RNA干扰YAP基因对人膀胱癌T24细胞株增殖和迁移能力的影响

摘要

Background and purpose:Urothelial carcinoma of the bladder (UCB) is the most common cancer in urinary system. Yes associated protein (YAP) gene is closely associated with urothelial carcinoma of the bladder. The study was aimed to explore the effect of siRNA targeting the YAP gene on cell proliferation and migration of T24 cells. Methods:Small interfering RNA (siRNA) was transfected together with LipofectamineTM2000 in T24 human bladder cancer cells to block the YAP signal pathway. The effect of siRNA on cell proliferation and invasiveness was assessed by cell counting kit-8 (CCK-8) assay, Transwell migration assay and wound healing assay. Quantitative real time-Polymerase chain reaction (qRT-PCR) and Western blot analysis were used to conifrm the successful suppression of YAP gene and protein by siRNA. Results:Expression of YAP gene and protein was successfully suppressed after transfected with siRNA which verified by qRT-PCR and Western blot(RNA:F=93.91, P<0.000 1; Protein: F=4.62, P<0.05). As CCK-8 test showed, the proliferation of T24 bladder cancer cells was successfully restrained by inhibition of YAP gene compared with blank control and negative control(12 h: F=6.00, P=0.037;24 h: F=41.72, P=0.000 3;36 h:F=462.8, P<0.000 1;48 h:F=236.6, P<0.000 1;72 h:F=140.5, P<0.000 1). Transwell and wound healing test were performed after YAP gene was interfered by siRNA. The result demonstrated that migration of T24 bladder cancer cells was signiifcantly inhibited (Transwell: F=43.55, P<0.05;Wound healing: F=43.55, P<0.05). Conclusion:This study suggested that YAP gene was an important enhancer for the proliferation and migration of bladder cancer cells.%背景与目的：膀胱尿路上皮癌是泌尿统系最常见的肿瘤，术后易复发及转移，Yes相关蛋白(Yes associated protein，YAP)基因与膀胱癌关系密切，本研究探讨通过RNA干扰技术沉默膀胱癌T24细胞中YAP基因的表达，观察YAP基因沉默后对人膀胱癌T24细胞增殖、迁移能力的影响。方法：用阳离子脂质体转染试剂LipofectamineTM2000将靶向沉默YAP基因的小干扰RNA(small interfering RNA，siRNA)序列转染至膀胱癌T24细胞株中，采用实时定量逆转录聚合酶链反应(quantitative real time-polymerase chain reaction， qRT-PCR)、蛋白质印迹法(Western blot)检测转染后T24细胞中YAP基因及蛋白的表达水平，细胞增殖活性检测试剂盒(cell counting kit-8，CCK-8)、Transwell迁移试验以及划痕实验观察siRNA在体外对人膀胱癌T24细胞增殖、迁移能力的影响。结果：转染siRNA后，YAP RNA和蛋白表达量同空白对照以及阴性对照组相比被显著抑制(RNA：F=93.91，P＜0.0001；蛋白：F=4.62，P＜0.05)，CCK-8增殖活性实验结果显示，RNAi干扰YAP表达可以显著抑制膀胱癌T24细胞的增殖活性(12 h：F=6.00，P=0.037；24 h：F=41.72，P=0.0003；36 h：F=462.8，P＜0.0001；48 h：F=236.6，P＜0.0001；72 h：F=140.5，P＜0.0001)，通过Transwell实验和划痕试验发现，RNAi干扰YAP表达显著抑制膀胱癌T24细胞的迁移能力(Transwell：F=43.55，P＜0.05；划痕：F=43.55，P＜0.05)。结论：YAP基因是膀胱肿瘤增殖及迁移的重要调控因子。