Role of HNF4A-AS1/HNRNPC-mediated HNF4A ubiquitination protection against ritonavir-induced hepatotoxicity

Ritonavir (RTV) is an important drug for anti-human immunodeficiency virus treatment and is mainly metabolized by cytochrome P450 (CYP) 3A4. Clinically, the most common side effect of RTV treatment is hepatoxicity. We previously showed that the long noncoding RNA hepatocyte nuclear factor 4 alpha (HNF4A) antisense 1 (HNF4A-AS1) negatively regulated CYP3A4 expression and participated in RTV-induced hepatotoxicity in vitro, but the mechanism has not been well understood. In this study, similar results were observed in the mouse, where liver-specific knockdown of Hnf4aos (homolog of human HNF4A-AS1) led to increased serum aspartate (∼1.8-fold) and alanine transaminase (∼2.4-fold) levels and enlarged and degenerated hepatocytes 24 hours after RTV administration. Meanwhile, endoplasmic reticulum stress markers GRP78, PDI, and XBP-1 increased about 2.4-fold, 2.1-fold, and 2.7-fold, respectively. The aggravated liver injury correlated with Hnf4aos knockdown, attributable to heightened Cyp3a11 (homolog of human CYP3A4) expression (mRNA and protein levels were 1.8-fold and 2.5-fold, respectively). Importantly, in vitro studies revealed the underlying mechanism that HNF4A-AS1 mediated the interaction between heterogeneous nuclear ribonucleoprotein C and HNF4A, whereas heterogeneous nuclear ribonucleoprotein C promoted HNF4A degradation through the ubiquitination pathway, thereby decreasing CYP3A4 expression and alleviating RTV-induced liver injury. Overall, our findings unveil a novel mechanism by which HNF4A-AS1 regulates CYP3A4 expression to influence RTV-induced liver injury. SIGNIFICANCE STATEMENT: HNF4A-AS1 negatively regulates the expression of CYP3A4, whose overexpression is highly correlated with ritonavir (RTV)-induced liver injury. In this study, the role of Hnf4aos (homolog of human HNF4A-AS1) in RTV-induced hepatotoxicity was confirmed in mice. We found that HNF4A-AS1 and HNRNPC form a complex and facilitate the ubiquitination and degradation of HNF4A protein, thereby decreasing CYP3A4 expression and alleviating RTV hepatotoxicity.