目的 探讨N-myc下游调节基因2(N-myc downstream regulated gene 2,NDRG2)在膀胱癌细胞凋亡中对信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)信号通路的影响.方法 2014年12月至2015年8月采用蛋白质印迹法检测人膀胱癌细胞BIU-87和膀胱上皮永生化细胞SV-HUC-1中NDRG2的表达水平.分别用空载体(pcDNA3.1)、过表达NDRG2的载体(pcDNA3.1/NDRG2)、NDRG2小干扰RNA(siRNA-NDRG2)和对照siRNA转染NDRG2细胞,并分别命名为pcDNA3.1组、pcDNA3.1/NDRG2组、siRNA-NDRG2组和siRNA对照组.培养48h后,蛋白质印迹法检测NDRG2、Cleaved caspase 3、STAT3、p-STAT3、JAK2、p-JAK2表达水平,MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡情况.BIU-87与45μmol/L的STAT3信号通路抑制剂AG490作用48h后,MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,蛋白质印迹法检测Cleaved caspase 3、STAT3、p-STAT3、JAK2、p-JAK2表达水平.结果 NDRG2在膀胱癌细胞中的表达水平(0.016 ±0.001)低于膀胱上皮永生化细胞(0.096±0.003).pcDNA3.1/NDRG2组细胞存活率(40.32 0.06)%显著低于pcDNA3.1组(100.00±0.02)%,差异有统计学意义(P<0.01).siRNA-NDRG2组细胞存活率(135.74±0.04)%高于siRNA对照组(100.01±0.05)%.pcDNA3.1/NDRG2组细胞凋亡率(28.64±0.05)%高于pcDNA3.1组(11.21±0.03)%,差异有统计学意义(P<0.01).siRNA-NDRG2组细胞凋亡率(6.31±0.05)%低于siRNA对照组(10.32±0.07)%,差异有统计学意义(P<0.01).pcDNA3.1/NDRG2组细胞中p-STAT3(0.11 ±0.04)、p-JAK2 (0.13 ±0.03)的表达水平低于pcDNA3.1组(0.29±0.06、0.32 ±0.03),差异有统计学意义(P<0.01).AG490作用后BIU-87的细胞存活率和凋亡率与转染pcDNA3.1/NDRG2后的趋势一致.结论 NDRG2能够促进膀胱癌细胞凋亡,作用机制与STAT3信号通路有关.
Objective To investigate the effect of N-myc downstream regulated gene 2 (NDRG2) on the apoptosis of bladder cancer cells by regulating the signal transducer and activator of transcription 3(STAT3) signaling pathway.Methods The expression level of NDRG2 in human bladder cancer cell BIU-87 and immortalized cell SV-HUC-1 was detected by Western blot.NDRG2 over expression vector and empty vector control (pcDNA3.1),siRNA-NDRG2,siRNA control were transfected into BIU-87 cells.After transfected 48 h,the expression level of NDRG2,Cleaved caspase 3,STAT3,p-STAT3,JAK2,p-JAK2 were detected by Western blot,the cell proliferation and apoptosis were measured by MTT and flow cytometry.After adding inhibitor AG490 of 45 μmol/L in cultured BIU-87 cells,MTT assay was used to detect cell proliferation and flow cytometry was used to detect the cell apoptosis,Western blot to detect the expression level of Cleaved caspase 3,STAT3,p-STAT3,JAK2,p-JAK2.Results The expression level of NDRG2 in bladder cancer cells was higher than that in bladder epithelial cells.The cell survival rate of pcDNA3.1/NDRG2 group was lower than that of pcDNA3.1 group,the difference was statistically significant (P < 0.01).The cell survival rate of siRNA-NDRG2 group was higher than that of siRNA control group (P< 0.01).The apoptosis rate of pcDNA3.1/NDRG2 group was higher than that of pcDNA3.1 group (P < 0.01).The apoptosis rate of NDRG2 siRNA group was lower than that of siRNA control group (P < 0.01).The level of p-STAT3 and p-JAK2 in pcDNA3.1/NDRG2 group was lower than that in pcDNA3.1 group (P< 0.01).The survival rate and apoptosis rate of BIU-87 cells cultured with AG490 were in agreement with the trend of pcDNA3.1/NDRG2 after transfection.Conclusions NDRG2 could promote the apoptosis of bladder cancer cells,and its mechanism may be related to STAT3 signaling pathway.