南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (03): 370-375.doi: 10.12122/j.issn.1673-4254.2020.03.12

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孕烷X受体可上调HepG2细胞内PDCD4的表达

王少兰，李 涛，杜 婧，韩 曼，鞠 迪   

  • 出版日期:2020-04-08 发布日期:2020-03-20
  • 基金资助:

Pregnane X receptor promotes programmed cell death protein 4 expression in HepG2 cells

  

  • Online:2020-04-08 Published:2020-03-20

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摘要： 目的 探究孕烷X受体（PXR）对HepG2细胞程序性细胞死亡因子（PDCDs）的调控作用及其分子机制。方法 HepG2细胞给予不同的PXR激动剂刺激24 h，分为利福平（10 μmol/L）组、SR12813（1 μmol/L）组和DMSO对照组；采用持续激活型PXR的腺病毒感染HepG2细胞（VP-PXR组和Mock对照组）36 h。应用qRT-PCR技术检测PDCD2、PDCD4、PDCD5、PDCD6基因以及miRNA21的表达变化，采用Western blot技术检测PDCD4的蛋白表达水平。运用生物信息学方法预测PDCD4启动子区存在的潜在PXR结合反应元件（PXREs）。 结果 qRT-PCR结果显示利福平与对照组相比，PDCD2的表达显著下调（t=-2.875，P< 0.05），PDCD4表达则显著上调（t=4.209，P<0.01），而 PDCD5和PDCD6无明显差异。SR12813与对照组相比，PDCD4表达明显升高（t=4.574，P<0.01），PDCD2、PDCD5和 PDCD6均无明显变化。同时，利福平、SR12813组与对照组相比，PXR经典靶基因MDR1的表达显著升高（P<0.05）；VP-PXR与Mock对照组相比，PDCD2和PDCD6的基因表达无明显差异，PDCD4基因的表达则显著上调（t=3.343，P<0.05），MDR1的表达也显著升高（t=3.343，P<0.01）；给予利福平刺激后，PDCD4的蛋白表达比对照组显著升高（t=2.779，P<0.05）；PDCD4的蛋白在VP-PXR组也显著高于Mock组（t=3.066，P<0.05）；机制研究发现利福平或VP-PXR腺病毒刺激细胞后与各自对照组相比，PDCD4上游负调控因子miRNA21表达均无明显差异。利用生物信息学软件分析后发现，PDCD4启动子区存在PXREs。结论 PXR活化上调HepG2细胞内PDCD4的表达，但不依赖于miRNA21，PDCD4在HepG2细胞可能是PXR的靶基因。

Abstract: Objective 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. Methods 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. Results The expressions of PDCD5 and PDCD6 mRNA did not differ significantly between rifampicin-treated and the control cells, while PDCD4 mRNA expression increased (t=4.209, P=0.008) and PDCD2 mRNA decreased significantly (t=-2.875, P=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 (t=4.574, P=0.006). The PXR target gene MDR1 also increased significantly in the rifampicin- and SR12813- treated cells compared with the control cells (P=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 (t=3.343, P=0.000; t=3.343, P=0.024, respectively) without causing obvious changes in PDCD2 and PDCD6 mRNA expressions. The protein level of PDCD4 increased significantly in both rifampicin (t= 2.779, P=0.025) group and VP- PXR group (t=3.066, P=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. Conclusion 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.