Mechanisms For Transcriptional Inhibition Of CYP2D6 And CYP3A4 In Liver Cancer

Liver cancer is a major public health problem and considered to be the third leading cause of all cancer-related death worldwide.Hepatocellular Carcinoma(HCC),accounting for 90%of primary liver cancer,is a common cancer in China.Most HCC was diagnosed in later stage,leading to high incidence and mortality rate.The cause of HCC is complicated,including infection with Hepatitis B or C virus,alcoholic liver diseases,non-alcoholic fatty liver and aflatoxins.The therapeutic effect of anti-cancer drugs is always restricted by unable to predict the exact expression level of drug-metabolizing enzymes and the toxicity of chemotherapeutics.CYP2D6 and CYP3A4 are two major isoforms of phase I drug metabolizing enzyme,participating in a number of clinical drugs(including anti-cancer drugs)biotransformation.Based on the data analysis from OncomineTM and TCGA,we found CYP2D6 and CYP3A4 were significantly decreased in liver cancer.We determined the mRNA level of CYP2D6 and CYP3A4 in 58 paired liver cancer tissues and tumor-adjacent tissues.The results were consistent with the data analysis from microarray.Decreased expression of CYP2D6 and CYP3A4 were found in 86.2%(50/58)and 94.8%(55/58)liver cancer tissues.Protein level of CYP2D6 and CYP3A4 were also dramatically reduced in 6 tumor tissues,determined by western blot.Then we determined the various transcription factors and nuclear receptor in 10-16 paired liver cancer tissues and tumor-adjacent tissues,and made a Spearman Correlation analysis.The results showed that CYP2D6 was positive related to HNF4α,C/EBPα,KLF9,FXR and SHP,while CYP3A4 was positive related to HNF4α,PXR,CAR,FXR,SHP and CYP3A4,indicating reduced expression of transcription factors and nuclear receptors in liver cancer tissues were partial cause of decreased expression of CYP2D6 and CYP3A4.Epigenetic modifications play an important role in regulating gene expression,including DNA methylation and histone modifications.The development of liver cancer was accompanied with the whole change of epigenetic modifications.UCSC predicted that there was a CpG island in CYP2D6 genebody,containing 95 CpG sites.There were sporadic CpG sites around the transcription factors binding sites.We identified that high methylation frequency of these CpG sites on CYP2D6 in tumors,resulting in low recruitment of RNA pol Ⅱ.Treatment with DNMT inhibitor,Decitabine(DAC),in four HCC cell lines could induce CYP2D6 in a dose-dependent manner,while only in HepG2 cell showed a dose-dependent induction of CYP3A4.Meanwhile,DAC could induce C/EBPα,HNF4α,KLF9,CAR and PXR,suggesting CYP2D6 and CYP3A4 induction of DAC might be correlated with induction of these transcriptional factors and nuclear receptors.Knockdown DNMT1 and MeCP2,MBD2 could slightly induce CYP2D6 expression.In liver cancer tissues,the expression of DNMT1,DNMT3a and DNMT3b were elevated,with high expression level of MeCP2 and MBD2.Histone methylation and acetylation were two common histone modifications,playing a dominant role in the regulation of gene expression.Histone acetylation is generally considered with activation of transcription.While,depending on the methylated sites and the level of methylation(mono-,di-,tri-methylation)occurs,histone methylation by methyltransferases may lead to different effects on gene transcription.H3Ac,H3K4me3 and H3K27me3 were associated with the expression of CYP2D6 and CYP3A4 as reported.ChIP-qPCR in tissue samples indicated alternation of H3K4me3 might be a key factor in altered expression of CYP2D6 and CYP3A4 in liver cancer.The modification of H3K4me3 was decreased on CYP2D6 and CYP3A4 in liver cancer.In addition,H3K27me3 modification was increased in CYP3A4 regulaton area with decreased CYP3A4 expression level.However,the enrichment of H3Ac was lower on CYP2D6 and CYP3A4 in both cancer and paired tumor-adjant tissues.Further investigation of TSA and SAHA treatment in four HCC cell lines indicated histone acetylation had no relationship with the expression of CYP2D6 and CYP3A4.Non-coding RNA(ncRNA)plays a critical role in regulation of gene expression,including chromatin modification,transcription and post-transcriptional processing.We got miRNA and lncRNA profile through RNA-seq and found a majority of differential expression of miRNAs.After TargetScan and miRanda prediction,we selected hsa-miR-493-5p as a candidate to regulate CYP2D6.By dual luciferase reporter assay and overexpression experiments,hsa-miR-493-5p was identified to negative regulate CYP2D6 expression in mRNA and protein level.Competing endogenous RNA(ceRNA)was a crucial way for lncRNA regulation.According to lncRNA-seq,we found LINC00261 was decreased in liver cancer tissues.Besides,there were multiple hsa-miR-27b-3p and hsa-miR-660-5p binding sites on LINC00261.However,only hsa-miR-27b-3p has been identified to have exact binding sites by dual luciferase reporter assay,indicating LINC00261 might serve as a miRNA sponge in order to regulate CYP3A4.In summary,the improved knowledge on epigenetic regulation in regulating CYP2D6 and CYP3A4 in HCC could make a supplement to mechanisms for CYP2D6 and CYP3A4 transcriptional regulation.In addition,the important altered epigenetic modifications and ncRNA indentified in this study might be used as novel diagnosis and/or prognostic markers and therapeutic targets,as well as be used to evaluate the expression level of individual drug metabolizing enzymes,improving individual design for the rational use of drugs in HCC treatment.