摘要目的 用RNA干扰技术阻断人膀胱癌T24细胞中Yes相关蛋白1(YAP1)基因的表达,观察细胞增殖、迁移和侵袭能力的变化.方法 检测人膀胱癌T24细胞中YAP1表达水平;采用真核转录质粒pGreenPuro构建针对YAP1基因的重组转染质粒.将4种不同序列的YAP1短发卡RNA(shRNA)质粒以及含与YAP1无关序列的对照质粒(YAP1 shRNA NC)分别转染T24细胞,48 h后行实时定量反转录聚合酶链反应(RT-qPCR)检测其YAP1表达水平,筛选YAP1基因沉默效果最好的shRNA质粒干扰T24细胞;使用噻唑蓝(MTT)检测shRNA质粒对T24细胞增殖能力的影响、划痕实验检测其对迁移能力的影响、Transwell小室体外侵袭实验检测侵袭能力的影响.结果 RT-qPCR检测T24细胞阳性表达YAP1基因,用Lipofectamine 2000成功将不同YAP1 shRNA质粒转染入T24细胞;RT-qPCR检测发现YAP1 shRNA3质粒沉默T24细胞YAP1表达效果最显著(抑制率为75.5％),随后用YAP1 shRNA3质粒干扰T24细胞,MTT检测结果显示转染YAP1 shRNA3质粒组T24细胞增殖能力小于空白对照组(干扰组增殖率为空白对照组的85.9％,P=0.000);转染YAP1 shRNA3的T24细胞迁移能力较空白对照组低[24h平均划痕宽度分别为(218.50±3.50) μm和(86.5±4.50) μm,P=0.003];转染YAP1 shRNA3的T24细胞侵袭能力较空白对照组弱(穿透Transwell小室的平均细胞数62∶ 345,P=0.000).结论 用RNA靶向干扰能明显降低人膀胱癌T24细胞内的YAP1基因表达,并抑制肿瘤细胞的增殖、迁移和侵袭能力.

abstractsObjective To block the expression of Yes-associated protein 1 (YAP1) gene in human bladder cancer cell line T24 using RNA interference,and to observe the changes of cell proliferation,migration and invasion.Methods The YAP1 expression was detected in T24 cells.The recombinant plasmid targeting YAP1 gene with eukaryotic transcription plasmid pGreenPuro.YAPlshort hairpin RNA (shRNA) plasmids of 4 different sequences and YAP1 shRNA normal control (NC) were transfected into T24 cells respectively.After 48 h,the expression level of YAP1 was detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR),and the shRNA plasmid with the best silencing effect of YAP1 gene was screened for following research.The methyl thiazol tetrazolium (MTT) assay was used to detect the effect of shRNA plasmid on the proliferation of T24 cells,the effect of shRNA plasmid on cell migration ability was detected by scratch test,and the invasion ability of T24 cells was analyzed by Transwell assay.Results RT-qPCR detection showed that T24 cells expressed YAP1 gene.YAP1 shRNA plasmid was successfully transfected into T24 cells by Lipofectamine 2000.RT-qPCR detection showed that YAP1 shRNA3 plasmid had the most significant effect on silencing YAP1 of T24 cells (the ratio of silencing was 75.5％),and then T24 cells were interfered with YAP1 shRNA3 plasmid.MTT assay showed that the proliferation ability of T24 cells transfected with YAP1 shRNA3 plasmid was less than control group (the proliferation ratio of interference group was 85.9％ of the control group,P =0.000).The migration ability of T24 cells transfected with YAP1 shRNA3 was weaker than that in control group [24-h scratch width was (218.50 ± 3.50) μm and (86.5 ± 4.50) μm respectively,P =0.003],and the invasion ability was also weaker than that in control group (the mean number of cells penetrating Transwell was 62 ∶ 345,P =0.000).Conclusion RNA interference can significantly reduce the expression of YAP1 gene in human bladder cancer cell line T24,and inhibit the proliferation,migration and invasion of tumor cells.