Effects Of CYP3A4*1G On CYP3A Expression And Enzyme Activity In Gene-edited HepG2 Cells

Background and ObjectivesCytochrome P450 3A4(CYP3A4)is the most important drug metabolic enzyme in adults,which can catalyze the metabolism of more than 50% of the commonly used clinical prescription drugs.There is a significant individual difference in the expression of the gene CYP3A4 encoding the enzyme in the liver(up to 40 times).The resulting individual difference in CYP3A4 enzyme activity is one of the important factors leading to individual differences in efficacy and adverse reactions of patients.The complexity of drug metabolic substrates,drug interactions and toxicant accumulation caused by CYP3A4 enzyme activity have become serious hidden dangers in clinical drug use.Among the various factors that cause the individual differences in enzyme activity(such as age,sex,drugs,environmental chemicals,genetic variation of transporter genes related to CYP3A4 metabolic drugs,etc.),about 90% are caused by genetic variation of CYP3A4 enzyme genes,among which single nucleotide polymorphism(singlenucleotidepolymorphism,SNP)is the most common genetic variation.Therefore,the search for SNP biomarkers that cause individual differences in CYP3A4 expression and enzyme activity is of great significance for individual drug use.At present,it has been found that the functional SNP of CYP3A4 exons and their regulatory regions play a limited role in explaining the individual differences in CYP3A4 enzyme activity,but there are few studies on the function and mechanism of CYP3A4 intron region SNP and its effect on CYP3A4 expression and enzyme activity.The single nucleotide variation of CYP3A4*1G(G>A)rs2242480 is located in intron 10 of the CYP3A4 gene,and the allele frequency is 27.6% in the Han population,and the allele frequency is also higher in the Roma population and the Japanese population.The results of a number of in vivo studies and liver tissue level studies at home and abroad show that the change of CYP3A4 activity caused by CYP3A4*1G polymorphism affects the metabolism of different drugs such as fentanyl and Atto vastatin,resulting in individual differences in efficacy or adverse reactions,but the results of CYP3A4 enzyme activity changes(increase,decrease and no effect)caused by CYP3A4*1G variation in the metabolism of different drugs are inconsistent or even contradictory.Because of the high substrate similarity between CYP3A5 and CYP3A4 enzyme protein,that is,the substrate catalyzed by CYP3A4 is also likely to be metabolized by CYP3A5 enzyme at the same time,and there is a high linkage disequilibrium between CYP3A4*1G and CYP3A5*3(A>G)which affects the expression of CYP3A5,the change of CYP3A5 enzyme activity caused by CYP3A5*3 polymorphism may interfere with the change of CYP3A4 enzyme activity caused by CYP3A4*1G polymorphism.The results of more in vivo studies in different ethnic groups show that in the carriers of wild type An allele(wild type homozygote and wild type heterozygote)expressing CYP3A5 enzyme,CYP3A4*1G mutation affects the pharmacokinetics of tacrolimus in Japanese renal transplant patients.In the G variant homozygote(mutant homozygote)that does not express CYP3A5 enzyme,CYP3A4*1G polymorphism affects the pharmacokinetics of Atto vastatin in Chinese Han patients with coronary heart disease.In vitro double reporter gene experiment,He and other double reporter gene experiments in HepG2 cells showed that CYP3A4*1G A allele had a stronger transcriptional enhancement effect on SV40 promoter than G allele.In our double fluorescence report experiment,it was found that CYP3A4*1G allele regulated the activity of enhancer and promoter of intron 10 of CYP3A4 in HepG2 cells in a dependent manner,and the activity of wild type was higher than that of mutant type.A fragment of intron 10 of CYP3A4 containing CYP3A4*1G SNP site affects the expression of CYP3A4 in double reporter gene experiment.The effect of CYP3A4*1G on CYP3A4 expression and enzyme activity is indirectly reflected by reporter gene or eukaryotic expression vector at extracellular level,but it is not clear whether CYP3A4*1G affects CYP3A4 expression and enzyme activity at intracellular level.In vitro experiments,it is difficult to obtain normal liver tissues and primary liver cells with only different genotypes of CYP3A4*1G when the genetic variation of other linked genes is the same,which seriously hinders the research process of the mechanism of CYP3A4*1G affecting CYP3A4 expression.In addition,there are no homozygous cell lines or cell lines of CYP3A4*1G.CRISPR/Cas9 gene editing technology has the advantages of high specificity and accuracy.It has been reported that the CYP3A5*3,of human hepatoma cell line Hu H-7 can be successfully edited by CRISPR/Cas9 to study the relationship between gene variation and drug metabolism.This study confirmed that gene variation can be transformed into cell lines to study gene expression and the activity of related enzymes at the cellular level.HepG2 cells are CYP3A4*1G(GA),which can be used for CYP3A4*1G homozygous gene editing.In summary,the purpose of this study is to(1)to establish CYP3A4*1G wild type homozygote(GG)and mutant homozygote(AA)genotype HepG2 cell lines by gene editing of CYP3A4*1G site in CYP3A4*1G mutant heterozygote(GA)HepG2 cell genome by CRISPR/Cas9 technique.(2)using the above three different genotypes of CYP3A4*1G HepG2 cells to directly verify and explore the effects of CYP3A4*1G variation on CYP3A4 and CYP3A5 gene expression and CYP3A4 enzyme activity at the cellular level in vivo.(3)to analyze the effects of rifampicin on the expression of CYP3A4,CYP3A5 and PXR mRNA in HepG2 cells with different CYP3A4*1G genotypes.It provides an ideal cell model for directly verifying whether CYP3A4*1G regulates CYP3A4 expression by affecting the binding of transcription factors in vivo,and to explain the individual differences in CYP3A4 activity from the point of view of SNP-regulated gene expression in CYP3A4 intron,so as to provide a new theoretical basis for individual medication with CYP3A4*1G as a biomarker in Chinese Han population.Materials and Methods1.Construction of recombinant vector: sg RNA and reporter sequences were designed and synthesized according to the CYP3A4*1G mutation in CYP3A4 gene of HepG2 cells,and inserted into px330 and pmcherry EGFP vectors respectively to construct px330-sg RNA and pmcherry EGFP-reporter recombinant vectors.sg RNA with high cleavage efficiency was screened out in HEK293 T cells.2.CYP3A4*1G wild type homozygous(GG)and mutant homozygous(AA)HepG2 cell lines:(1)the constructed recombinant vectors and the designed and synthesized repair templates were transfected into CYP3A4*1G mutant heterozygous(GA)HepG2 cells for flow sorting and monoclonal cell culture.Genomic DNA was extracted and sequenced after PCR amplification.CYP3A4*1G GG and AA monoclonal HepG2 cell lines were identified and screened.(2)according to the sg RNA(CYP3A4*1G sg RNA-2W,CYP3A4*1G sg RNA-3M)sequences used in CYP3A4*1G GG and AA HepG2 cell lines established successfully,the missed sites were found,and the corresponding primers were designed,and the PCR,sequencing was performed to verify the miss sites with the mono-clone cell DNA as the substrate.3.CYP3A4*1G mutation on CYP3A4 and CYP3A5 mRNA,protein and CYP3A4 enzyme activity in HepG2 cells: the expression levels of CYP3A4 and CYP3A5 mRNA in three different genotypes of CYP3A4*1G(GG/GA/AA)HepG2 cells were detected by RT-q PCR,and the expression levels of CYP3A4 and CYP3A5 proteins in CYP3A4*1G GG/AA/GA HepG2 cells were detected by Western blot method.Indirect detection of CYP3A4 enzyme activity in CYP3A4*1G GG/AA/GA HepG2 cells by P450-GLO CYP3A4 enzyme activity kit4.Effects of rifampicin induction on the expression of CYP3A4,CYP3A5 and PXR mRNA in HepG2 cells with different CYP3A4*1G genotypes: after rifampicin induced CYP3A4*1G(GG/GA/AA)HepG2 cells for 48 hours,the expression levels of CYP3A4,CYP3A5 and PXR mRNA were detected by RT-q PCR.5.The monoclonal CYP3A4*1G GG/AA HepG2 cells were detected by mRNA-seq.6.Bioinformatics analysis of transcription factors at CYP3A4*1G SNP site in HepG2 cells.Result1.The recombinant vector px330-sg RNA and pmcherry EGFP-reporter were successfully constructed.2.CYP3A4*1G wild-type homozygous(GG)and mutant homozygous(AA)HepG2 cell lines were successfully established.3.In different genotypes of HepG2 cells,compared with CYP3A4*1G wild type homozygous(GG)HepG2 cells,the expression levels of CYP3A4,CYP3A5 mRNA and protein in CYP3A4*1G mutant homozygous(AA)HepG2 cells were lower(P<0.01).The CYP3A4 enzyme activity of CYP3A4*1G mutant homozygote(AA)and wild type homozygote(GG)HepG2 cells was higher than that of CYP3A4*1G mutant heterozygote(GA)HepG2 cells(P<0.01).4.Experimental results of rifampicin induction:(1)Compared with CYP3A4*1G wild type homozygous(GG)HepG2 cell line,the expression levels of CYP3A4,CYP3A5 and PXR mRNA in CYP3A4*1G mutant homozygous(AA)HepG2 cells decreased before rifampicin induction.(2)Rifampicin increased the expression of CYP3A4,CYP3A5 and PXR mRNA in different genotypes of HepG2 cells,and the expression levels of CYP3A4,CYP3A5 and PXR in CYP3A4*1G mutant homozygous(AA)HepG2 cells induced by rifampicin were lower than those of CYP3A4*1G wild homozygote(GG)genotypes HepG2 cells(P < 0.01).5.mRNA-seq results:The main results were as follows:(1)the expression levels of CYP3A4,CYP3A5 and PXR mRNA in CYP3A4*1G mutant homozygote(AA)HepG2 were lower than those in CYP3A4*1G wild type homozygote(GG)HepG2 cells,which were consistent with the results of RT-PCR.(2)the mRNA expression of transcription factors DMRT(DMRTA2,DMRTC2,etc.)changed in CYP3A4*1G GG and AA HepG2 cells.6.Bioinformatics results: DMRT3 is likely to be the target transcription factor of CYP3A4*1G A allele binding.Conclusion1.Wild type homozygous(GG)and mutant homozygous(AA)HepG2 cell lines of CYP3A4*1G were successfully constructed by CRISPR/Cas9 technique.2.In CYP3A4*1G GG/GA/AA HepG2 cells,(1)CYP3A4*1G regulates CYP3A4 mRNA,protein expression and enzyme activity,mainly at the transcriptional level,allele-dependent regulation(decrease)of CYP3A4 mRNA expression.(2)CYP3A4*1G also regulates the expression of CYP3A5 mRNA and protein,mainly at the post-transcriptional level,allele-dependent regulation(decrease)of CYP3A5 protein expression.3.Rifampicin has a significant CYP3A4*1G allele dependence on the expression level of CYP3A4 mRNA in HepG2 cells.To some extent,rifampicin,together with CYP3A4*1G mutation and rifampicin,affects the mRNA expression level of PXR,and thus participates in the regulation of CYP3A4 mRNA expression level.4.CYP3A4*1G A allele binds more transcription factors than G allele.It is speculated that there may be transcription factors in the DMRT family that bind to CYP3A4*1G A alleles in HepG2 cells.