The Epigenetic Regulation Of PXR-mediated Induction Of Drug-metabolizing Enzyme CYP3A4

ObjectiveCytochrome P450 3A4(CYP3A4)is an important drug-metabolizing enzyme in human body and is responsible for the metabolism of about 60% of clinically used drugs.The expression and enzyme activity of CYP3A4 show high interindividual variation and are difficult to be predicted or estimated.Over the past years,a large number of studies have proved that many drugs and compounds could induce or inhibit the expression of CYP3A4.The induction of CYP3A4 is an important factor that causes its interindividual variation and drug-drug interactions as well as adverse reactions,which bring many troubles to drug discovery and safety medication.Studies have pointed out that genetic variations could affect CYP3A4 expression and its interindividual difference,like single nucleotide polymorphisms(SNP).However,only 10~30% of the variations can be explained by genetic polymorphisms in genes encoding drug-metabolizing enzymes or transcription regulators.In recent years,epigenetic modifications have been showed to play important roles in the regulation of drug-metabolizing enzymes,such as DNA methylation,histone modifications and non-coding RNAs.Histone modifications are involved in the regulation of cell biological process and are related to the transcriptional activation or repression of most genes.Pregnane X receptor(PXR)is an important nuclear receptor in human liver,which mediates several CYPs genes expression,including CYP3A4.Although several genes encoding drug-metabolizing enzymes have been demonstrated to be regulated by histone modifications,the exact role of histone modifications in PXR-mediated regulation and induction of CYP3A4 is still unclear.The hypothesis of this study is that the iduction of CYP3A4 by inducers is through the activation of PXR and recruit coactivators like nuclear receptor coactivator 6(NCOA6),histone acetylase p300 to change the histone modifications in PXR binding regions of CYP3A4 promoter.Besides,microRNAs(miRNAs)are a family of small,noncoding RNAs of appropriately 22 nucleotides that repress target gene expression at post-transcriptional level through binding to complementary regions of target sequences,mostly in the 3’ untranslated regions(3’-UTR).CYP3A4 was found to be targeted by miRNAs and was down-regulated via direct targeting by miRNAs or indirect ways like transcriptional factors.To date,there is no direct evidence to indicate that miRNAs are involved in the induction of CYP3A4 by drugs.The potential mechanisms are remained undiscovered.Accordingly,the aim of this study is to reveal the epigenetic regulation of rifampicin-induced expression of CYP3A4.In the present study,we explored:(1)The role of histone methylation and acetylation in CYP3A4 promoter in rifampicin-induced expression of CYP3A4;(2)The effect of miRNAs in rifampicin-induced expression of CYP3A4;(3)The associations between miRNA/histone modifications and CYP3A4 expression in human liver tissues.This study provides new explanations to the interindividual difference of CYP3A4 and will benefit drug discovery and safe and rational drug therapy.Materials and Methods 1.Liver samplesAll used liver tissues were obtained from the First Affiliated Hospital of Zhengzhou University.Samples were taken from patients with hepatic hemangioma,or gallbladder carcinoma,or hepatolithiasis after hepatectomy,conjoint normal tissues were collected and stored in liquid nitrogen until used.Samples were excluded with hepatitis or infectious diseases.All patients were informed before hepatectomy and signed informed consent form.This study was approved by The Medical Ethics Committee of The First Affiliated Hospital of Zhengzhou University2.The measurement of CYP3A4 expression in rifampicin-induced cells and human liver tissuesLS174T and HepaRG cell lines were used in this study to explore the induction of CYP3A4 by rifampicin.Total RNA were isolated by trizol reagent and reverse-transcribed into cDNA.The expression level of CYP3A4 mRNA was detected by real-time quantitative PCR(qPCR)using SYBR method.The protein of CYP3A4 were measured by Western Blot.3.RNA interference The down-regulation of PXR,NCOA6 and p300 were performed using shRNA plasmids by stable transfection.The decreased expression of PXR,NCOA6 and p300 were measured by qPCR and Western Blot.The effects of RNA interference on CYP3A4 expression and induction were analyzed by qPCR.4.Chromatin immunoprecipitation(ChIP)analysis of histone modifications in the promoter region of CYP3A4 geneCells were crosslinked by 1% formaldehyde and sonicated to obtain DNA fragments range from 100 to 1000 bp.Immunoprecipation was performed using different antibodies followed by washes and DNA was eluted and purified.The enrichment of histone mofications was analyzed by qPCR.5.Immunofluorescence(IF)and Co-immunoprecipitation(Co-IP)assayThe slides of treated LS174 T cells were photographed using laser scanning confocal microscope after immunofluorescent staining with different antibodies.Total protein was isolated and immunoprecipitation was performed to study the interactions between PXR and NCOA6/p300 protein.6.miRNA expression analysisTotal RNA was isolated from treated HepaRG cells using mirVanaTM miRNA isolation kit,human miRNA array V4.0 microarray was used to detect mi RNA expression profile.Bioinformatic prediction was employed to screen miRNAs that could target CYP3A4 and followed by qPCR validation.Dual luciferase reporter gene assay and site-specific mutagenesis were performed to study the exact effect of miRNAs.7.The expression of miRNAs and histone modification levels in human liver tissuesThe expression of candidate miRNAs were tested in liver samples using qPCR,ChIP-qPCR analysis was used to explore the enrichment levels of histone modifications in the promoter region of CYP3A4 gene in liver samples.Statistical analysisData are shown as mean±SD,statistical analysis was performed by SPSS.t-test was used to analyze the difference between two groups and ANOVA was used for comparation between three or more groups followed by Bonferroni’s post hoc test or Dunnett’s test.Spearman correlation analysis were performed to analyze the correlation.A P value of less than 0.05 was considered to be statistically significant.Results 1.The effect of histone modifications in rifampicin-induced expression of CYP3A4(1)Rifampin induced the expression of CYP3A4 in LS174 T cellsBoth of the mRNA and protein expression of CYP3A4 in LS174 T cells were increased(4~15fold for mRNA,1.6~2.9 fold for protein)by rifampicin treatment and showed a time-dependent manner.Knockdown the expression of PXR significantly decreased the induction,which indicates that the induction of rifampicin was mediated by PXR.(2)The effect of rifampicin on histone modifications levels and PXR,NCOA6,and p300 binding in the promoter of CYP3A4 geneRifampicin increased the levels of H3K4me3 and H3 acetylation,and decreased the levels of H3K27me3 in PXR binding regions in the promoter of CYP3A4 gene when compared with control group(P<0.05).The changes were consistent with CYP3A4 induction.Besides,the binding of PXR,NCOA6,and p300 were also increased significantly in PXR binding regions of CYP3A4 gene promoter(above 1.5 fold,P<0.05).These results show that histone methylation and acetylation are involved in the rifampicin-induced expression of CYP3A4.(3)The effect of silencing NCOA6 and p300 on rifampicin-induced expression of CYP3A4 and histone modificationsSignificant decrease of CYP3A4 basal expression were observed after knockdown of NCOA6 or p300(65% and 31%,respectively)as well as induction by rifampicin(above 70%,P<0.05).Moreover,the enrichment levels of H3K4me3 and H3 acetyaltion were also decreased and H3K27me3 levels were increased regardless of rifampicin treatment.Thus indicating that NCOA6 and p300 were indispensable in the induction of CYP3A4 by rifampicin.(4)The histone modifications and NCOA6,p300 binding were mediated through activation of PXR by rifampicinThe effect of rifampicin on histone modification changes was attenuated significantly after knockdown of PXR(P<0.05).The binding of NCOA6 and p300 in the promoter of CYP3A4 gene were also reduced significantly(more than 50%,P<0.05).Then,we performed Co-IP assay and results showed that PXR interact with NCOA6 and p300 through protein interactions,which were enhanced by rifampicin.Besides,the IF results showed that,rifampicin increased the nuclear accumulation of PXR,NCOA6 and p300,and promoted the colocalization of PXR and NCOA6/p300.2.The role of miRNAs in rifampicin-induced expression of CYP3A4(1)Rifampicin altered the expression of miRNAs in HepaRG cellsThe expression of CYP3A4 in HepaRG cells was increased by 30 fold after rifampicin treatment,and the expression of 65 miRNAs were altered: 47 miRNAs were down-regulated well 18 mi RNAs were up-regulated.(2)Bioinformatic prediction of miRNA target9 miRNAs were screened out to target the 3’-UTR of CYP3A4 and qPCR analysis validated that 7 of them were significantly suppressed by rifampicin: miR-641,miR-628-3p,miR-342-5p,miR-2392,miR-4289,mi R-4461,miR-766-5p(above 2 fold,P<0.05).These results show that the down-regulation of miRNAs was involved in CYP3A4 induction.(3)The effect of miRNAs on the 3’-UTR reporter gene activity of CYP3A4Co-transfection of miR-641 or miR-628-3p showed significantly decrease of the luciferase activities of CYP3A4 3’-UTR reporter gene,but no effect of miR-766-5p or miR-342-5p was observed(P>0.05).After mutation of miRNA binding sites in 3’-UTR sequence,the suppression of luciferase activities were no longer affected by miR-641 or mi R-628-3p.This indicates that miR-641 and miR-628-3p inhibit the expression of CYP3A4 via direct targeting to the 3’-UTR.(4)The effect of miR-641 and miR-628-3p on the basal and induced expression of CYP3A4We examined the effect of miR-641 and miR-628-3p on the m RNA expression of CYP3A4 in HepaRG cells through transient transfection,and found that the CYP3A4 mRNA was decreased after transfection(decreased by 38% and 55%,respectively).Moreover,the induction effect of rifampicin was also reduced(decreased by 27% and 35%,respectively).3.The correlation between miRNAs/histone modifications and CYP3A4 expression in human liver tissues(1)The expression of CYP3A4 mRNA and protein in human liver tissuesSignificant differences of CYP3A4 expression were observed in 18 liver samples,the mRNA levels range from 0.06 to 2.59 fold(median 0.72,relative to GAPDH mRNA),the protein levels range from 0.10 to 2.33 fold(median 0.49,relative to GAPDH protein).No significant correlation between mRNA and protein levels was observed(P>0.05),which suggests that the expression of CYP3A4 is regulated by post-transcriptional regulation.(2)The correlation between histone modifications and CYP3A4 mRNA in liver tissuesThe H3K4me3 levels in proximal PXR binding region(-263~-108 bp)were found to be correlated with CYP3A4 mRNA levels(r=0.574,P<0.05,n=10),H3K27me3 levels in distal PXR binding region(-8015~-7793 bp)were correlated with CYP3A4 mRNA(r=-0.638,P<0.05,n=10).No significant correlation between H3 acetylation and CYP3A4 mRNA was observed.(3)The correlation between miR-641/miR-628-3p and CYP3A4 expression in liver tissueBoth of miR-641 and miR-628-3p were found to be correlated with CYP3A4 expression in liver samples.miR-641 was negatively associated with CYP3A4 protein levels(r=-0.525,P<0.05,n=10)while miR-628-3p was negatively related with mRNA levels(r=-0.502,P<0.05,n=10).These results suggest that CYP3A4 was post-transcriptional regulated by miR-641 and miR-628-3p in human liver.Conclusions 1.The induction of CYP3A4 by rifampicin is associated with the changes of histone modifications in its promoter.Upon activated by rifampicin,coactivator NCOA6 and p300 are recruited to the promoter of CYP3A4 gene by PXR,and facilitate the histone modifications to promote CYP3A4 gene transcription.2.miR-641 and miR-628-3p could target the 3’-UTR of CYP3A4 and decrease its expression,rifampicin could repress the expression of miR-641 and miR-628-3p.3.The expression of CYP3A4 in human liver tissues are correlated with high levels of H3K4me3 and low levels of H3K27me3 in the promoter of CYP3A4 gene.CYP3A4 expression is post-transcriptional regulated by miR-628-3p and miR-641.