MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity

Pritmohinder Gill,Harsh Dweep,Sandra Mccullough,Lynda Letzig,Sudeepa Bhattacharyya,Laura James,Chunqiao Luo,Prasun J Mishra

doi:10.1038/s41598-017-11811-y

Abstract

MicroRNAs (miRNAs) that regulate the cytochrome P-450 isoforms involved in acetaminophen (APAP) toxicity were examined in HepaRG cells treated with APAP (20 mM). In-vitro studies found that APAP protein adducts were increased at 1 h, followed by ALT increases at 12 and 24 h. CYP1A2, CYP3A4 and CYP2E1 mRNA levels were decreased, while miRNAs were increased for miR-122-5p, miR-378a-5p, miR-27b-3p at 6 h and miR-125b-5p at 12 h and miR-27b-3p at 24 h. Putative miRNA binding sites on the 3′UTRs of the CYPs were identified in-silico. Overexpression of miR-122-5p and miR-378a-5p in cells suppressed protein expression of CYP1A2, CYP3A4 and CYP2E1. Luciferase reporter assays confirmed the interaction between miR-122 and the 3′UTR of the CYP1A2 and CYP3A4. Thus, the in-vitro experiments showed that miR-122-5p and miR-378a-5p upregulation were associated with translational repression of CYPs. Serum samples of children with APAP overdose had significant elevation of miR-122-5p, miR-378a-5p, miR-125b-5p and miR-27b-3p, compared to healthy controls and receiver operator curves of the miRNAs had AUCs of 91 to 100%. Collectively, the data suggest that miRNA elevations in APAP toxicity represent a regulatory response to modify CYP1A2, CYP3A4 and CYP2E1 translation due to cellular stress and injury.

Highlights

MicroRNAs are 18-25-nucleotides short non-coding RNAs which regulate gene expression[1,2,3] through mRNA degradation or the inhibition of protein translation[4,5]
Data were normalized for HepaRG cell culture medium with Let-7d and spiked-in C. elegans miR-39; *p < 0.05 compared to controls. (E and F) Describe CYP1E2, CYP1A2 and CYP3A4 mRNA levels determined by quantitative real-time PCR (qRT-PCR)
The present study used HepaRG cells and clinical samples to examine the expression of miRNAs identified to regulate cytochrome P450 (CYP) known to be involved in the metabolism and subsequent development of APAP toxicity[35,36,37]

Summary

Introduction

MicroRNAs (miRNAs, miRs) are 18-25-nucleotides short non-coding RNAs which regulate gene expression[1,2,3] through mRNA degradation or the inhibition of protein translation[4,5]. A number of laboratories using murine models of APAP toxicity have reported the upregulation of liver enriched miR-122 in association with hepatotoxicity[9,10,11,12] and several researchers have reported elevation of miR122 in clinical samples of subjects with acetaminophen toxicity[13,14,15,16,17,18] Despite this literature, the relationship of miRNAs to DMEs relevant to APAP toxicity (i.e., CYP1A2, CYP3A4 and CYP2E1)[19,20,21,22,23] is not well understood. Levels of miRNAs were examined in clinical samples obtained from APAP overdose subjects and compared to common clinical indicators of liver injury and APAP toxicity

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Results

Conclusion