Knockdown of Long Noncoding RNAs Hepatocyte Nuclear Factor 1α Antisense RNA 1 and Hepatocyte Nuclear Factor 4α Antisense RNA 1 Alters Susceptibility of Acetaminophen-Induced Cytotoxicity in HepaRG Cells

Acetaminophen (APAP) is a commonly used over-the-counter drug for its analgesic and antipyretic effects. However, APAP overdose leads to severe APAP-induced liver injury (AILI) and even death as a result of the accumulation of N-acetylp-benzoquinone imine, the toxic metabolite of APAP generated by cytochrome P450s (P450s). Long noncoding RNAs HNF1 alpha antisense RNA 1 (HNF1 alpha-AS1) and HNF4 alpha antisense RNA 1 (HNF4 alpha-AS1) are regulatory RNAs involved in the regulation of P450 expression in both mRNA and protein levels. This study aims to determine the impact of HNF1 alpha-AS1 and HNF4 alpha-AS1 on AILI. Small hairpin RNAs were used to knock down HNF1 alpha-AS1 and HNF4 alpha-AS1 in HepaRG cells. Knockdown of these lncRNAs altered APAP-induced cytotoxicity, indicated by MTT and LDH assays. Specifically, HNF1 alpha-AS1 knockdown decreased APAP toxicity with increased cell viability and decreased LDH release, whereas HNF4 alpha-AS1 knockdown exacerbated APAP toxicity, with opposite effects in the MTT and LDH assays. Alterations on gene expression by knockdown of HNF1 alpha-AS1 and HNF4 alpha-AS1 were examined in several APAP metabolic pathways, including CYP1A2, CYP2E1, CYP3A4, UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Knockdown of HNF1 alpha-AS1 decreased mRNA expression of CYP1A2, 2E1, and 3A4 by 0.71-fold, 0.35-fold, and 0.31-fold, respectively, whereas knockdown of HNF4 alpha-AS1 induced mRNAs of CYP1A2, 2E1, and 3A4 by 1.3fold, 1.95-fold, and 1.9-fold, respectively. These changes were also observed in protein levels. Knockdown of HNF1 alpha-AS1 and HNF4 alpha-AS1 had limited effects on the mRNA expression of UGT1A1 , UGT1A9, SULT1A1 , GSTP1, and GSTT1. Altogether, our study suggests that HNF1 alpha-AS1 and HNF4 alpha-AS1 affected AILI mainly through alterations of P450-mediated APAP biotransformation in HepaRG cells, indicating an important role of the lncRNAs in AILI. SIGNIFICANCE STATEMENT The current research identified two lncRNAs, hepatocyte nuclear factor 1 alpha antisense RNA 1 and hepatocyte nuclear factor 4 alpha antisense RNA 1, which were able to affect susceptibility of acetaminophen (APAP)-induced liver injury in HepaRG cells, possibly through regulating the expression of APAP-metabolizing cytochrome P450 enzymes. This discovery added new factors, lncRNAs, which can be used to predict cytochrome P450-mediated drug metabolism and drug-induced toxicity.