Association Between Genetic Polymorphisms Of Cytochrome P450 Oxidoreductase And Hepatocellular Carcinoma Susceptibility

Hepatocellular carcinoma(HCC)ranks the third leading cause of cancer-related death in the world.The main causes of HCC include hepatitis virus infection,chemical carcinogens,alcohol,and obesity-related metabolic disorders.These factors may cause chronic inflammation and immunosuppression,and ultimately lead to the occurrence of HCC.In addition,as an intrinsic factor,genetic polymorphisms may mediate the occurrence of HCC by influencing individual HCC susceptibility.Therefore,the main mechanism of HCC occurrence can be summarized into four aspects: inflammation,metabolism,immunity,and polymorphism.Cytochrome P450 oxidoreductase(POR)is the only electron donor of liver microsomal cytochrome P450 enzymes(CYPs),and the POR is highly polymorphic.It has been reported that different POR single nucleotide polymorphism(SNP)loci can affect different CYPs activities,which may lead to disease susceptibility.For example,POR genetic polymorphisms were associated with breast cancer and bladder cancer susceptibilities,probably due to the changes of activities of steroid-metabolizing CYPs.CYP1,2,and 3 families belong to drug-metabolizing CYPs.They mainly mediate the metabolism of exogenous substances such as clinical drugs,carcinogens,and procarcinogens,and are closely related to liver diseases such as hepatitis,cirrhosis,and HCC.We previously carried out a lot of studies on the relationships between CYPs andHCC,and found that the higher CYP2E1 activity is a risk factor for HCC.In addition,we separately analyzed the effects of mutant-heterozygotes and mutant-homozygotes on the CYPs activities in multiple POR SNP loci in HCC patients,filling the gap in the field of HCC researches.Based on the previous results,we further explore the association between POR genetic polymorphisms and HCC susceptibility,and the CYPs that probably participate in this process.At present,the literatures about genetic polymorphisms paid more attentions to the relationships with disease susceptibility,but the susceptibility mechanisms of SNP loci were not discussed in depth.Protein is the base of life activities.The changes of genotype affect the protein phenotype and protein interactions,which finally affect the physiological functions and pathological status of the body.With the rapid development of proteomics,the multi-omics application strategy with genomics,transcriptomics,and proteomics has been applied to explore the association and molecular mechanisms from gene aspect to protein aspect.Thus,proteomics provides a reliable and feasible method for exploring the mechanism of HCC occurrence involved in susceptibility loci.In this study,105 normal human liver samples and 102 para-cancerous liver samples of HCC patients were collected to investigate the association between POR genetic polymorphisms and HCC susceptibility.And then,we explored the possible mechanisms of susceptibility SNP loci by Label-free quantitative proteomics method.The aim of the research is to discover new SNP loci of HCC susceptibility and provide new strategies and evidence for the studies of POR genetic polymorphisms and HCC occurrence mechanisms in the future.1 Methods 1.1 Human liver samples and clinical data collection 105 normal human liver samples and 102 para-cancerous liver samples of HCC patients were collected,and basic information such as gender,age,history of smoking and drinking,and clinical diagnosis were included.In addition,the clinical parameters including alanine aminotransferase(ALT),aspartate aminotransferase(AST),gammaglutamyl transferase(GGT),total protein(TP),and globulin(GLO)were recorded.For HCC patients,follow-up was conducted after surgical resection for 42~54 months.The experimental scheme of our research was permited by the Ethics Committee of Zhengzhou University,and all subjects had signed the informed consent.1.2 Human liver microsomes preparation The human liver microsomes were prepared by the differential centrifugation method.1.3 Liver microsomal protein concentration determination The microsomal protein concentrations were determined by the BCA method.1.4 CYP2E1 activity determination The chlorzoxazone was used as the CYP2E1 probe substrate,and the metabolite was the 6-hydroxy chlorzoxazone.The 100 ?L incubation system includes liver microsomal protein,chlorzoxazone solution,and phosphate buffer.The NADPH was added to start the reaction,and the reaction was terminated in an ice bath.The mixture was extracted by ethyl acetate,and dried by nitrogen.Determination of samples by high performance liquid chromatography(HPLC)after re-dissolution.The enzyme kinetic parameters were calculated,including Michaelis-menten constant(Km),maximum velocity(Vmax)and intrinsic clearance(CLint).1.5 POR genetic polymorphisms determination The distribution frequencies of POR rs10954732(G>A),rs2286822(C>T),rs1135612(A>G),and rs1057868(C>T)were more than 1% in Chinese population.Human liver tissue DNA was extracted,and genotyping was performed using the SNP Mass ARRAY method.1.6 Hepatic tissue proteins identification by Label-free quantitative proteomics The tissues proteins were extracted and digested to obtained the peptide solution.The components were eluted and collected by high-p H reversed-phase chromatography and detected by mass spectrometry(MS).Max Quant software was used to data analyze.The values of intensity-based absolute protein quantification(i BAQ)indicated the protein expression.The screening criteria of differentially expressed proteins was that the expression difference was greater than 1.2-fold and P < 0.05.The 41 and 71 samples were collected randomly based on random number table from above tissues for samples preparation and MS analyses.According to quality control standards of proteomic data,34 normal and 61 para-cancerous liver tissues were finally selected for differentially expressed proteins screening and subsequent analyses.1.7 Statistical analyses Statistical analyses and graphs were performed by SPSS 22 software and Graph Pad Prism 7 software.Parametric test is used for normal distribution data,otherwise non-parametric test is used.A binary Logistic regression model was adopted to explored the association between POR SNP loci and HCC susceptibility,which corrected confounding factors including age,gender,smoking,and drinking.KaplanMeier method was selected for survival analysis.Using ROC analysis to describe the HCC predictor value.P < 0.05 was considered statistically significance(two-tailed).2 Results 2.1 Association between POR genetic polymorphisms and HCC susceptibility 2.1.1 Association between POR genetic polymorphisms and the risk of HCC For POR rs10954732(G>A),compared with the GG genotype population,the risk of HCC was lower in the GA genotype population(OR = 0.30,95% CI:0.11~0.78,P = 0.014)and GA+AA genotype(genetic variant)population(OR = 0.35,95% CI:0.14~0.88,P = 0.026),respectively.No HCC susceptibility was found between G and A alleles,GG and AA genotypes,or GG+GA and AA genotypes.The results suggested that the POR rs10954732 variant may reduce the risk of HCC.The genetic polymorphisms of POR rs2286822,rs1135612,and rs1057868 have no significant association with the risk of HCC.2.1.2 Relationships between POR rs10954732 genetic polymorphism and clinical parameters.In the HCC group,ALT and AST expression levels were lower in population with variant(P = 0.027;P = 0.005),indicating that the POR rs10954732 variant may improve liver function to a certain extent.There was no significant relationship between ALT,AST expression levels and POR rs10954732 genetic polymorphism in normal population.2.1.3 Relationships between POR rs10954732 genetic polymorphism and CYP2E1 activity.In the HCC group,V2E1 and CL2E1 level were lower in population with variant(P = 0.038;P = 0.017),indicating that the protective effect of POR rs10954732 variant may be related to the decrease of CYP2E1 activity.There was no significant relationship between CYP2E1 activity and POR rs10954732 genetic polymorphism in normal population.2.2 HCC susceptibility proteomics study of POR rs10954732 2.2.1 Differentially expressed proteins identification The 1360 differentially expressed proteins were identified between 34 normal subjects and 61 HCC patients.According to significance of difference,the number of differentially expressed protein of P < 0.01,0.01 ? P < 0.03,and 0.03 ? P < 0.05 was 640,425,and 295,respectively.Compared with normal group,there were 814 upregulated proteins and 546 down-regulated proteins in HCC group.The HCC group was divided into GG and GA+AA subgroups to identify differentially expressed proteins,including 16 and 44 cases,respectively.And one case was deleted for failed genotype detection.The 345 differential proteins were identified.According to significance of difference,the number of differentially expressed protein of P < 0.01,0.01 ? P < 0.03,and 0.03 ? P < 0.05 was 80,144,and 121,respectively.Compared with GG subgroup,there were 315 up-regulated proteins and 30 downregulated proteins in GA+AA subgroup.2.2.2 Gene ontology(GO)analysis of subgroup differentially expressed proteins The 345 differentially expressed proteins of the subgroup with variant were subjected to GO analysis from three aspects: cell composition,molecular function,and biological process.For cell composition aspect,subgroup differentially expressed proteins were mainly located in organelle inner membrane,endoplasmic reticulum lumen,cytosolic ribosome,anchoring junction,and lysosome.For molecular function aspect,subgroup differentially expressed proteins were mainly involved in oxidoreductase activity oxidoreductase activity,structural constituent of ribosome,cell adhesion molecule binding,glucosyltransferase activity,protein binding involved in heterotypic cell-cell adhesion,and electron transfer activity.For biological process aspect,subgroup differentially expressed proteins were mainly involved in protein targeting,mitochondrial respiratory chain complex assembly,protein folding,cytochrome complex assembly,cellular protein catabolic process,regulation of response to endoplasmic reticulum stress,monocarboxylic acid metabolic process,T cell mediated immunity,and lipid catabolic process.2.2.3 Screening and analysis of candidate differentially expressed proteins 2.2.3.1 Classification and screening of differentially expressed proteins Further,the proteins that may be directly associated with the protective effect of POR rs10954732 variant were screened.There were 70 intersecting proteins of differentially expressed proteins of HCC group and variant subgroups.According to GO,Gene Cards,and NCBI Pub Med databases,these proteins were classified into five categories: metabolism,inflammation,immunity,polymorphism,and others.Each protein could be classified into several categories.Finally,52 proteins were included.Among them,11 proteins participated in metabolic process,25 proteins participated in inflammatory process,20 proteins participated in immune process,8 proteins whose SNP loci have cancer susceptibilities,and 31 proteins participated in cancer process in mechanisms other than the above four aspects.All of the 52 proteins were up-regulated proteins in the GA+AA subgroup.The 6 candidate proteins were screened out from 52 proteins,which could simultaneously satisfy the down-regulated expression in HCC group and have anti-cancer effects reported in literatures.They were serine protease hepsin(HPN),coxsackievirus and adenovirus receptor(CXADR),short-chain dehydrogenase/reductase 3(DHRS3),Rasrelated protein Rap-2c(RAP2C),enoyl-Co A hydratase 1(ECH1),and desmoplakin(DSP).2.2.3.2 Relationships between candidate proteins expression and clinical parameters.HPN Compared with normal subjects,the HPN expression was significantly down-regulated in HCC(P = 1.00×10-6).In HCC group,HPN expression was upregulated in variant population(P = 0.014),indicating that the protective effect of POR rs10954732 may be related to the increase of HPN expression.There was no significant relationship between HPN expression and POR rs10954732 genetic polymorphism.The HCC group was divided into low and high expression groups according to HPN expression,the levels of AST and GGT were lower in the high expression group(P = 0.008;P = 0.006),and ALT and GLO also showed a decreased trend(P = 0.072;P = 0.074),indicating that HPN may improve liver function to a certain extent.The KaplanMeier survival analysis showed that the survival time was significantly prolonged with higher expression of HPN in HCC patients(Plog-rank = 0.0057),suggesting that the HPN may inhibit the development of HCC.The area under ROC curve was 0.792(P = 3.00×10-6),which indicated that the HPN had certain value of predictor for HCC.CXADR Compared with normal subjects,the CXADR expression was downregulated in HCC(P = 0.015).In HCC group,CXADR expression was up-regulated in variant population(P = 0.011),indicating that the protective effect of POR rs10954732 may be related to the increase of CXADR expression.There was no significant relationship between CXADR expression and POR rs10954732 genetic polymorphism.The HCC group was divided into low and high expression groups according to CXADR expression,the levels of CL2E1 was lower in the high expression group(P = 0.043),and V2E1 also showed a decreased trend(P = 0.100),indicating that the decrease of CYP2E1 activity may be related to the increase of CXADR expression.The area under ROC curve was 0.650(P = 0.016),which indicated that CXADR had certain value of predictor for HCC.DHRS3 Compared with normal subjects,the DHRS3 expression was downregulated in HCC(P = 0.0004).In HCC group,DHRS3 expression was up-regulated in variant population(P = 0.012),indicating that the protective effect of POR rs10954732 may be related to the increase of DHRS3 expression.There was no significant relationship between DHRS3 expression and POR rs10954732 genetic polymorphism.The area under ROC curve was 0.710(P = 0.0007),which indicated that DHRS3 had certain value of predictor for HCC.RAP2 C Compared with normal subjects,the RAP2 C expression was downregulated in HCC(P = 0.010).In HCC group,RAP2 C expression was up-regulated in variant population(P = 0.011),indicating that the protective effect of POR rs10954732 may be related to the increase of RAP2 C expression.There was no significant relationship between RAP2 C expression and POR rs10954732 genetic polymorphism.The area under ROC curve was 0.659(P = 0.010),which indicated that RAP2 C had certain value of predictor for HCC.ECH1 Compared with normal subjects,the ECH1 expression was downregulated in HCC(P = 0.011).In HCC group,ECH1 expression was up-regulated in variant population(P = 0.030),indicating that the protective effect of POR rs10954732 may be related to the increase of ECH1 expression.There was no significant relationship between ECH1 expression and POR rs10954732 genetic polymorphism.The area under ROC curve was 0.657(P = 0.011),which indicated that ECH1 had certain value of predictor for HCC.DSP Compared with normal subjects,the DSP expression was down-regulated in HCC(P = 0.017).In HCC group,DSP expression was up-regulated in variant population(P = 0.023),indicating that the protective effect of POR rs10954732 may be related to the increase of DSP expression.There was no significant relationship between DSP expression and POR rs10954732 genetic polymorphism.The area under ROC curve was 0.648(P = 0.017),which indicated that DSP had certain value of predictor for HCC.Conclusion 1.POR rs10954732(G>A)variant reduced the risk of HCC,and its protective effect may be related to decreased activity of CYP2E1 and increased expression of HPN,CXADR,DHRS3,RAP2 C,ECH1,and DSP.Among them,the CYP2E1 activity was decreased in HCC patients with higher expression of CXADR.2.The survival time was prolonged in HCC patients with higher expression of HPN.