孕烷X受体通过微RNA21抑制乳腺癌MCF-7细胞内程序性细胞死亡因子4的表达  

Pregane X receptor inhibits expression of programmed cell death 4 in MCF-7 cells via miRNA21

作　　者：王少兰[1] 李涛[1] 韩曼 刘晓华 Wang Shaolan;Li Tao;Han Man;Liu Xiaohua(Department of Histology and Embryology,Shaanxi University of Chinese Medicine,Xi’an 712046,China;Department of Physiology,School of Basic Medicine,Shaanxi University of Chinese Medicine,Xi’an 712046,China)

机构地区：[1]陕西中医药大学基础医学院组织学与胚胎学教研室,西安712046 [2]陕西中医药大学基础医学院生理学教研室,西安712046

出　　处：《中华乳腺病杂志（电子版）》2020年第4期200-206,共7页Chinese Journal of Breast Disease(Electronic Edition)

基　　金：国家自然科学基金资助项目(81904038);陕西省教育厅科研项目(16JK1208);陕西省自然科学基础研究计划青年项目(2017JQ8023);陕西中医药大学创新团队项目(2019-YS06)。

摘　　要：目的探讨孕烷X受体(PXR)活化对人乳腺癌细胞株MCF-7内程序性细胞死亡因子(PDCDs)的调控作用及其分子机制。方法用PXR激动剂利福平(10μmol/L)处理MCF-7细胞24 h后作为利福平组,并以DMSO处理的MCF-7细胞作为其对照组(DMSO对照组),同时,为了排除PXR激动剂的非特异性,采用持续激活型PXR的腺病毒感染MCF-7细胞36 h后作为VP-PXR组,并以Mock处理的MCF-7细胞作为其对照组(Mock对照组)。对以上各组样品均采用实时荧光定量反转录PCR(qRT-PCR)检测PDCD2、PDCD4、PDCD5、PDCD6以及PXR经典靶基因CYP3A4和MDR1的mRNA表达,采用Western blot检测PDCD4的蛋白表达,并采用qRT-PCR检测PDCD4上游负调控因子微RNA(miRNA)21的表达,用Western blot检测miRNA21下游靶基因PTEN的蛋白表达。正态分布的数据用±s表示,偏态分布的数据用M(P 25~P 75)表示。2组间各指标的比较采用两独立样本t检验或两独立样本非参数秩和检验。结果(1)qRT-PCR结果显示:利福平组与DMSO对照组相比,PDCD4和PDCD6的mRNA表达更低(0.896±0.069比1.262±0.103,t=-2.961,P=0.012;0.708±0.085比0.963±0.029,t=-2.829,P=0.029),而PDCD2和PDCD5的mRNA表达差异无统计学意义(0.834±0.148比1.040±0.086,t=-1.210,P=0.254;0.896±0.142比0.946±0.110,t=-0.281,P=0.786),同时,PXR经典靶基因CYP3A4和MDR1的mRNA表达均更高(2.192±0.418比1.000±0.071,t=2.809,P=0.045;2.112±0.397比1.000±0.071,t=2.758,P=0.048);VP-PXR组与Mock对照组相比,PDCD2和PDCD4的mRNA表达均更低(0.721±0.085比0.975±0.035,t=-2.767,P=0.033;0.766±0.131比1.635±0.284,t=-2.775,P=0.017),PDCD6和CYP3A4的mRNA表达差异无统计学意义[2.053(0.932~2.653)比1.000(0.796~2.091),Z=0.314,P=0.753;1.844±0.397比1.000±0.071,t=2.097,P=0.100],而MDR1的mRNA表达则更高(3.323±0.600比1.000±0.071,t=3.846,P=0.017)。(2)Western blot检测结果显示:利福平组与DMSO对照组相比,PDCD4的蛋白表达更低(0.865±0.062比1.080±0.060,t=-2.490,P=0.026);VP-PXR组与Mock对照组相比,PDCD4的蛋白�Objective To investigate the potential role of activated pregane X receptor(PXR)in the regulation of programmed cell death proteins(PDCDs)in MCF-7 cells and explore the mechanism involved.Methods MCF-7 cells were treated with PXR agonist rifampicin(10μmol/L)for 24 h as rifampicin group,and the cells treated with DMSO served as the control group(DMSO control group).Meanwhile,to rule out the nonspecific effect of the PXR agonist,MCF-7 cells were infected with continuously activated PXR adenovirus for 36 h(VP-PXR group),which served as VP-PXR group,and the cells infected with Mock adenovirus served as the control group(Mock control group).The mRNA expressions of PDCD2,PDCD4,PDCD5,PDCD6 and the PXR target genes(CYP3A4 and MDR1)in aforementioned groups were detected by real-time fluorescent quantitative reverse transcriptase PCR(qRT-PCR).The protein level of PDCD4 was assessed by Western blot.The expression of microRNA(miRNA)21(the negative regulatory factor of PDCD4)was determined by qRT-PCR,and protein level of PTEN(a target gene of miRNA21)was detected by Western blot.The data of normal distribution were expressed as±s and the data of skewed distribution were expressed as M(P 25-P 75).The t test of two independent samples or nonparametric rank sum test of two independent samples were used to compare the parameters between two groups.Results(1)The qRT-PCR result demonstrated that mRNA expressions of PDCD4 and PDCD6 in rifampicin group were significantly lower than those in DMSO control group(0.896±0.069 vs 1.262±0.103,t=-2.961,P=0.012;0.708±0.085 vs 0.963±0.029,t=-2.829,P=0.029);the mRNA expressions of PDCD2 and PDCD5 showed no significant difference(0.834±0.148 vs 1.040±0.086,t=-1.210,P=0.254;0.896±0.142 vs 0.946±0.110,t=-0.281,P=0.786).Meanwhile,mRNA expressions of CYP3A4 and MDR1,the known PXR target genes,were significantly increased in rifampicin group compared with DMSO control group(2.192±0.418 vs 1.000±0.071,t=2.809,P=0.045;2.112±0.397 vs 1.000±0.071,t=2.758,P=0.048).The mRNA expressions of PDC

关 键 词：乳腺肿瘤 抗药性 肿瘤 细胞死亡 微RNAS 孕烷X受体 

分 类 号：R737.9[医药卫生—肿瘤]