Human CYP2E1 is regulated by miR-378

doi:10.1016/j.bcp.2009.11.015

Biochemical Pharmacology

Volume 79, Issue 7, 1 April 2010, Pages 1045-1052

https://doi.org/10.1016/j.bcp.2009.11.015 Get rights and content

Abstract

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.

Graphical abstract

Introduction

Human cytochrome P450 (CYP) 2E1 catalyzes the metabolism of numerous low molecular-weight xenobiotics including drugs (e.g., acetaminophen, isoniazid), organic solvents (e.g., ethanol, acetone, carbon tetrachloride), and procarcinogens (e.g., N-nitrosodimethylamine) [1]. CYP2E1 is induced by its own substrates such as isoniazid, ethanol, and acetone, resulting in the enhancement of their metabolism [2]. It should be noted that the induction of CYP2E1 protein by these chemicals was not necessarily accompanied by an increase of CYP2E1 mRNA level [3]. The proposed mechanisms for the induction of CYP2E1 are the stabilization of mRNA [4] or protein [5]. Previously, Sumida et al. [6] reported that the CYP2E1 mRNA levels in 15 human liver samples were not positively correlated with the chlorzoxazone 6-hydroxylase activities, which is a probe activity of CYP2E1. In addition, special attention should be paid to the fact that CYP2E1 is the most abundant isoform among all P450s in human liver (56% of total P450) at the mRNA level, followed by CYP2C9, CYP2C8 and CYP3A4 (8–11% of total P450) [7], whereas it is the fourth most abundant isoform (about 7% of total P450) at the protein level after CYP3A (30% of total P450), CYP2C (20% of total P450), and CYP1A2 (about 13% of total P450) [8]. Collectively, the post-transcriptional regulation would be responsible for not only the inducible but also the constitutive expression of CYP2E1 in liver. However, the molecular basis of the human CYP2E1 regulation largely remains unknown, in contrast to the other human P450s for which much progress has been made in understanding the regulation mechanisms at the transcriptional level.

To uncover the molecular mechanism of the post-transcriptional regulation of CYP2E1, we sought to determine whether microRNA (miRNA) might be involved in the regulation of CYP2E1. MiRNAs, an evolutionarily conserved class of endogenous ∼22-nucleotide non-coding RNAs, recognize the 3′-untranslated region (3′-UTR) of the target mRNA and cause translational repression or mRNA degradation [9]. The regulation by miRNAs is involved in diverse biological processes, including development, cell proliferation, differentiation, apoptosis, and cancer initiation and progression [10], [11], [12]. The human genome may contain up to 1000 miRNAs and 30% of human mRNAs are predicted to be targets of miRNAs [13]. However, the targets of miRNAs largely remain to be identified. Based on the evidence of the post-transcriptional regulation, we investigated whether miRNAs might be involved in the regulation of human CYP2E1.

Section snippets

Chemicals and reagents

Chlorzoxazone, 6-hydroxychlorzoxazone, and coumarin were from Sigma–Aldrich (St. Louis, MO). NADP⁺, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were purchased from Oriental Yeast (Tokyo, Japan). The pGL3-promoter vector, phRL-TK plasmid, pTARGET vector, and a dual-luciferase reporter assay system were purchased from Promega (Madison, WI). LipofectAMINE2000 and LipofectAMINE RNAiMAX were from Invitrogen (Carlsbad, CA). Pre-miR miRNA Precursors for miR-378 and negative control #1

A miR-378 complementary sequence on the 3′-UTR of human CYP2E1 mRNA

The length of the 3′-UTR of human CYP2E1 is 152 bp. Computational prediction using miRBase Target database (http://microrna.sanger.ac.uk/) [17] indicated that 24 miRNAs including miR-378, miR-607, miR-223, and miR-105 share complementarity with sequences in the 3′-UTR. Meanwhile, when a Targetscan (http://www.targetscan.org/) was used, 6 miRNAs were found to share complementarity. The common miRNAs predicted in both web sites were only miR-378 and miR-607. We focused on miR-378 because it showed

Discussion

Earlier studies have reported that the induction of CYP2E1 seems to be regulated at the post-transcriptional or post-translational levels by the stabilization of mRNA [4] or protection against the rapid degradation of protein [5], [19]. To obtain a clue towards understanding the mechanisms, we investigated the possibility that miRNAs may be involved in the regulation of human CYP2E1. As the results, we found that the miR-378 is involved in the post-transcriptional regulation of CYP2E1.

The

Acknowledgements

This work was supported in part by Grant-in-Aid for Scientific Research (B) from Japan Society for the Promotion of Science. We acknowledge Mr. Brent Bell for reviewing the manuscript.

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Human CYP2E1 is regulated by miR-378

Abstract

Graphical abstract

Introduction

Section snippets

Chemicals and reagents

A miR-378 complementary sequence on the 3′-UTR of human CYP2E1 mRNA

Discussion

Acknowledgements

Free Radic Biol Med

J Biol Chem

J Biol Chem

Biochem Biophys Res Commun

Cell

Cell

J Biol Chem

Cell

J Biol Chem

J Biol Chem

Hepatology

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals: consequences for occupational and environmental medicine

Int Arch Occup Environ Health

Stabilization of cytochrome P450j messenger ribonucleic acid in the diabetic rat

Mol Endocrinol