NDRG2通过抑制Bcl-2表达增加膀胱癌细胞对顺铂的敏感性

NDRG2 increases bladder cancer cell sensitivity to cisplatin by inhibiting Bcl-2 expression

背景与目的:N-Myc下游调节基因2(N-Myc down stream-regulated gene 2,NDRG2)是一种新发现的抑癌基因,在细胞增殖和凋亡中发挥着重要作用,膀胱癌的化疗耐药是临床治疗失败的主要原因之一。阐明NDRG2蛋白在膀胱癌中的表达及顺铂(cisplatin,CDDP)抗性形成的机制。方法:将膀胱癌T24细胞通过CDDP浓度递增的方式连续传代,持续6个月,获得CDDP抗性T24/CR细胞株;通过质粒转染方式使得T24/CR中NDRG2过表达,采用MTT法测定细胞活力,采用凋亡和transwell侵袭实验等比较NDRG2过表达对T24细胞CDDP抗性的影响,随后用Bcl-2-siRNA瞬时转染敲低T24细胞中NDRG2的表达,并通过蛋白质印迹法(Westernblot)检测两者之间的表达变化,从而了解两者之间的相关性,采用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)检测NDRG2 mRNA的表达。结果:NDRG2的表达水平在CDDP抗性膀胱癌细胞中显著下调。外源性NDRG2过表达,增强了CDDP的敏感性并且在CDDP存在下抑制膀胱癌细胞的活力和侵袭。下调NDRG2的表达对Bcl-2的表达无明显影响,而下调Bcl-2的表达可有效解除NDRG2低表达对膀胱癌细胞活力和凋亡的影响,并恢复CDDP对膀胱癌细胞的敏感性。结论:NDRG2/Bcl-2的失衡可能是晚期膀胱癌中CDDP抗性形成的关键机制,NDRG2有望成为CDDP抗性或难治性膀胱癌治疗的新靶点。

Background and purpose:N-Myc down stream-regulated gene 2(NDRG2)is a new type of tumor suppressor gene,which plays an important role in cell proliferation and apoptosis.The chemotherapy resistance of bladder cancer is the main cause for the failure of clinical treatment;therefore,this study aimed to clarify the expression of NDRG2 and its potential role in the pathogenesis of cisplatin(CDDP)resistance in bladder cancer cells.Methods:Bladder cancer T24 cells were continuously passaged by increasing the CDDP concentration for 6 months to obtain CDDP-resistant T24/CR cell lines.Lentiviral transfection was used to achieve high expression of NDRG2 in T24/CR.Cell viability was measured by MTT,apoptosis and transwell invasion assay were performed.The effect of NDRG2 overexpression on T24 cell resistance to CDDP was compared.Bcl-2-siRNA transient transfection was used to knock down the expression of NDRG2 in T24 cells,and then the expression in different groups was detected by Western blot.Real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)was used to detect the expression level of NDRG2 mRNA.Results:The expression level of NDRG2 was significantly down-regulated in CDDP-resistant bladder cancer cells.Exogenous overexpression of NDRG2 enhanced the sensitivity to CDDP and inhibited the viability and invasion of bladder cancer cells in the presence of CDDP.Knockdown of NDRG2 expression had no significant effect on the expression of Bcl-2.Knockdown of Bcl-2 could effectively eliminate the effect of low NDRG2 expression on the viability and apoptosis of bladder cancer cells,and could restore the sensitivity to CDDP in bladder cancer cells.Conclusion:The unbalance of NDRG2/Bcl-2 maybe the key mechanism of CDDP resistance in advanced bladder cancer.Enhanced expression of NDRG2 might be used as a candidate for the treatment of CDDP-resistant or refractory bladder cancer.