Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans

Abstract

Acetaminophen (APAP) overdose is the leading cause of acute liver failure. Yet the mechanisms underlying adaptive tolerance toward APAP-induced liver injury are not fully understood. To better understand molecular mechanisms contributing to adaptive tolerance to APAP is an underpinning foundation for APAP-related precision medicine. In the current study, the mRNA and microRNA (miRNA) expression profiles derived from next generation sequencing data for APAP-treated (5 and 10 mM) HepaRG cells and controls were analyzed systematically. Putative miRNAs targeting key dysregulated genes involved in APAP hepatotoxicity were selected using in silico prediction algorithms, un-biased gene ontology, and network analyses. Luciferase reporter assays, RNA electrophoresis mobility shift assays, and miRNA pull-down assays were performed to investigate the role of miRNAs affecting the expression of dysregulated genes. Levels of selected miRNAs were measured in serum samples obtained from children with APAP overdose (58.6-559.4 mg/kg) and from healthy controls. As results, 2758 differentially expressed genes and 47 miRNAs were identified. Four of these miRNAs (hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p) suppressed drug metabolizing enzyme (DME) levels involved in APAP-induced liver injury by downregulating HNF1A, HNF4A and NR1I2 expression. Exogenous transfection of these miRNAs into HepaRG cells effectively rescued them from APAP toxicity, as indicated by decreased alanine aminotransferase levels. Importantly, hsa-miR-320a and hsa-miR-877-5p levels were significantly elevated in serum samples obtained from children with APAP overdose compared to health controls. Collectively, these data indicate that hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p suppress DME expression involved in APAP-induced hepatotoxicity and they contribute to an adaptive response in hepatocytes.

Keywords: Acetaminophen; Adaptation; Drug metabolizing enzymes; MicroRNAs; Pharmacogenomics.

Fig. 1

Deregulated global gene and miRNA expression in HepaRG cells exposed to APAP. a Distribution of log-fold changes of 2758 genes (fold change > 2, left panel), and heat map of 339 dramatically deregulated genes (fold change > 10, right panel). b Top 10 pathways of 339 deregulated genes listed in IPA analysis. The y-axis (left) indicates the ratio of number of deregulated genes mapping to specific pathway, and the y-axis (right) indicates the P value calculated by Benjamini–Hochberg multiple testing correction. c Distribution of log-fold changes (left panel), and heat map (right panel) of 47 miR-NAs (fold change > 2). d Venn diagram of the overlap of deregulated genes (fold change > 10) regulated by miRNAs and NRs

Fig. 2

The “shut-down” of APAP-metabolism system in HepaRG cells under APAP exposure. a RNA-seq data for genes encoding metabolic activation (CYP2E1, CYP3A4, and CYP2A6), glutathione conjugation (GSTM1, and GSTT1), glucuronidation (UGT1A1, UGT1A6, UGT1A9 and UGT2B15), and sulfation (SULT1A1, SULT1A4 and SULT2A1) were extracted and reanalyzed. The “shut-down” of APAP-metabolism system were also identified using qRT-PCR (b) and western blot (c) in HepaRG samples obtained at multiple time points after APAP exposure

Fig. 3

miRNAs interacted with the cognate target mRNAs in vitro and in vivo. a RNA EMSAs using 5′-dyed miRNA or mRNA oligonucleotide, cytoplasmic extracts, and antibodies against Ago1-4. Asterisk represents specific gene or miRNA labeled at the left of each panel; arrow represents the miRNA/ mRNA complex; hollow triangle represents miRNA/mRNA/ protein complex; solid triangle represents supershift complex. b The mRNAs of CYP3A4, HNF1A, HNF4A or NR1I2 was enriched by streptavidin pull-down for biotinylated hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, or hsa-miR-877-5p, respectively. **P < 0.001

Fig. 4

miRNAs inhibited endogenous DMEs expression, directly (a) or indirectly (b). The siRNAs specific for HNF1A, HNF4A and NR1I2, or mimics and inhibitors of hsa-miR-224-5p, hsa-miR-320a, hsa-miR-449a, or hsa-miR-877-5p were transfected into the differentiated HepaRG cells, respectively. Each assay was carried out in triplicate. Data are shown as relative mRNA and protein levels normalized by GAPDH levels. *P < 0.05; **P < 0.001; NC miRNA negative control

Fig. 5

miRNAs served as serum biomarkers to predict APAP-induced hepatotoxicity. a Time-dependent deregulation of miRNAs in HepaRG cells or medium, respectively. b Increased serum miRNA levels of hsa-miR-320a, or hsa-miR-877-5p in APAP overdose group, compared to the control and/or therapeutic dose group. *P < 0.05; **P < 0.001. c Exogenous transfection of hsa-miR-320a, hsa-miR-449a, and hsa-miR-877-5p reduced ALT levels induced by APAP