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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2020, Vol. 14 ›› Issue (04) : 200 -206. doi: 10.3877/cma.j.issn.1674-0807.2020.04.002

所属专题： 总编推荐； 文献；

论著

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
王少兰1,†(), 李涛1, 韩曼2, 刘晓华2   
  1. 1. 712046　西安，陕西中医药大学基础医学院组织学与胚胎学教研室
    2. 712046　西安，陕西中医药大学基础医学院生理学教研室
  • 收稿日期:2018-12-08 出版日期:2020-08-01
  • 通信作者: 王少兰
  • 基金资助:
    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)

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

Shaolan Wang1,†(), Tao Li1, Man Han2, Xiaohua Liu2   

  1. 1. Department of Histology and Embryology, Shaanxi University of Chinese Medicine, Xi’an 712046, China
    2. Department of Physiology, School of Basic Medicine, Shaanxi University of Chinese Medicine, Xi’an 712046, China
  • Received:2018-12-08 Published:2020-08-01
  • Corresponding author: Shaolan Wang
  • About author:
    Corresponding author: Wang Shaolan, Email:
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引用本文：

王少兰, 李涛, 韩曼, 刘晓华. 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[J]. 中华乳腺病杂志(电子版), 2020, 14(04): 200-206.

Shaolan Wang, Tao Li, Man Han, Xiaohua Liu. Pregane X receptor inhibits expression of programmed cell death 4 in MCF-7 cells via miRNA21[J]. Chinese Journal of Breast Disease(Electronic Edition), 2020, 14(04): 200-206.

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(10 μmol/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(P25～P75)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的蛋白表达也更低(0.901±0.065比1.130±0.045，t＝-2.921，P＝0.019)。(3)机制研究发现：利福平组与DMSO对照组相比，PDCD4上游负调控因子miRNA21的表达更高(1.641±0.227比1.029±0.070，t＝2.576，P＝0.032)，VP-PXR组miRNA21的表达也显著高于Mock对照组(1.920±0.251比1.188±0.113，t＝2.657，P＝0.028);而且miRNA21下游靶基因PTEN的蛋白表达在利福平组和VP-PXR组均明显低于各自的对照组(0.694±0.057比0.875±0.038，t＝-2.630，P＝0.030；0.713±0.035比0.859±0.020，t＝-3.661，P＝0.006)。

结论

PXR可能通过miRNA21抑制MCF-7细胞PDCD4的表达，从而影响乳腺癌细胞的耐药性。

关键词: 乳腺肿瘤, 抗药性, 肿瘤, 细胞死亡, 微RNAs, 孕烷X受体
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 (10 μmol/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(P25-P75). 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 PDCD2 and PDCD4 in VP-PXR group were significantly reduced compared with Mock control group (0.721±0.085 vs 0.975±0.035, t=-2.767, P=0.033; 0.766±0.131 vs 1.635±0.284, t=-2.775, P=0.017), and the mRNA expressions of PDCD6 and CYP3A4 showed no significant difference [2.053(0.932-2.653) vs 1.000(0.796-2.091), Z=0.314, P=0.753; 1.844±0.397 vs 1.000±0.071, t=2.097, P=0.100], while the MDR1 mRNA expression in rifampicin group was significantly increased (3.323±0.600 vs 1.000±0.071, t=3.846, P=0.017). (2) Western blot analysis demonstrated that the protein expression of PDCD4 in rifampicin group was significantly decreased compared with DMSO control group (0.865±0.062 vs 1.080±0.060, t=-2.490, P=0.026), and PDCD4 protein expression in VP-PXR group was significantly lower compared with Mock control group (0.901±0.065 vs 1.130±0.045, t=-2.921, P=0.019). (3) miRNA21 expression in rifampicin group was significantly higher than that in DMSO control group (1.641±0.227 vs 1.029±0.070, t=2.576, P=0.032), and miRNA21 in VP-PXR group was significantly higher than that in Mock control group (1.920±0.251 vs 1.274±0.161, t=2.657, P=0.028). In addition, the protein expression of PTEN in the rifampicin or VP-PXR group was significantly lower than that in DMSO or Mock control group (0.694±0.057 vs 0.875±0.038, t=-2.630, P=0.030; 0.713±0.0353 vs 0.859±0.020, t=-3.661, P=0.006).

Conclusion

PXR can inhibit the expression of PDCD4 via miRNA21 in MCF-7 cells, thus promoting the drug resistance of breast cancer cells.

Key words: Breast neoplasms, Drug resistance, neoplasm, Cell death, MicroRNAs, Pregane X receptor
表1 实时荧光定量反转录PCR引物序列
引物名称 正向引物 反向引物
PDCD2 5′-GCATCTTCCTCTTCTGCTGC-3′ 5′-TAAACAGCCACAAACCCTGC-3′
PDCD4 5′-TGGATTAACTGTGCCAACCA-3′ 5′-TCTCAAATGCCCTTTCATCC-3′
PDCD5 5′-AACAGGAAGCAAAGCACAGG-3′ 5′-GTTGTCCATATCTTGCCATCTGT-3′
PDCD6 5′-TCTCTGACCAGTTCCACGAC-3′ 5′-TGGCTCTTTCCATGTTGTGC-3′
MDR1 5′-CTTGTTAGACAGCCTCATA-3′ 5′-TCATACAGTCAGAGTTCAC-3′
CYP3A4 5′-AATCCTAGCAGTTTGGGAGGCTGA-3′ 5′-TGAGATTACAGGCGAGTCCACCAT-3′
GADPH 5′-ACCACAGTCCATGCCATCAC-3′ 5′-TCCACCACCCTGTTGCTGTA-3′
miRNA21 5′-TAGCTTATCAGACTGATGTTGA-3′ 5′-GAGGTATTCGCACTGGATACG-3′
U6 5′-CTCGCTTCGGCAGCACA-3′ 5′-AACGCTTCACGAATTTGCGT-3′
表2 利福平组与DMSO对照组各基因mRNA表达的差异(±s)
组别 实验次数 PDCD2 PDCD4 PDCD5 PDCD6 CYP3A4 MDR1
DMSO对照组 6 1.040±0.086 1.262±0.103 0.946±0.110 0.963±0.029 1.000±0.071 1.000±0.071
利福平组 6 0.834±0.148 0.896±0.069 0.896±0.142 0.708±0.085 2.192±0.418 2.111±0.397
t值   -1.210 -2.961 -0.281 -2.829 2.809 2.758
P值   0.254 0.012 0.786 0.029 0.045 0.048
表3 VP-PXR组与Mock对照组各基因mRNA表达的差异
组别 实验次数 PDCD2(±s) PDCD4(±s) PDCD6 [M(P25～P75)] CYP3A4(±s) MDR1(±s)
Mock对照组 5 0.975±0.035 1.635±0.284 1.000(0.796～2.091) 1.000±0.071 1.000±0.071
VP-PXR组 5 0.721±0.085 0.766±0.131 2.053(0.932～2.653) 1.844±0.397 3.323±0.600
检验值   t＝-2.767 t＝-2.775 Z＝0.314 t＝2.097 t＝3.846
P值   0.033 0.017 0.753 0.100 0.017
图1 采用Western blot检测乳腺癌MCF-7细胞中PDCD4和VP16的蛋白表达　a图所示利福平组与DMSO对照组PDCD4的蛋白表达；b、c图分别所示VP-PXR组与Mock对照组的PDCD4和VP16蛋白表达
表4 DMSO对照组与利福平组MCF-7细胞中miRNA21表达的差异
组别 实验次数 miRNA21
DMSO对照组 6 1.029±0.070
利福平组 6 1.641±0.227
t值   2.576
P值   0.032
表5 Mock对照组与VP-PXR组MCF-7细胞中miRNA21表达的差异
组别 实验次数 miRNA21
Mock对照组 5 1.188±0.113
VP-PXR组 5 1.920±0.251
t值   2.657
P值   0.028
图2 采用Western blot检测乳腺癌MCF-7细胞中PTEN蛋白表达　a、b图分别所示利福平组与DMSO对照组间、VP-PXR组与Mock对照组间PTEN的蛋白表达
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