Transcriptional Regulation Of The Human CYP3A4 Expression By Epigenetic Mechanisms

Background and Objective:CYP3A4 is the most abundant P450 expressed in human liver and small intestine, where it comprises more than 50% of the total cytochrome P450, contributes to the metabolism of over 60% of all currently used drugs. Individual differences in CYP3A4 activity and expression exist among normal human liver tissues. Investigations over the past 20 years have largely focused on identifying polymorphisms in genes that encode drug metabolism enzymes. However, the molecular basis for individual variation in CYP3A4 activity has been remained elusive.A considerable number of reports have demonstrated that genetic polymorphisms are associated with the individual differences of CYP3A4 expression, and many single-nucleotide polymorphism (SNP) sites were identified in CYP3A4 gene. However, ethnic differences in allele frequency of CYP3A4 gene have been reported, with frequencies of less than 1% in the populations studied, thus cannot be the main factors affecting CYP3A4 expression. Because the pregnane X receptor (PXR) is a major regulator of CYP3 A-inducible expression, a consensus is building that human variation in CYP3A4 activity is caused by regulatory polymorphisms rather than structural polymorphisms in the CYP3A4 gene. But human PXR sequence variants are so rare that they cannot explain variation in CYP3 A expression.In recent years, a cutting-edge field of genetics called epigenetics, providing people a new idea. It does not involve changes in DNA sequence, but of heritable changes in gene expression, study how to activate or inhibit the expression of certain genes selectively using genomic information. At present, epigenetics research mainly focus on DNA methylation, histone modification, chromatin folding and regulatory noncoding RNAs. Studies have shown that some CYP members may be miRNA target genes, suggested miRNA may play an important role in the regulation of CYP expression. This study through the bioinformatics software is to predict CYP3A4 mRNA 3'-UTR area are complementary binding sites for miRNA, after a score in the final select miRNA-526b and miRNA-361-3p for the study, and examine miRNA-526b and miRNA-361-3p to the influence of CYP3A4 gene expression. In addition, it's reported that Trichostatin A treatment increased the expression of the CYP3A4 gene in HepG2 cells. We supposed that histone modification seems to be the most likely reason for the transcription factors combined with the CYP3 A4 gene. So, the objective of this study is to evaluate the influence of histone modification on CYP3A4 gene expression.Materials and methods:1. Subjects and tissue preparationLiver tissue samples were obtained from the normal liver tissues of 6 patients with hepatectomy, which contained five cases of patients with hepatic hemangioma and one case with liver cancer. The numbers of 1-6 represented the time order of obtaining the samples. The study design was approved by the Committee on Human Research of Zhengzhou University.2. Observed indicators The following indices were detected:①HPLC method was used to determine the individual differences of CYP3A4 activity.②Western blot was used to determinate CYP3A4 protein expression.③The CYP3A4, PXR mRNA level and the expression of miRNA were determined by Real time PCR.④Histone modifications in the CYP3A4 proximal ER6 element and the DR-3 and ER-6 elements from the enhancer region were determined using ChIP-qPCR.3. Statistical analysisThe analyses were performed by SPSS 13.0 statistical software. Data are presented as mean±SD. Correlation analyses were performed by Pearson correlation analysis. P values of less than 0.05 were taken as significant.Results:1. CYP3A4 enzyme activity, protein expression and mRNA level1.1 Methodological studies have shown that the method of determining midazolam was validated by specificity, accuracy, reliability and resolution analysis, which comply with the standard of determining biological samples. Hence, the method can be used to quantity the content of midazolam in human liver microsomes. The indicator of enzyme activity was the turnover of midazolam. The CYP3A4 activity of the six samples were 465.84,1180.04,791.45,851.52,606.86 and 1328.39μmol·min-1·mg-1 respectively.1.2 The relative expression level of CYP3A4 protein was represented by the band density of CYP3A4/GAPDH. In six samples, the values were 0.3159,0.9023,0.8368, 0.7574,0.7075 and 1.0611, respectively.1.3 After comparison, between each specimen, it could be concluded that in this study the CV of CYP3A4 mRNA level was 0.6096. The mRNA level of sample 6 was 5.8 times to sample 1. And the CV of PXR mRNA level was 0.6096. The mRNA level of sample 6 was 8.2 times to sample 1.1.4 Compared with sample 1, the CYP3A4 activity, protein expression and mRNA level of sample 6 were 2.8 times,3.4 times and 5.8 times, respectively. The mRNA expression of CYP3A4 consistented with the protein quantity, enzyme activity and the mRNA expression of PXR. 2. Relationship between CYP3A4 mRNA and miRNA levelsThe expression of miRNA-526b was negatively correlated with the mRNA expression of CYP3A4 (r=-0.575, P>0.05). The expression of miRNA-361-3p was also negatively correlated with the mRNA expression of CYP3A4(r=-0.617,P>0.05).3. Determination of histone modificationsIt could be concluded from Chip-qPCR results that the proximal promoter region of histone acetylation were difference and the expression level consistent with CYP3A4.Conclusions:1. The mRNA expression of CYP3A4 consistent with the protein quantity, enzyme activity and the mRNA expression of PXR.2. The expression of miRNA-526b and miRNA-361-3p are negatively correlated with the mRNA expression of CYP3A4, which hints that CYP3A4 mRNA is the target mRNA of miRNA-526b and miRNA-361-3p.3. Transcription factors PXR influence CYP3A4 gene expression, which is related to histone modification. Histone acetylation can increase the expression of CYP3 A4 gene.