Wang Shaolan, Li Tao, Du Jing, Han Man, Ju Di
School of Basic Medicine, Shaanxi University of Chinese Medicine, Xi'an 712046, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2020 Mar 30;40(3):370-375. doi: 10.12122/j.issn.1673-4254.2020.03.14.
To investigate the role of pregnane X receptor (PXR) in the regulation of programmed cell death proteins (PDCDs) in HepG2 cells and explore the underlying molecular mechanism.
HepG2 cells were treated with PXR agonist rifampicin (10 μmol/L) or SR12813 (1 μmol/L) for 24 h, using DMSO as the negative control. HepG2 cells were infected with constitutively activated PXR adenovirus (VP-PXR) for 36 h, with the cells infected with Mock as the negative control. The mRNA levels of PDCD2, PDCD4, PDCD5, and PDCD6 and the expression of miRNA21 were detected using qRT-PCR, and the protein level of PDCD4 was detected with Western blotting. Bioinformatic analysis was performed to predict the potential PXRresponsive elements (PXREs) motifs in the promotor region of human PDCD4.
The expressions of PDCD5 and PDCD6 mRNA did not differ significantly between rifampicin-treated and the control cells, while PDCD4 mRNA expression increased (=4.209, =0.008) and PDCD2 mRNA decreased significantly (=-2.875, =0.017) in rifampicin-treated cells. The mRNA expressions of PDCD2, PDCD5 and PDCD6 showed no significant difference between SR12813-treated cells and the control cells, while PDCD4 mRNA expression increased obviously in SR12813-treated cells (=4.574, =0.006). The PXR target gene MDR1 also increased significantly in the rifampicin- and SR12813-treated cells compared with the control cells (=0.020 and 0.01, respectively). Infection of the cells with VP-PXR adenovirus resulted in significantly increased expression of PDCD4 and MDR1 mRNA as compared with Mock group (=3.343, =0.000; =3.343, =0.024, respectively) without causing obvious changes in PDCD2 and PDCD6 mRNA expressions. The protein level of PDCD4 increased significantly in both rifampicin (= 2.779, =0.025) group and VP- PXR group (=3.066, =0.012). The expression of miRNA21, the negative regulatory factor of PDCD4, did not differ significantly between PXR agonist group and the control group. Informatic analysis revealed the presence of putative PXREs in the 5'-flanking region of PDCD4 gene.
Our findings demonstrate that PXR agonism in HepG2 cells increases the expression of PDCD4, which is independent of miRNA21 pathway, and PDCD4 may be a target gene of PXR in HepG2 cells.
探讨孕烷X受体（PXR）在调控HepG2细胞程序性细胞死亡蛋白（PDCDs）中的作用，并探究其潜在的分子机制。
将HepG2细胞用PXR激动剂利福平（10 μmol/L）或SR12813（1 μmol/L）处理24 h，以二甲基亚砜（DMSO）作为阴性对照。将HepG2细胞用组成型激活的PXR腺病毒（VP-PXR）感染36 h，以感染空载体的细胞作为阴性对照。采用qRT-PCR检测PDCD2、PDCD4、PDCD5和PDCD6的mRNA水平以及miRNA21的表达，并用蛋白质印迹法检测PDCD4的蛋白水平。进行生物信息学分析以预测人PDCD4启动子区域潜在的PXR反应元件（PXREs）基序。
利福平处理组与对照组细胞中，PDCD5和PDCD6 mRNA的表达无显著差异，而利福平处理组细胞中PDCD4 mRNA表达增加（=4.209，=0.008），PDCD2 mRNA显著降低（=-2.875，=0.017）。SR12813处理组与对照组细胞中，PDCD2、PDCD5和PDCD6的mRNA表达无显著差异，但SR12813处理组细胞中PDCD4 mRNA表达明显增加（=4.574，=0.006）。与对照组相比，利福平及SR128l3处理组细胞中PXR靶基因MDR1也显著增加（分别为=0.020和0.01）。与空载体组相比，用VP-PXR腺病毒感染细胞导致PDCD4和MDR1 mRNA表达显著增加（分别为=3.343，=0.000；=3.343，=0.024），而PDCD2和PDCD6 mRNA表达无明显变化。利福平组（=2.779，=0.025）和VP-PXR组（=3.066，=0.012）中PDCD4的蛋白水平均显著增加。PDCD4的负调控因子miRNA21在PXR激动剂组与对照组之间表达无显著差异。信息学分析显示在PDCD4基因的5'侧翼区域存在假定的PXREs。
我们的研究结果表明，HepG2细胞中PXR激动作用可增加PDCD4的表达，这一过程独立于miRNA21途径，且PDCD4可能是HepG2细胞中PXR的靶基因。
