NDRG1过表达对去势抵抗性前列腺癌耐药细胞株C4-2/ENZA耐药性的影响及其机制  

Effect of over-expression of NDRG1 on resistance of castration-resistant prostate cancer resistant cell line C4-2/ENZA and its mechanism

作　　者：张鹰 万朝辉 蒋先训 ZHANG Ying;WAN Zhaohui;JIANG Xianxun(Department of Critical Medicine,Affiliated Second Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China;Department of Emergency,Affiliated Second Hospital,Hengyang Medical School,University of South China,Hengyang 421001,China)

机构地区：[1]南华大学衡阳医学院附属第二医院重症医学科,湖南衡阳421001 [2]南华大学衡阳医学院附属第二医院急诊科,湖南衡阳421001

出　　处：《吉林大学学报（医学版）》2024年第3期708-717,共10页Journal of Jilin University：Medicine Edition

基　　金：湖南省卫健委卫生科研项目(D202304058812,D202213014836)。

摘　　要：目的:探讨N-myc下游调节基因1(NDRG1)对去势抵抗性前列腺癌(CRPC)恩杂鲁胺(ENZA)耐药的影响,并阐明其作用机制。方法:体外培养人CRPC C4-2细胞和ENZA耐药株C4-2/ENZA细胞,采用实时荧光定量PCR(RT-qPCR)法检测C4-2/ENZA细胞及其亲本C4-2细胞中NDRG1 mRNA表达水平,Western blotting法检测C4-2/ENZA细胞及其亲本C4-2细胞中NDRG1、雄激素受体(AR)和前列腺特异性抗原(PSA)蛋白表达水平,以验证细胞转染效率。将C4-2/ENZA细胞分为空白组(正常培养不进行处理)、阴性对照慢病毒(Lv-NC)组(转染Lv-NC)、Lv-NDRG1组(转染Lv-NDRG1)、Lv-NC+ENZA组(转染Lv-NC后加入ENZA处理)、Lv-NDRG1+ENZA组(转染Lv-NDRG1后加入ENZA处理)、Lv-NDRG1+表皮生长因子(EGF)组(转染Lv-NDRG1后加入EGF处理)和Lv-NDRG1+EGF+ENZA组(转染Lv-NDRG1后加入EGF和ENZA处理)。采用噻唑蓝(MTT)法检测各组细胞半数抑制浓度(IC_(50))、耐药指数(RI)和细胞增殖活性,流式细胞术检测各组细胞凋亡率,RT-qPCR法检测各组细胞中NDRG1 mRNA表达水平,Western blotting法检测各组细胞中NDRG1、AR、第213位丝氨酸磷酸化雄激素受体(p-ARSer^(213))、第81位丝氨酸磷酸化雄激素受体(p-ARSer^(81))和PSA蛋白表达水平。结果:与C4-2细胞比较,C4-2/ENZA细胞中NDRG1 mRNA和蛋白表达水平均明显降低(P<0.01),AR和PSA蛋白表达水平明显升高(P<0.01),提示ENZA耐药株C4-2/ENZA中NDRG1低表达;与Lv-NC组比较,LvNDRG1组细胞中NDRG1 mRNA和蛋白表达水平均明显升高(P<0.01),提示成功构建NDRG1基因过表达C4-2/ENZA耐药细胞株。MTT法,与C4-2细胞比较,C4-2/ENZA细胞IC_(50)明显升高(P<0.01),RI为17.78;与Lv-NC组比较,Lv-NDRG1组C4-2/ENZA细胞IC_(50)明显降低(P<0.01)。EGF处理24 h,与Lv-NC组比较,Lv-NC+EGF组C4-2/ENZA细胞IC_(50)明显升高(P<0.01);与Lv-NDRG1组比较,Lv-NDRG1+EGF组C4-2/ENZA细胞IC_(50)明显升高(P<0.01)。与ENZA处理前比较,不同浓度ENZA处理24 h,C4-2和C4-2/ENZA细胞增殖活性均逐渐降低(F=2Objective:To discuss the effect of N-myc downstream-regulated gene 1(NDRG1) on the enzalutamide(ENZA) resistance in the castration-resistant prostate cancer(CRPC),and to clarify its mechanism.Methods:The human CRPC C4-2 cells and ENZA-resistant strain C4-2/ENZA cells were cultured in vitro.The expression levels of NDRG1 mRNA in the C4-2/ENZA cells and their parental C4-2cells were detected by real-time fluorescence quantitative PCR(RT-qPCR) method.The expression levels of NDRG1,androgen receptor(AR),and prostate-specific antigen(PSA) proteins in the cells were detected by Western blotting method to verify the transfection efficiency of the cells.The C4-2/ENZA cells were divided into blank group(normally cultured without treatment),negative control lentivirus(Lv-NC) group(transfected with Lv-NC),Lv-NDRG1 group(transfected with Lv-NDRG1),Lv-NC+ENZA group(transfected with Lv-NC followed by ENZA treatment),Lv-NDRG1+ENZA group(transfected with Lv-NDRG1 followed by ENZA treatment),Lv-NDRG1+epidermal growth factor(EGF) group(transfected with Lv-NDRG1 followed by EGF treatment),and Lv-NDRG1+EGF+ENZA group(transfected with Lv-NDRG1 followed by EGF and ENZA treatment).The half-maximal inhibitory concentration(IC_(50)),resistance index(RI),and proliferation activity of the cells were detected by MTT assay;the apoptotic rates of the cells in various groups were detected by flow cytometry;RTqPCR method was used to detect the expression levels of NDRG1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of NDRG1,AR,phosphorylated AR at serine213(p-ARSer~(213)),phosphorylated AR at serine81(p-ARSer~(81)),and PSA proteins in the cells in various groups.Results:Compared with C4-2 cells,the expression levels of NDRG1 mRNA and protein in the C4-2/ENZA cells were significantly decreased(P<0.01) and the expression levels of AR and PSA proteins were increased(P<0.01),indicating low expression of NDRG1 in the ENZA-resistant C4-2/ENZA strain.Compared with Lv-NC group,the expression levels

关 键 词：去势抵抗性前列腺癌 N-myc下游调节基因1 恩杂鲁胺 耐药性 雄激素受体 

分 类 号：R737.25[医药卫生—肿瘤]