白质印迹法(Western blot)分别检测转染72h后细胞株中FAP3、Ki67的mRNA和蛋白质的表达情况;采用软琼脂克隆集落形成实验检测siRNA-FAP3对肺癌CSCs侵袭能力的影响。结果:转染48h和72h后,siRNA-FAP3组的细胞增殖抑制率[(24.67±1.08)%,(53.98±3.75)%]大于阴性对照组[(3.76±0.88)%,(4.53±1.01)%]及空白对照组[(2.34±0.41)%,(3.28±0.65)%],差异均有统计学意义(P分别<0.05和0.01)。转染72h后,siRNA-FAP组细胞中FAP3和Ki67基因的mRNA表达水平(0.32±0.06,0.18±0.02)显著低于阴性对照组(1.03±0.12,0.99±0.17)和空白对照组(1.02±0.09,0.97±0.11),差异有统计学意义(P<0.01)。转染72h后,siRNA-FAP组细胞中FAP3和Ki67蛋白表达水平(0.16±0.05,0.25±0.09)显著低于阴性对照组(0.49±0.26,0.47±0.13)和空白对照组(0.59±0.27,0.63±0.22),差异有统计学意义(P<0.05)。siRNA-FAP组细胞克隆形成率[(13.09±5.21)%]显著低于阴性对照组[(77.59±8.29)%]和空白对照组[(83.86±10.11)%],差异有统计学意义(P<0.01)。结论:转染siRNA可有效抑制肺癌CSCs中FAP3蛋白的表达,并且还可抑制CSCs的体外增殖和侵袭能力。Objective：To investigate the effect of siRNA-mediated down-regulation of fibroblast activation protein 3（FAP-3） on the proliferation and invasion of human lung cancer stem cells（CSCs）.Methods：The CD44＋/CD133＋ human lung CSCs were sortted from A549 human lung cancer cell line by flow cytometry（FCM）.The lung CSCs were divided into 3 groups.In blank control group,no plasmids have been transfected into the cells.In transfected group,plasmids containing FAP3 siRNA have been transfected.In negative control group,plasmids containing siRNA composed of random sequences have been transfected.MTT assay was used to study the influence of siRNA-FAP3 on the proliferation of human lung CSCs at 12,24,48 and 72 h after siRNA-FAP3 plasmids were transfected.Real-time fluorescent quantitative polymerase chain reaction and Western blot were used to determine the expression levels of FAP3 and Ki67 in CSCs at 72 h after siRNA-FAP3 plasmids were transfected.The soft agar colony formation assay was used to detect the invasion of human lung CSCs after siRNA-FAP3 plasmids were transfected.Results： At 48 and 72 h after transfection,the proliferation of human lung CSCs in transfected group was significantly inhibited [（24.67±1.08）%,（53.98±3.75）%],compared with negative control group[（3.76±0.88）%,（4.53±1.01）%]and blank control group[（2.34±0.41）%,（3.28±0.65）%],P0.05 and P0.01.At 72 h after transfection,the expression levels of Ki67 and FAP3 mRNA in transfected group[（0.32±0.06）%,（0.18±0.02）%] were lower than those in negative control group[（1.03±0.12）%,（0.99±0.17）%] and blank control group[（1.02±0.09）%,（0.97±0.11）%],P0.01.At 72 h after transfection,the expression levels of Ki67 and FAP3 protein in transfected group（0.16±0.05,0.25±0.09） were lower than those in negative control group（0.49±0.26,0.47±0.13） and blank control group（0.59±0.27,0.63±0.22）,P0.05.Colony forming efficiency of cells in transfected group [（13.09±5.21）%] was obviously lo

关 键 词：小干扰RNA 成纤维细胞激活蛋白3 肺癌干细胞 增殖 侵袭 

分 类 号：R734.2[医药卫生—肿瘤]