沉默Notch-1基因影响人膀胱癌RT4细胞生物学行为的体外实验研究  

Effect of Notch-1 Gene Silencing on Biological Behavior of Human Bladder Cancer RT4 Cells in vitro

作　　者：郑克文[1] 潘悦[1] 黄航[1] 陈洪德[1] 卢湧湧[1] 吴可明 邓哲宪[1] 翁志梁[1] 余志贤[1] 陈伟[1] ZHENG Ke-wen;PAN Yue;HUANG Hang(Department of Urology,the First Affiliated Hospital of Wenzhou Medical University,the First Clinical College of Wenzhou Medical University,Wenzhou(325000),China)

机构地区：[1]温州医科大学附属第一医院温州医科大学第一临床学院泌尿外科

出　　处：《中国中西医结合外科杂志》2019年第6期868-875,共8页Chinese Journal of Surgery of Integrated Traditional and Western Medicine

基　　金：温州市公益性科技计划项目（Y20160336）;浙江省自然科学基金（LQ17H050002)

摘　　要：目的:研究沉默Notch-1基因对人膀胱癌RT4细胞增殖、凋亡、侵袭和上皮间质转化等生物学行为的影响。方法:设计并构建Notch-1 shRNA,选取RT4细胞作为研究对象分别进行转染。实验细胞分为Notch-1阴性对照组(shRNA NC),Notch-1干扰组(Notch-1 shRNA)。细胞转染48 h后,MTT法检测细胞增殖水平变化;Transwell检测细胞侵袭力的改变;流式细胞术检测细胞凋亡水平情况;RT-PCR和Western blot检测凋亡相关蛋白(Bax、Bid、Bcl-2)及上皮间充质转化(EMT)因子(E-cadherin、N-cadherin、Vimentin)在mRNA和蛋白水平上的改变。结果:与shRNA NC组相比,Notch-1 shRNA组细胞增殖率明显下降(t=4.629,P=0.010),侵袭力减弱(t=8.193,P=0.001),细胞凋亡率明显升高(t=–18.441,P<0.001),Bad(t=–12.521,P<0.001;t=–6.983,P=0.002)、Bid(t=–9.231,P<0.001;t=–8.871,P=0.001)、E-cadherin(t=–13.457,P<0.001;t=–5.610,P=0.005)mRNA和蛋白表达量增多,Bcl2(t=10.651,P<0.001;t=6.973,P=0.002)、N-cadherin(t=6.505,P<0.001;t=10.132,P=0.001)、Vimentin(t=9.135,P<0.001;t=5.630,P=0.005)mRNA和蛋白表达量显著降低。结论:沉默Notch-1的表达能够抑制人膀胱癌RT4细胞的增殖和侵袭迁移,促进其细胞凋亡,其机制可能与诱导Bad、Bid、E-cadherin,抑制Bcl2、N-cadherin、Vimentin的表达有关。Objective To study the effect of Notch-1 gene silencing on biological behavior such as proliferation,apoptosis,invasion and epithelial-mesenchymal transformation of human bladder cancer RT4 cells.Methods Notch-1 shRNA was designed and constructed.RT4 cells were selected for transfection.The cell experiments were divided into 2 groups:shRNA negative control(shRNA NC)and Notch-1 shRNA group.After 48 h of cell culture,MTT was used to detect the changes of cell proliferation,Transwell was used to detect the changes of cell invasiveness,flow cytometry was used to detect the changes in cell apoptosis.RTPCR and Western blot were used to detect the changes of apoptosis-related proteins(Bax,Bid and Bcl2)and epithelial mesenchymal transition(EMT)factors(E-cadherin,N-cadherin and Vimentin)at mRNA and protein levels.Results Compared with shRNA NC group,Notch-1 shRNA group showed a significant decrease in cell proliferation rate(t=4.629,P=0.010)and invasiveness(t=8.193,P=0.001),increased apoptotic rate(t=–18.441,P<0.001),increased mRNA and proteins expression of Bad(t=–12.521,P<0.001;t=–6.983,P=0.002),Bid(t=–9.231,P<0.001;t=–8.871,P=0.001)and E-cadherin(t=–13.457,P<0.001;t=–5.610,P=0.005),and significantly decreased mRNA and proteins expression of Bcl2(t=10.651,P<0.001;t=6.973,P=0.002),N-cadherin(t=6.505,P<0.001;t=10.132,P=0.001)and Vimentin(t=9.135,P<0.001;t=5.630,P=0.005).Conclusion Silencing Notch-1 expression can inhibit the invasion and migration of human bladder cancer RT4 cells and promote their apoptosis.The mechanism may be related to inducing Bad,Bid and E-cadherin and inhibiting the expression of Bcl2,N-cadherin and Vimentin.

关 键 词：NOTCH-1 膀胱癌 凋亡 侵袭 上皮间质转化 

分 类 号：R737.14[医药卫生—肿瘤]