Pregnane X receptor (PXR) is a major transcription factor regulating the inducible expression of a variety of transporters and drug-metabolizing enzymes, including CYP3A4 (cytochrome P450 3A4). We first found that the PXR mRNA level was not correlated with the PXR protein level in a panel of 25 human livers, indicating the involvement of post-transcriptional regulation. Notably, a potential miR-148a recognition element was identified in the 3-untranslated region of human PXR mRNA. We investigated whether PXR might be regulated by miR-148a. A reporter assay revealed that miR-148a could recognize the miR-148a recognition element of PXR mRNA. The PXR protein level was decreased by the overexpression of miR-148a, whereas it was increased by inhibition of miR-148a. The miR-148a-dependent decrease of PXR protein attenuated the induction CYP3A4 mRNA. Furthermore, the translational efficiency of PXR (PXR protein/PXR mRNA ratio) was inversely correlated with the expression levels of miR-148a in a panel of 25 human livers, supporting the miR-148a-dependent regulation of PXR in human livers. Eventually, the PXR protein level was significantly correlated with the CYP3A4 mRNA and protein levels. In conclusion, we found that miR-148a post-transcriptionally regulated human PXR, resulting in the modulation of the inducible and/or constitutive levels of CYP3A4 in human liver. This study will provide new insight into the unsolved mechanism of the large interindividual variability of CYP3A4 expression.
Most of the biological effects of 1a, 25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) are elicited by the binding to vitamin D receptor (VDR), which regulates gene expression. Earlier studies reported no correlation between the VDR protein and mRNA levels, suggesting the involvement of posttranscriptional regulation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression through translational repression or mRNA degradation. A potential miR-125b recognition element (MRE125b) was identified in the 3 0 -untranslated region of human VDR mRNA. We investigated whether VDR is regulated by miR-125b. In luciferase assays using a plasmid containing the MRE125b, the antisense oligonucleotide for miR-125b significantly increased (130% of control) the reporter activity in KGN cells, whereas the precursor for miR-125b significantly decreased (40% of control) the reporter activity in MCF-7 cells, suggesting that miR-125b functionally recognized the MRE125b. By electrophoretic mobility shift assays, it was demonstrated that the overexpression of miR-125b significantly decreased the endogenous VDR protein level in MCF-7 cells to 40% of control. 1,25(OH) 2 D 3 drastically induced the CYP24 mRNA level in MCF-7 cells, but the induction was markedly attenuated by the overexpression of miR-125b. In addition, the antiproliferative effects of 1,25(OH) 2 D 3 in MCF-7 cells were significantly abolished by the overexpression of miR-125b. These results suggest that the endogenous VDR level was repressed by miR125b. In conclusion, we found that miR-125b posttranscriptionally regulated human VDR. Since the miR-125b level is known to be downregulated in cancer, such a decrease may result in the upregulation of VDR in cancer and augmentation of the antitumor effects of 1,25(OH) 2 D 3 .
Human CYP2E1 is one of the pharmacologically and toxicologically important cytochrome P450 isoforms. Earlier studies have reported that the CYP2E1 expression is extensively regulated by post-transcriptional and post-translational mechanisms, but the molecular basis remains unclear. In the present study, we examined the possibility that microRNA may be involved in the post-transcriptional regulation of human CYP2E1. In silico analysis identified a potential recognition element of miR-378 (MRE378) in the 3'-untranslated region (UTR) of human CYP2E1 mRNA. Luciferase assays using HEK293 cells revealed that the reporter activity of the plasmid containing the MRE378 was decreased by co-transfection of precursor miR-378, indicating that miR-378 functionally recognized the MRE378. We established two HEK293 cell lines stably expressing human CYP2E1 including or excluding 3'-UTR. When the precursor miR-378 was transfected into the cells expressing human CYP2E1 including 3'-UTR, the CYP2E1 protein level and chlorzoxazone 6-hydroxylase activity were significantly decreased, but were not in the cells expressing CYP2E1 excluding 3'-UTR. In both cell lines, the CYP2E1 mRNA levels were decreased by overexpression of miR-378, but miR-378 did not affect the stability of CYP2E1 mRNA. In a panel of 25 human livers, no positive correlation was observed between the CYP2E1 protein and CYP2E1 mRNA levels, supporting the post-transcriptional regulation. Interestingly, the miR-378 levels were inversely correlated with the CYP2E1 protein levels and the translational efficiency of CYP2E1. In conclusion, we found that human CYP2E1 expression is regulated by miR-378, mainly via translational repression. This study could provide new insight into the unsolved mechanism of the post-transcriptional regulation of CYP2E1.
We found that PPARα in human liver is regulated by miRNAs.
Human vitamin D 3 hydroxylase (CYP24) catalyzes the inactivation of 1␣,25-dihydroxyvitamin D 3 (calcitriol), which exerts antiproliferative effects. CYP24 has been reported to be overexpressed in various cancers in which microRNA levels are dysregulated. In silico analysis identified a potential miR-125b recognition element (MRE125b) in the 3Ј-untranslated region of human CYP24 mRNA. We investigated whether CYP24 is regulated by miR-125b. In luciferase assays using a reporter plasmid containing MRE125b, transfection of the antisense oligonucleotide (AsO) for miR-125b increased the reporter activity in KGN cells, and transfection of precursor miR-125b decreased the reporter activity in MCF-7 cells. The endogenous CYP24 protein level was also increased by AsO for miR-125b in KGN cells and was decreased by precursor miR-125b in MCF-7 cells. These results suggested that human CYP24 is regulated by miR-125b. Immunohistochemical analysis revealed that the CYP24 protein levels in human breast cancer were higher than in adjacent normal tissues, without an accompanying CYP24 mRNA increase. On the other hand, the expression levels of miR-125b in cancer tissues were significantly (P Ͻ 0.0005) lower than those in normal tissues. It is noteworthy that the CYP24 protein levels in cancer tissues were inversely associated with the cancer/normal ratios of the miR-125b levels, indicating that the decreased miR-125b levels in breast cancer tissues would be one of the causes of the high CYP24 protein expression. In conclusion, this study clearly demonstrates that miR-125b post-transcriptionally regulates the CYP24, which serves as a possible mechanism for the high CYP24 expression in cancer tissues.Human CYP24 is a key enzyme involved in the inactivation of 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ; calcitriol].
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