Cytochrome P450 2D6 (CYP2D6) participates in the metabolism of approximately 20–25% of prescribed drugs. Genetic polymorphisms influence the expression and/or activity of CYP2D6, and inter-individual differences in drug activation and elimination caused by CYP2D6 genetic variants were reported. However, little is known about the potential modulation of CYP2D6 expression by microRNAs (miRNAs). In the current study, by using in silico prediction of the stabilities of miRNA/mRNA complexes, we screened 38 miRNA candidates that may interact with the transcript of CYP2D6. An inverse correlation between the expression of miRNA hsa-miR-370-3p and the expression of CYP2D6 was observed in human liver tissue samples. Electrophoretic mobility shift assays confirmed that hsa-miR-370-3p was able to directly bind to its cognate target within the coding region of the CYP2D6 transcript. The transfection of hsa-miR-370-3p mimics into the HepG2CYP2D6 cell line, a genetically modified cell line that overexpresses exogenous CYP2D6, was able to suppress the expression of CYP2D6 significantly at both mRNA and protein levels. The transfection of hsa-miR-370-3p mimics was also able to inhibit endogenous mRNA expression and/or protein production of CYP2D6 in HepaRG cells. Furthermore, in HepaRG, HepG2, and Huh7 cells, dexamethasone-induced expression of CYP2D6 was inhibited by hsa-miR-370-3p mimics. To investigate whether the miRNA mediated suppression is caused by inhibiting protein translation or promoting mRNA degradation, an actinomycin D assay was used to measure the stability of CYP2D6 transcripts. The results indicated that hsa-miR-370-3p mimics facilitated significantly the degradation of CYP2D6 mRNA. In addition, proteomics analyses of proteins isolated from the miRNA/mRNA/protein complex suggested that a group of multifunctional proteins facilitated the interaction between hsa-miR-370-3p and CYP2D6, thereby promoting mRNA degradation.
Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3′-untranslated region (3′-UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa-miR-214-3p and hsa-miR-942-5p. In a HepG2-derived CYP2E1 over-expression cell model, hsa-miR-214-3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa-miR-942-5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa-miR-214-3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa-miR-214-3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa-miR-214-3p mimics partially blocked ethanol-dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1-overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa-miR-214-3p on CYP2E1 expression.
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) Hep-aRG 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.
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