LncRNA HNF4A-AS1 Is Involved In The Regulation Of HNF4α Mediated Expression Of Cytochrome CYP450s

Background and ObjectiveCytochrome P450 enzymes(CYPs)are important phase-Ⅰ drug-metabolizing enzymes in the human body,distributing in livers,intestines,and kidneys,and participate in the metabolism of most endogenous and exogenous substances.CYPs are mainly composed of three subfamilies of CYP1(Al/B1),CYP2(A6/B6/C8/C9/C19/D6/E1/J2),and CYP3(A4/A5/A7),metabolizing approximately 75%of the total drugs of human body.CYP3A subfamily has the highest expression in the entire CYPs,of which CYP3A4 is the most important metabolic enzyme in the liver,and 60%of clinical drugs are metabolized by it.There are obvious individual differences in the basic expression and enzyme activity of CYPs among individuals,which leads to inter-individual differences in drug metabolism and clearance and seriously affects the safety and effectiveness of the clinical medication.Therefore,it has significant importance in the area of precision medicine in Clinical Practice that elucidating the molecular mechanism of regulation of CYPs expression and finding the main reasons for individual differences in CYPs.Multiple studies have demonstrated that the genetic polymorphisms in CYPs enzymes can only explain about 10-30%of individual differences.Many studies have shown that the expression of CYPs is also regulated by many nuclear transcription factors,such as hepatocyte nuclear factor 4α(HNF4α),hepatocyte nuclear factor la(HNF1α),androgen receptor(CAR),aryl hydrocarbon receptor(AHR),and pregnane X nuclear receptor(PXR).In recent years,more and more studies have shown that individual differences in CYPs expression are regulated by histone modifications,DNA methylation,and microRNAs.However,little is known about the mechanism of long-noncoding RNAs(lncRNAs)in regulating CYPs expression.Based on the previous work of our research group,it is explored in this project that the molecular mechanism of lncRNA HNF4A-AS1 involved in the transcription factor-mediated regulation of CYPs expression.The human liver cancer cell line Huh7 cells are used in this research.This study is divided into the following three parts,(1)to detect the regulatory effects of HNF4α on the basis of HNF4A-AS1,CYPs,transcription factors and rifampicin-induced expression;(2)to detect the regulatory effects of HNF4A-AS1 on the basal and rifampicin-induced expression of CYPs and transcription factors by gain-and loss-of-function analyses;(3)to explore the effect of HNF4A-AS1 on histone modification in the promoter region of CYP3A4 gene.This study expounds the epigenetic regulation mechanism of individual differences in CYPs expression from the perspective of lncRNAs regulation,and provides a new explanation for individual differences in CYPs,intending to guide the clinical safety and rational drug usage.Methods1.Human liver cancer cell line Huh7 cell cultureHuman liver cancer cell line(Huh7)was cultured in a sterile incubator at 37℃,5%CO2 and sufficient saturation humidity.The culture medium contained 10%fetal bovine serum(FBS),100 U/ml penicillin-streptomycin and 2 mmol/L L glutamic acid in high glucose DMEM medium.2.RNA silencing and overexpression experimentsThe siRNA silencing method was used to knock down the expression of HNF4αand HNF4A-AS1,respectively,to perform a functional deletion experiment;an overexpression plasmid HNF4A-AS1 was constructed to perform a function acquisition experiment.3.RT-qPCR to detect mRNA expression of related genes in basal and induction levelsThe basal expression levels of CYPs and transcription factors and the expression levels induced by rifampicin(RIF,10 μM)were detected by RT-qPCR after siRNA interference(HNF4α or HNF4A-AS1)or overexpression HNF4A-AS1 for 48 hours.4.Western-Blot to detect protein expression of related geneAfter silencing of HNF4α and HNF4A-AS1,the protein expression levels of HNF4α,PXR,and CYP3A4 were measured by Western-Blot.5.Chromatin immunoprecipitation(ChIP)-qPCRThe effect of siRNA interference HNF4A-AS1 on histone modification and HNF4α enrichment in HNF4α binding region of CYP3A4 gene promoter was investigated by ChIP-qPCR.Results1.Effect of HNF4α on the basal and drug-induced expression of HNF4A-AS1,CYPs and transcription factors(1)Effect of siRNA silencing of HNF4α on the basal expression levels of HNF4A-AS1,CYPs and transcription factorsIn Huh7 cell line,after siRNA silencing of HNF4α for 48h,compared with the control group,HNF4A-AS1 expression was reduced(P<0.05,n=3),CYP(1A2,2E1,2B6,2D6,2C8,2C9,2C19,and 3A4)mRNA expression and transcription factor(HNF1α,CAR,AHR,and PXR)mRNA expression were significantly reduced(P<0.05,n=3).However,HNF4α,PXR and CYP3A4 protein expression levels were consistent with their mRNA expression levels(P<0.05,n=3).(2)Effect of siRNA silencing of HNF4α on drug-induced expression levels of HNF4A-AS1,CYPs and transcription factorsTransfected with siRNA targeting HNF4α exposed to RIF(10 μM)for 48 hours,si-NC+RIF compared to si-NC,HNF4A-AS1,CYP(1A2,2E1,2B6,2D6,2C8,2C9,2C19,and 3A4)and transcription factor(HNF1α,CAR,AHR,and PXR)mRNA expression levels were significantly increased(P<0.05,n=3),si-HNF4A-AS1+RIF compared to si-NC+RIF,HNF4A-AS1,CYP(1A2,2E1,2B6,2D6,2C8,2C9,2C19,and 3A4)and transcription factors HNF1α mRNA.The expression level was significantly lower than that of si-NC+RIF(P<0.05,n=3).2.Effect of HNF4A-AS1 on the basal and drug-induced expression of CYPs and transcription factors(1)Effect of siRNA silencing of HNF4A-AS1 on the basal expression levels of CYPs and transcription factorsIn Huh7 cell line,after siRNA silencing of HNF4A-AS1 for 48 h,compared with the control group,mRNA expression of CYP(1A2,2C8,2C9,2C19,and 3A4)and transcription factor HNF4α,PXR significantly increased(P<0.05,n=3).While,HNF4α,PXR and CYP3A4 protein expression levels were consistent with their mRNA expression levels(P<0.05,n=3).(2)Effect of siRNA silencing of HNF4A-AS1 on drug-induced expression levels of CYPs and transcription factorsInterference of HNF4A-AS1 Huh7 cell line exposed to RIF(10 μM)for 48 hours,si-NC+RIF compared to si-NC,HNF4A-AS1,CYP(1A2,2E1,2B6,2D6,2C8,2C9,2C19,and 3A4)and transcription factor(HNF1α,CAR,AHR,and PXR)mRNA expression levels were significantly increased(P<0.05,n=3).si-HNF4A-AS1+RIF compared to si-NC+RIF CYP(1A2,2C8,2C9,2C19,and 3A4)and transcription factors HNF4α,PXR mRNA expression levels are significantly higher than si-NC+RIF(P<0.05,n=3).3.Overexpression of HNF4A-AS1 on the basal and drug-induced expression of CYPs and transcription factors(1)Effect of overexpression of HNF4A-AS1 on basal expression levels of CYPs and transcription factorsIn Huh7 cell line,after over-expressing HNF4A-AS1 for 48 hours,compared with the control group,the mRNA expression of CYP(1A2,2E1,2B6,2C8,2C9,2C19,and 3A4)and the transcription factors CAR,AHR,and PXR were significantly reduced(P<0.05,n=3).(2)Effect of overexpression of HNF4A-AS1 on drug-induced expression levels of CYPs and transcription factorsAfter over-expressing HNF4A-AS1 Huh7 cell line exposed to RIF(10μM)for 48 hours,si-NC+RIF compared to si-NC,HNF4A-AS1,CYP(1A2,2E1,2B6,2D6,2C8,2C9,2C19,and 3A4)and transcription factor(HNF1α,CAR,AHR,and PXR)mRNA expression levels were significantly increased(P<0.05,n=3).The mRNA expression levels of CYP(1A2,2C8,2C9,2C19,and 3A4)and transcription factor PXR were significantly lower than those of OE-NC+RIF(P<0.05,n=3).4.Effect of siRNA silencing of HNF4A-AS1 on histone modification and BNF4α binding in HNF4α binding region of CYP3A4 promoterIn Huh7 cells,48 hours after siRNA effectively interfered with HNF4A-AS1,the H3K4me3 as well as HNF4α enrichment level in the HNF4α binding region of the CYP3A4 promoter was significantly increased(P<0.05,n=3).Conclusion1.HNF4A-AS1 reverse-regulates the basal and RIF-induced expression of CYPs and transcription factors mediated by HNF4α in human liver cancer Huh7 cell line.2.The reverse regulation of CYP3A4 expression by HNF4A-AS1 is related to the changes of histone modification H3K4me3 and HNF4α enrichment in the promoter region of CYP3A4.