Role And Mechanism Of HNF4A-As1 Involving In Ritonavir-Induced Liver Injury By Negatively Regulating HNF4A/PXR/CYP3A4 Pathway

Background and ObjectiveThe combination of ritonavir（RTV,used to treat HIV infection）and rifampicin（RIF,an anti-tuberculosis drug）can cause liver injury.RIF is an activator of human pregnane X receptor（PXR）and a strong inducer of CYP3A4.CYP3A4 plays an important role in the biological activation of RTV.Therefore,the excessive expression of CYP3A4 induced by RIF through activation of PXR may be an important cause of liver injury caused by RTV.In recent years,long noncoding RNAs（lnc RNAs）have been shown to be crucial for the regulation of CYPs.Our previous results showed that the hepatocyte nuclear transcription factor 4A（HNF4A）antisense RNA 1（HNF4A-AS1）negatively regulates the expression of PXR and CYP3A4 in hepatocytes.Whether HNF4A-AS1 affects RTV-induced hepatotoxicity by regulating the expression of PXR/CYP3A4 through HNF4A remains unclear.The purpose of this study is to investigate,（1）the role of PXR activation in liver injury induced by RTV in mice;（2）the role of HNF4A-AS1 in RTV-induced liver injury and the mechanism of HNF4A-AS1 regulating HNF4A/PXR/CYP3A4expression;（3）the role of Hnf4aos in RTV-induced liver injury in mice.For PXR activation varies in species,RIF and pregnenolone-16α-carbonitrile（PCN）were used as typical activators of PXR in human hepatocytes and mice,respectively,to observe the effect of PXR activation on RTV-induced liver injury.This study will expand the research field of PXR-mediated metabolic drug interactions and provide a new strategy for the prevention and treatment of drug-induced liver injury.Methods1.The role of Pxr activation in liver injury induced by RTVWild-type（WT）and Pxr knockout(Pxr^-/-)C57BL/6 mice were used as model animals to explore the effect of Pxr activation on liver injury induced by RTV.WT mice were randomly divided into four groups:negative control（NC）,PCN,RTV and PCN+RTV group（n=6/group）.NC group was treated with corn oil（10m L/kg,qd,i.p.）for 4 consecutive days.PCN group was treated with PCN（100 mg/kg,qd,i.p.）for 4 consecutive days.RTV group was treated with corn oil（10 m L/kg,qd,i.p.）for 4 consecutive days,and RTV（50 mg/kg,bid,i.p.）on the fourth day.The PCN+RTV group was treated with PCN（100 mg/kg,qd,i.p.）for 4 consecutive days,and RTV（50 mg/kg,bid,i.p.）on the fourth day.Pxr^-/-mice were randomly divided into NC,PCN and PCN+RTV group（n=6/group）.The dosing protocol was ibid.The levels of aspartate transaminase（AST）and alanine transaminase（ALT）in serum were detected.H&E staining in liver was used to detect the degree of liver injury.The expression levels of Pxr,Cyp3a11,markers associated with endoplasmic reticulum（ER）stress and apoptosis-related protein in mouse liver tissues were detected by quantitative real-time polymerase chain reaction（q RT-PCR）and Western Blot.Mouse liver microsome（MLM）protein was extracted and RTV was added into the MLM system for incubation.Ultra-performance liquid chromatography-mass spectrometry was used to analyze the main reactive metabolites.2.Mechanism of HNF4A-AS1 regulating HNF4A/PXR/CYP3A4 and involving in hepatotoxicity induced by RTVIn this part,Huh7 and Hep G2 cell lines were used.The intracellular localization of HNF4A-AS1 and HNF4A was detected by nucleo-cytoplasmic separation assay.After overexpression or knockdown of HNF4A-AS1,HNF4A or PXR,the expression of HNF4A-AS1,HNF4A,PXR and CYP3A4 were detected by q RT-PCR and Western Blot,respectively.Chromatin immunoprecipitation（Ch IP）-q PCR was used to detect the enrichment of PXR and histone modifications in the CYP3A4 promoter.Cells were treated with different concentrations of RTV.The hepatotoxicity was detected by lactate dehydrogenase（LDH）assay and reactive oxygen species（ROS）.The effect of HNF4A-AS1 and HNF4A on RTV-induced hepatotoxicity was investigated.The proteins binding to HNF4A-AS1 were identified by RNA pull-down combined with mass spectrometry.The interactions of HNF4A-AS1 and other proteins were verified by RNA binding protein immunoprecipitation（RIP）and co-immunoprecipitation（Co-IP）techniques.The effect of HNRNPC on the stability of HNF4A protein was observed through actinomycin（CHX）and MG132 experiments.3.The role of Hnf4aos in RTV-induced liver injury in miceWT and Pxr^-/-C57BL/6 mice were used in this part.The homologous lnc RNA Hnf4aos of mice was specifically knocked down in liver by adeno-associated virus through tail vein injection.The expression of Hnf4aos,Hnf4a,Pxr and Cyp3a11 was detected by q RT-PCR and Western Blot.WT-AAV-Hnf4aos and WT-AAV-Control mice were divided into RTV group and PCN+RTV group.Pxr^-/--AAV-Hnf4aos and Pxr^-/--AAV-Control mice were all PCN+RTV group（n=4～5/group）.Drug administration protocol,detection index and method were the same as above.Results1.Pxr activation is involved in RTV-induced liver injury in miceIn WT mice,the serum AST and ALT levels of PCN+RTV group were significantly increased compared with NC,PCN or RTV group.The hepatocytes degeneration was observed.The expression of ER stress markers CHOP,GRP78,PDI and XBP-1s in the liver treated with PCN combined with RTV group was significantly increased.The ratio of Bax/Bcl-2 was significantly increased.The expression of liver Pxr protein,Cyp3a11 m RNA and protein in PCN group was significantly higher than that in NC group.Metabolite analysis in vitro showed that,after PCN pretreatment,the main reactive metabolites（M1,M12 and M17）of RTV produced by MLM were significantly increased.The liver weight coefficient of PCN+RTV group was significantly higher than NC group.In Pxr^-/-mice,PCN could not induce the expression of Pxr and Cyp3a11 in liver.The reactive metabolites of RTV were significantly lower than those of WT-PCN group.There was no significant change of serum AST and ALT levels,morphology of hepatocytes,liver weight coefficient,expression of ER stress markers and ratio of Bax/Bcl-2 in PCN+RTV group compared with NC group.2.Mechanism of HNF4A-AS1 regulating HNF4A/PXR/CYP3A4 and involving in hepatotoxicity induced by RTV2.1 HNF4A-AS1 negatively regulates the expression of HNF4A/PXR/CYP3A4The results of subcellular fractionation showed that HNF4A-AS1 and HNF4A were mainly located in the nucleus of Huh7 and Hep G2 cells.Knockdown of HNF4A-AS1 increased the protein expression of HNF4A,as well as the m RNA and protein expression of PXR and CYP3A4.Knockdown or overexpression of HNF4A down-regulated or increased the expression of HNF4A-AS1,PXR and CYP3A4,respectively.Knockdown or overexpression of PXR decreased or increased the expression of CYP3A4,but had no effect on the expression of HNF4A and HNF4A-AS1.Ch IP-q PCR results showed that,after knockdown of HNF4A-AS1,enrichment of PXR and gene activation-related histone modification H3K4me3 increased,while enrichment of histone modification H3K27me3 associated with gene suppression was reduced in the CYP3A4 promoter.2.2 HNF4A-AS1 is involved in hepatotoxicity induced by RTVKnockdown of HNF4A-AS1 or overexpression of HNF4A increased RTV-induced hepatotoxicity,while knockdown of PXR alleviated the effect.Overexpression of HNF4A-AS1 reduced hepatotoxicity induced by RIF combined with RTV.2.3 HNF4A-AS1 mediates the interaction between HNF4A and HNRNPC to regulate the expression of PXR/CYP3A4RNA pull-down combined with mass spectrometry identified HNRNPC as the binding protein of HNF4A-AS1.After knockdown of HNRNPC,the protein expression of HNF4A,PXR and CYP3A4 was increased.RIP and Co-IP experiments showed that,HNRNPC,HNF4A-AS1 and HNF4A interact with each other.After RNA degradation or knockdown of HNF4A-AS1,the interaction between HNRNPC and HNF4A was weakened.After knockdown of HNRNPC,the degradation of HNF4A protein was slowed down and the half-life was prolonged.In addition,the down-regulation of PXR and CYP3A4 expression induced by overexpression of HNF4A-AS1 could be reversed by knockdown of HNRNPC or overexpression of HNF4A.3.Hnf4aos plays an important role in RTV-induced liver injury in miceThe m RNA expression of Pxr,Cyp3a11,and the protein expression of Hnf4a,Pxr and Cyp3a11 was significantly increased after Hnf4aos knockdown in WT mice.Compared with AAV-Control-RTV group,the serum AST and ALT levels were elevated,hepatocyte enlargement and degeneration,expression levels of GRP78,PDI and XBP-1s increased,and Bax/Bcl-2 ratio increased in AAV-Hnf4aos-RTV group.After Hnf4aos knockdown,the serum AST level was elevated,hepatocyte enlargement was more obvious and Bax/Bcl-2 ratio increased in PCN+RTV group compared with RTV group;compared with AAV-Control-PCN+RTV group,the serum AST level was also significant increased.In Pxr^-/-mice,knockdown of Hnf4aos did not aggravate liver injury induced by PCN combined with RTV.Conclusions1.PXR activation promotes the bioactivation of ritonavir,which causes liver endoplasmic reticulum stress and liver injury.2.HNF4A-AS1 negatively regulates HNF4A/PXR/CYP3A4 pathway and participates in ritonavir-induced hepatotoxicity.3.HNF4A-AS1 mediates the interaction between HNRNPC and HNF4A,and regulates the expression of PXR/CYP3A4 by affecting the stability of HNF4A protein.4.Hnf4aos plays an important role in ritonavir-induced liver injury in mice.