www.nature.com/scientificreports OPEN MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity Received: 4 May 2017 Pritmohinder Gill1,2, Sudeepa Bhattacharyya1,2, Sandra McCullough1,2, Lynda Letzig1,2, Accepted: 30 August 2017 Prasun J. Mishra3, Chunqiao Luo2, Harsh Dweep4 & Laura James1,2 Published: xx xx xxxx 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 identifiedin-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. MicroRNAs (miRNAs, miRs) are 18-25-nucleotides short non-coding RNAs which regulate gene expression1–3 through mRNA degradation or the inhibition of protein translation4,5. Previous reports have described a role for microRNAs (miRNAs) in the regulation of drug metabolizing enzymes (DMEs)6–8. Characterizing miRNA profiles in relationship to cytochrome P450 (CYP) expression for DMEs relevant to APAP toxicity is important to further understanding mechanisms of toxicity and may have relevance for understanding individual variability in susceptibility to drug toxicity, as metabolism is an early, initiating event in the development of APAP hepato- toxicity. A number of laboratories using murine models of APAP toxicity have reported the upregulation of liver enriched miR-122 in association with hepatotoxicity9–12 and several researchers have reported elevation of miR- 122 in clinical samples of subjects with acetaminophen toxicity13–18. Despite this literature, the relationship of miRNAs to DMEs relevant to APAP toxicity (i.e., CYP1A2, CYP3A4 and CYP2E1)19–23 is not well understood. In the present study, the relationship of CYP-regulating miRNAs to CYP1A2, CYP3A4 and CYP2E1 gene expression in APAP exposed HepaRG cells was evaluated and compared to indicators of APAP toxicity and metabolism24,25. HepaRG cells have high expression and activity levels for DMEs26–28 and have been previously used in studies of APAP toxicity29–31. In addition, levels of miRNAs were examined in clinical samples obtained from APAP over- dose subjects and compared to common clinical indicators of liver injury and APAP toxicity. Results Time course and dose response; ALT, APAP protein adducts, and CYP-binding miRNAs in HepaRG cells. Dose and response studies were conducted in cells to evaluate the temporal relationships among toxicity (ALT elevation), APAP protein adducts (an indicator of oxidative metabolism) and miRNA pro- files. As shown in Fig. 1A, APAP protein adducts were increased at 12 and 24 h in media from cells exposed to APAP 5 mM (12.9 IU/L + 0.57 and 32.3 IU/L + 0.4, respectively; *p < 0.050), compared to control cells (6.4 1Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, 72202, USA. 2Arkansas Children’s Research Institute, Little Rock, AR, 72202, USA. 3Department of Biochemical and Cellular Pharmacology, Genentech, 1, DNA Way, South San Francisco, California, 94080, USA. 4The Wistar Institute, 3601 Spruce St, Philadelphia, Pennsylvania, 19104, USA. Pritmohinder Gill and Sudeepa Bhattacharyya contributed equally to this work. Correspondence and requests for materials should be addressed to P.G. (email: [email protected]) SCIENTIFIC REPORTS | 7: 12331 | DOI:10.1038/s41598-017-11811-y 1 www.nature.com/scientificreports/ Figure 1. Time and dose-dependent changes in HepaRG cells with APAP treatment for ALT, APAP protein adducts, miRNA (miR-122-5p, miR-378a-5p, miR-27b-3p, and miR-125b-5p and mRNA levels (CYP2E1, CYP3A4, and CYP1A2) levels. Effect of 5 and 20 mM APAP treatment of HepaRG cells at 1, 6, 12 and 24 h time points. (A and B) Show levels of ALT and APAP protein adducts in cell medium; *p < 0.05 compared to controls for APAP 5 mM and 10 mM. Parenthesis around the asterisk denotes significance of ALT for 5 mM APAP treatment at 12 and 24 h. (C and D) Depict miRNA expression in cell medium. 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 qRT-PCR. TaqMan assays were duplexed with GAPDH to normalize the mRNA expression. Parenthesis around the asterisk differentiate 24 h CYP3A4 from CYP1A2; *p < 0.05 compared to controls. “h” Denotes hour. Error bars represent Standard error of the mean. IU/L + 0.44). Consistent with previous data29, a dose response pattern was observed for APAP protein adducts in the APAP 5 and 20 mM exposed cells (Fig. 1B). Figure 1A,B demonstrates that ALT levels increased over time in both dose groups at 24 h (*p < 0.05). Cells treated with 5 or 20 mM APAP released miR-122-5p, miR-378a-5p, miR-27b-3p, and miR-125b-5p into media as a function of both APAP dose and time. Consistent with the ALT and adduct profiles described above, the most marked changes in miRNA profiles were observed in the APAP 20 mM cells. Figure 1C shows down-regulation of all miRNAs at 1 h in the APAP 5 mM cells, followed by elevation of miR-122-5p at 6 h, miR- 378a-5p at 6 h, and miR-27b-3p at 24 h. In contrast, the APAP 20 mM cells (Fig. 1D) had a significant elevation of miR-122-5p at 6 h, miR-378a-5p at 6 h, miR-125b-5p at 12 h, and miR-27b-3p at 24 h. Thus, the data demonstrate SCIENTIFIC REPORTS | 7: 12331 | DOI:10.1038/s41598-017-11811-y 2 www.nature.com/scientificreports/ Figure 2. Luciferase assay confirming direct suppression of CYP1A2 and CYP3A4 transcription through 3′UTR binding of miR-122. Effects of miR-122 mimic or inhibitor on luciferase activity in HEK293 cells co- transfected with a firefly luciferase reporter vector containing the CYP1A2 3′UTR-Luc (Fig. 2A) and CYP3A4 3′ UTR-Luc (Fig. 2B). Control plasmid pEZX-MT06 with no 3′UTR, miR-122 mimic (1 nM and 5 nM), miR-122 inhibitor (100 nM). A one-way analysis of variance (ANOVA) with post hoc Dunnett’s test, *p < 0.0196 and **p < 0.006. Values are mean of 2 independent experiments. RLU = relative luciferase units. that HepaRG cells generate dose-response and temporal data for toxicity and oxidative drug metabolism end- points known to be important in models of APAP toxicity. Furthermore, miRNA profiles were distinct between the 5 (“low dose”) and 20 mM (“high dose”) exposed cells. Effect of APAP treatment on CYP2E1, CYP1A2 and CYP3A4 mRNA expression. To understand temporal relationships between miRNA profiles and gene expression for DME important in APAP toxicity, gene expression of CYP2E1, CYP1A2 and CYP3A4 was examined in cells exposed to APAP 5 mM or 20 mM. As shown in Fig. 1E, CYP2E1 mRNA expression in cells treated with 5 mM APAP was down regulated by 2.2 fold (p < 0.05) at 24 h. Transient down-regulation of CYP1A2 mRNA expression was observed with 5 mM APAP at 1 h (p < 0.05; Fig. 1E), while increased expression was apparent at 24 h. CYP3A4 mRNA expression was increased at all-time points for the APAP 5 mM (Fig. 1E),while the APAP 20 mM cells had down regulation of CYP2E1 at 6, 12 and 24 h (*p < 0.05, Fig. 1F). In contrast, the 20 mM APAP dose produced marked down regulation of CYP1A2 at all-time points, with a 26 fold decrease by 12 h (*p < 0.05, Fig. 1F). Moreover, cells exposed to 20 mM APAP had decreased CYP3A4 mRNA expression at 12 and 24 h (*p < 0.05). miRNA target sites located on promoters, 5′UTR, CDS and 3′UTR of CYP genes. The miR- Walk2.0 in-silico meta-analysis showed that hsa-miR-122-5p, hsa-miR-378a-5p, hsa-miR-27b-3p and hsa-miR- 125b-5p have target sites within the 3′UTR region of CYP1A1, CYP1A2, CYP3A4 and CYP2E1 genes. Moreover, a total of 47 interactions were identified between the CYP1A1, CYP1A2, CYP3A4 and CYP2E1 genes and the 4 miRNAs (Table S1). Among these interactions, 15, 5, 16 and 11 were detected on the promoter, 5′ UTR, CDS and 3′ UTR regions, respectively. The mRNA-miRNA interactions suggest preferential control at transcriptional, as well as translational levels. The mRNA 5′ and 3′UTR regions were predicted to harbor several miRNA binding sites. Collectively, the in-silico analysis suggests cooperativity and multiplicity of miRNA binding sites on gene regulation during APAP toxicity. miR-122 targets 3′ UTR of CYP1A2 and CYP3A4.