| ObjectiveLiver transplantation is the most effective way to treat various end-stage liver diseases.Rejection is the most common complication after transplantation as one of the biggest barriers to the survival of transplanted organs.Therefore,the key to the success of organ transplantation is to suppress the host's immune system so as to prevent the occurrence of rejection reaction.The most effective method is the rational selection and use of immunosuppressants.At present,the commonly used triple-drug immunosuppressive therapy is composed of tacrolimus,corticosteroids and mycophenolate mofetil.Among them,tacrolimus dose needs to be adjusted by therapeutic drug monitoring(TDM)due to its pharmacokinetic and pharmacodynamic characteristics such as narrow therapeutic window,individual differences and severe adverse drug reactions.However,the incidence of acute rejection and drug toxicity after transplantation can only be reduced in a certain extent due to the significant time lag of TDM.Therefore,for the efficacy and safety use of tacrolimus application after transplantation,it is urgent to confirm the key factors affecting the metabolic process of tacrolimus and to identify them as biomarkers of determining the individual dose of different patient before administration.Present studies believe that phase-I metabolizing enzyme CYP3A5 is the major enzyme involved in the metabolic pathway of tacrolimus in human.The research on the causes of individual variant of tacrolimus blood concentration in TDM is only in pharmacogenetics,that is,gene polymorphism,and most of the conclusions are not consistent.It is unknown whether there are other metabolic pathways in the metabolism of tacrolimus in the human body besides CYP3A5,and if there are any other genetic or epigenetic factors regulating the expression or activity of drug metabolic enzymes or drug transporters.Meanwhile,as liver is the most important organ for drug metabolism,studies have suggested that donor liver played a more important role in the study of drug metabolism after liver transplantation.And tacrolimus initial blood concentration could reflect individual variant of donor livers in the early period after liver transplantation.Therefore,this study is based on the initial tacrolimus blood concentration after liver transplantation,aim to:(1)study the metabolic pathway of tacrolimus systematically;(2)identify the genetic and epigenetic factors of donor liver that may affect the metabolic process of tacrolimus after liver transplantation;(3)confirm the biomarkers for personalized tacrolimus dosage regimen.Chapter I Genetic factors of donor liver affecting tacrolimus blood concentrationMaterial and Methods1.Subject screening and donor liver tissue sample collectionIn this study,we retrospectively reviewed 230 liver transplants performed at the First Affiliated Hospital of Zhengzhou University between June 2016 and June 2018.Individual data of donors and receptors,the postoperative immunosuppressant administration protocols and tacrolimus initial blood concentration results were sorted and compared.A total of 78 patients were selected after screening which were divided into two groups with significant differences in blood concentration,and donor liver tissue of these 78 patients were obtained from the key organ transplantation laboratory of Henan Province.2.DNA extraction and gene polymorphism detection of 78 donor liver tissuesDNA was extracted from liver tissues,PCR was amplified for target DNA fragments,CYP3A5,CYP3A4,CYP3A7,UGT1A4,UGT2B7 and ABCB1 gene polymorphisms were detected based on the common SNP)sites in Asians.Then a comparative analysis was performed with tacrolimus initial blood concentration/dose(C/D)ratio to identify the genetic factors of drug metabolic enzymes or transporters affecting tacrolimus initial blood concentration after liver transplantation.3.RNA isolation and sequencing analysis of CYP3A5*3/*3 donor hepatic tissue specimensAccording to the genetic polymorphism results,23 of 36 donor liver samples with CYP3A5*3/*3 genotype which had a significant differences in tacrolimus C/D ratio were selected.Isolated RNA from tissue samples,then RNA-sequencing was conducted on an Illmina genome analyzer IIx using next-generation sequencing analysis.Significantly expressed genes which might be related to the tacrolimus metabolic were determined by p-value of multiple tests between high and low C/D ratio groups.Results1.Distribution of gene polymorphism and correlation with tacrolimus initial blood C/D ratio(1)Frequency of CYP variantsIn 78 donor livers,there was a significant difference in tacrolimus initial blood C/D ratio between CYP3A5 expressor(CYP3A5*1/*1,CYP3A5*1/*3)and CYP3A5 non-expressor(CYP3A5*3/*3)(P=0.0026).But the analysis of donors with CYP3A5*3/*3(activity loss)genotype had showed that there still exited a difference in tacrolimus initial blood concentration,indicating that CYP3A5*3 could not fully explain the individual variant in tacrolimus blood concentration after liver transplantation.In 78 donor livers,there was significant difference in tacrolimus initial blood C/D ratio between CYP3A4 non-expressor(CYP3A4*1/*1)and CYP3A4 expressor(CYP3A4*1/*1G+CYP3A4*1G/*1G)(P=0.0003),the SNP at this site is one of the causes of individual differences in tacrolimus blood concentration.The frequency of CYP3A7 genotypes in 78 donors showed that there was a significant difference between GG type and GC+CC type at rs2257401 site in tacrolimus initial blood C/D ratio(P=0.0028),and there was also a significant difference between AA and GG+GA donors at rs10211 site(P=0.041).(2)Frequency of UGT variantsIn 78 donor livers,the investigation of five SNP sites of UGT1A4 which were commonly mutated in Asian population showed that there were linkage disequilibrium,but there was no significant difference in the C/D ratio between different genotypes at each site(P=0.66).The data showed there were linkage disequilibrium between the five SNP site(rs7662029,rs7668258,rs7439366,rs7441750,rs7441774)of UGT2B7,but there were no significant differences in tacrolimus initial blood C/D ratio between different genotypes at each point(P--0.77).Further investigation was needed to check whether UGTs participated in the phase-II metabolic pathway of tacrolimus.(3)Frequency of ABCB1 variantsFrom the data of genotyping,the three genetic polymorphisms of ABCB1 showed no significant correlation with tacrolimus C/D ratio(P=0.29?0.60?0.50).2.Differentially expressed genes in RNA-sequencingThe results of RNA-sequencing demonstrated that there was no correlation between the gene expression of CYP3A4 or CYP3A7 and tacrolimus C/D ratio.But the gene expression of UGT2B7,UGT1A4,UGT1A1 and ABCB1 was significantly correlated with tacrolimus initial blood C/D ratio(r=-0.427?-0.432?-0.516?0.515,P<0.05).Meanwhile the gene expression of CYP3A7 was much lower the others.The results also showed that there was a high correlation between the gene expression of hepatic nuclear factor 4?(HNF4?)and tacrolimus C/D ratio,also with the gene expression of UGT2B7,UGT1Al and UGT1A4(r=-0.507?0.692?0.413?0.635,P<0.05).These results suggest that it is necessary to identify the UGT enzymes in the tacrolimus phase-? metabolic pathway.Chapter ? Effect of HNF4?/HNF4?-AS1/UGT1A4 on tacrolimus blood concentrationMaterial and Methods1.In vitro enzyme experiment for selecting UGT isoform towards tacrolimus(1)Recombinant human UGTs screening of tacrolimusA glucuronic acid binding reaction incubation system for tacrolimus was established,recombinant human UGT1A1?UGT1A3?UGT1A4?UGT1A6?UGT1A7?UGT1A8?UGT1A9?UGT1A10?UGT2B4?UGT2B7?UGT2B10?UGT2B15?UGT2B17 and human liver microsomes(HLM)were incubated with tacrolimus respectively.Liquid chromatography and mass spectrometry were employed for the detection of tacrolimus and its glucuronide.(2)Chemical inhibition experimentTo further determined the selectivity of UGT1A4 towards tacrolimus.Chemical inhibitors of UGT1A1,UGT1A4,UGT1A9 and UGT2B7 were used to incubate with tacrolimus in HLM,and the metabolites of tacrolimus glucuronide were detected and analyzed by mass spectrometry.2.Determination of UGT1A4,HNF4?,HNF4?-AS1 mRNA expression in CYP3A5*3/*3 donor liversThe mRNA expression levels of UGT1A4,HNF4?,and HNF4?-AS1 in 23 CYP3A5*3/*3 donor liver tissues with different tacrolimus blood concentrations were detected by quantitative real-time polymerase chain reaction(qPCR).Correlation analysis was performed between the mRNA expression level and tacrolimus C/D ratio.3.Loss-of-function approaches in human hepatocellular carcinoma cell line Huh7HNF4? and HNF4?-AS1 were knocked down in human hepatocellular carcinoma cell line Huh7 cells respectively by loss-of-function experiment,then mRNA and protein expression levels of HNF4? and UGT1A4 in the knock-down cells were detected by RT-qPCR and Western Blot for determining the regulatory effect of HNF4?-AS1/HNF4? pathway.Results1.In vitro enzyme experimentScreening of the 13 recombinant human UGTs showed that only UGT1A4 high selectively catalyzed the glucuronidation of tacrolimus.Chemical inhibition experiments indicated that Nilotinib and Hecogenin showed obvious inhibition to tacrolimus glucuronidation.The results indicated that in UGTs family,UGT1A4 was participated in phase-II metabolism pathway of tacrolimus.2.mRNA expression levels of UGT1A4,HNF4? and HNF4?-ASl in donor liver tissue with CYP3A5*3/*3 genotypeIn the 23 CYP3A5*3/*3 donor livers,the mRNA expression levels of UGT1A4 and HNF4? were negatively correlated with the tacrolimus initial blood concentration(r=-0.418,-0.453,P<0.05),and the mRNA expression levels of HNF4? and UGT1A4 were significantly positively correlated(r=0.614,P<0.05).The mRNA expression levels between HNF4?-AS1 and HNF4?,UGT1A4 were all positively correlated(r=0.505,0.416,P<0.05).These results suggested that HNF4?/HNF4?-AS1 pathway could affect tacrolimus blood concentration by regulating the expression of UGT1A4.3.Effect of HNF4?/HNF4?-As1 on the expression of UGT1A4 in Huh7 cellsThe results showed that after transfection with si-HNF4?,the mRNA and protein expression of HNF4?,HNF4?-AS1 and UGT1A4 were decreased significantly;after transfection with si-HNF4?-AS1,the mRNA and protein expression of HNF4? and UGT1A4 was all increased,compared with the control group.From this result,it was suggested that HNF4?-AS1 might act as a co-inhibitor and interact with HNF4? to localize on the binding site of the promoter region of UGT1A4 gene,so as to regulate the expression of UGT1A4.Chapter ? Effect of DNA methylation in ABCB1 promoter region on tacrolimus blood concentrationMaterial and Methods1.Detection of DNA methylation in CYP3A5*3/*3 donor hepatic tissueIllumina 850 methylation chip sequencing technology was used to screening the DNA methylation level of CpG sites in 15 of 23 donors with CYP3A5*3/*3 genotype(approximately 500ng of DNA per sample),the CpG sites of ABCB1 with significant methylation differences between high and low tacrolimus C/D ratios were screened out,and then verified by pyrosequencing in all 23 liver samples.2.ABCB1 mRNA expression in liver tissueThe mRNA expression level of ABCB1 in 23 donor livers with CYP3A5*3/*3 genotype was detected by qPCR,and the correlation between mRNA expression,tacrolimus C/D ratios and DNA methylation level was analyzed.3.Effect of DNA methylation on the expression of ABCB1 and tacrolimus concentration in HepG2 cellsAfter treating HepG2 cells with 0 or 10 ?mol/L 5-Aza-2dC for 72 h,the medium containing 60 ?mol/L tacrolimus was replaced.After 48 h changed back to normal medium,then cells were collected at 0,4,6,and 12 h after replacement.The ABCB1 mRNA and protein expression levels were detected by RT-qPCR and Western Blot,tacrolimus concentration intra-cellular were determined by liquid mass spectrometry,and the DNA methylation level were verified by pyrosequencing.Results1.Differences of ABCB1 CpG methylation in CYP3A5*3/*3 donor livers with different tacrolimus C/D ratiosGenome-wide methylation sequencing results showed that the methylation of three CpG sites(cg12501229,cg00634941,cg05496710)of ABCB1 had significant differences in groups with different tacrolimus C/D ratios.Further investigation by pyrosequencing showed that the methylation level of all three CpG sites of ABCB1 was negatively correlated with tacrolimus C/D ratio.2.ABCB1 mRNA expression level in donor livers with CYP3A5*3/*3 genotypeqPCR results showed that ABCB1mRNA expression and tacrolimus C/D ratios were positively correlated(r=0.458,P<0.05),and the methylation levels at three CpG sites of ABCB1 were negetively correlated with ABCB1 mRNA expression level(r=-0.273?-0.292?-0.195).This suggested that methylation of ABCB1 promoter region might be another important epigenetic factor affecting tacrolimus blood concentration.3.Effect of 5-Aza-2-dC on ABCB1 expression and metabolism of tacrolimus in HepG2 cellThe results of RT-qPCR and Western Blot were obviously increased after treated with 5-Aza-2-dC.And tacrolimus levels intra-cellular was significantly reduced compared to the control group.Meanwhile,the results of pyrosequencing on the methylation level of ABCB1 CpG sits had shown that the methylation level was greatly decreased after treatment with 5-Aza-2-dC.Thus,it was proved that the methylation of ABCB1 promoter affected the metabolism process of tacrolimus in the human by regulating the expression of ABCB1.Conclusions1.Donor CYP3A5*3 and CYP3A4*1G genotype is associated with tacrolimus initial blood concentration after liver transplantation,but cannot fully explain the individual differences.2.UGT1A4 is participated in phase-? metabolism pathway of tacrolimus in human.And HNF4?/HNF4?-AS1 pathway regulates the differential expression of UGT1A4 and thus affects tacrolimus blood concentration after liver transplantation.3.DNA methylation of donor ABCB1 promoter region plays a crucial role in regulating the expression of ABCB1 and thus affects tacrolimus blood concentration after liver transplantation. |
PDF Full Text Request