The Regulation And Its Mechanism Of CYP3A4 Gene Expressionin HepG2 Cell By CYP3A4*1G

Background and AimMany medicines are metabolized by cytochrome P450 enzymes. The variation of cytochrome P450 enzymes could result in adverse drug reactions, which may go against medication safety and patient compliance. CYP3A4, one of the most important members of the cytochrome P450 superfamily of enzymes and making up 30%~40% of the total P450 enzymes, is mainly found in the liver and in the intestine. CYP3A4*1G(G>A, rs2242480) is a Single Nucleotide Polymorphism located in intron 10 of CYP3A4 gene, which has different frequencies in different ethnic groups. The allele gene is rare in Caucasian, but has high frequency(over 20%) in Asian populations. Research results in vivo showed that CYP3A4*1G genetic polymorphism was associated with the dose, effect and pharmacokinetic changes of tacrolimus, atorvastatin, fentanyl and other therapeutic agents. Using dual luciferase reporter gene system, our previous results showed that CYP3A4 intron 10 has promoter activity as well as enhancer activity, which were regulated by CYP3A4*1G in Hep G2 cells. Besides, a certain inhibition element near CYP3A4*1G site was predicted. But it is unclear whether CYP3A4 intron 10 regulated by CYP3A4*1G can influence the expression of CYP3A4 gene. The aim of this research is to explore the influence of CYP3A4 intron 10 and CYP3A4*1G on the expression of CYP3A4 gene in Hep G2 cells. Transcription factors and their complexes are the major participants in the interaction between enhancers and promoters. We predicted one transcription factor could recognize and bind CYP3A4 intron 10 and further regulate CYP3A4 gene expression. Thus, another aim of this research is to explore the transcription factor, to uncover the molecular mechanism of individual differences in the CYP3A4 enzymatic activity from the viewpoint of intronic SNP and to provide theoretical basis for clinical individualized medication and forensic identification associated with drug using. MethodsA series of eukaryotic expression vectors were constructed and transfected into Hep G2 cells. 48 hours later, the expression level of CYP3A4 m RNA was measured with q PCR. The data were analyzed in terms of promoter function(including negative control group, positive control groupⅠ, wild promoter group and mutant promoter group), upstream enhancer function(including positive control group Ⅰ, wild enhancer group, mutant enhancer group, reverse wild enhancer group and reverse mutant enhancer group)and downstream enhancer function(including positive control groupⅡ, downstream wild enhancer group, downstream mutant enhancer group, downstream reverse wild enhancer group and downstream reverse mutant enhancer group)using SPSS Statistics 21.0. The one-way analysis of variance and LSD-t test were adopted to compare the expression level of CYP3A4 m RNA, with inspection level α=0.05.Nuclear proteins were extracted from Hep G2 cells, added with biotinylated CYP3A4*1G double-stranded oligonucleotide probes(the wild and the mutant). Streptavidin–Agarose Pull-down, SDS-PAGE, silver-staining were implemented and HPLC-MS/MS were applied to analyze proteins CYP3A4*1G and its flanking sequence binding. Results(1) The analysis results of CYP3A4 intron 10 promoter function: all of positive control groupⅠ, wild promoter group and mutant promoter group could promote the expression of CYP3A4 gene. There is no significant difference between positive control groupⅠand wild promoter group(P>0.05), the expression level of CYP3A4 m RNA in mutant promoter group was significantly lower than that in positive control groupⅠ(P<0.05)or in wild promoter group(P<0.01).(2) The analysis results of CYP3A4 intron 10 upstream enhancer function: the expression level of CYP3A4 m RNA was higher when CYP3A4 intron 10 was in the forward than in the reverse direction(P<0.01); the wild group showed higher CYP3A4 m RNA level than the mutant group(P<0.01); while the mutant enhancer group showed lower CYP3A4 m RNA level than the positive control groupⅠ.(3) The analysis results of CYP3A4 intron 10 downstream enhancer function: all the groups promoted by CYP3A4 promoter and regulated by intron 10 located downstream showed higher CYP3A4 m RNA level than the positive control groupⅡ, with the SNP CYP3A4*1G playing a peripheral role; the forward direction of CYP3A4 intron 10 showed better contribution to CYP3A4 promoter, but not very significantly.(4) The analysis results of transcription factors CYP3A4*1G and its flanking sequence binding: Polyacrylamide gel electrophoresis analysis showed that the mutant probe bound more proteins than the wild one at the species and quantities; both of the binding proteins reduced obviously when adding the homologous cold probe, but the disparity between them became larger. HPLC-MS/MS results showed that the mutant probe bound more proteins than the wild one at the species and quantities. Conclusions(1) The activity in promoting CYP3A4 m RNA transcription is similar to the wild CYP3A4 intron 10 and CYP3A4 promoter. The promoter activity of CYP3A4 intron 10 is regulated by CYP3A4*1G, and the wild type performs higher activity.(2) CYP3A4 intron 10 is also similar to but not the same as classical enhancer in function, for its depending on direction and location.(3) CYP3A4 intron 10 regulates CYP3A4 expression by CYP3A4*1G allele difference in combination of transcription factors.