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

doi:10.3877/cma.j.issn.1674-0807.2020.04.002

Abstract

Abstract:

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

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.

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References 24

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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′

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′

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

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

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

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