The Studies Between Aminoacid Metabolism Pathway Polymorphism Of High Incidence Family Of Hepatocellular Carcinoma In Guangxi And Genetic Susceptibility

Part?The study of screen and analysis key gene and pathway in hepatocellular carcinoma by bioinformatics methodsBACKGROUND Hepatocellular carcinoma is one of the most common malignancies.It has a high morbidity and mortality.The incidence of liver cancer has doubled or even more in the past 15 years.However,the molecular mechanism of liver cancer is still largely unknown.environmental effects and hereditary susceptibility contribute to the disease.The poor outcome of HCC treatment is attributed to the deficiency of effective therapeutic approaches and medicines,the complex structure of the liver,nonspecific clinical features,the difficulty of early diagnosis and variations in tumor histological types and differentiation.Therefore,there is an urgent requirement to identify specific molecular biomarkers for the early diagnosis of HCC.For above reasons,an increasing number of researches on liver cancer have been conducted in recent years.Different molecular mechanism and various biomarkers related to liver cancer have been identified.thus further researches are still needed to improve the prognosis of liver cancer.In addition,Previous studies in this field have been relatively small-scale,with only a few genes examined.Due to the complexity of cancer evolution,multiple genes and pathways deviated from normal condition should have been involved.An overall study would be more helpful to understand the pathogenesis and progression of hepatocellular carcinoma.OBJECTIVE Our knowledge about the development of hepatocellular carcinoma is still very limited.In order to acquire better understanding about the pathological mechanisms in this field.Therefore,in this study we compared the global expression profile of the samples from liver cancer patients,and found that key gene and pathway could play an important role in mediating the pathogenesis and progression of liver cancer.MATERIAL AND METHODS mi RNA expression data was obtained from published microarray data of the GSE33006 dataset from Gene Expression Omnibus(GEO),and the raw data were first transformed into probe-level data and the differentially expressed genes(DEGs)between tissues of patients with hepatocellular and normal specimen were identified using T-test in samr package of R language.Protein-protein interaction network analysis was conducted by STRING 9.1 and visualized by Cytoscape software,at the same time,genes with high degree were selected out.while functional enrichment[Gene Ontology(GO)biological process terms]and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were conducted by Gene Codis3 for the target genes.In total 966 DEGs,including 326 upregulated genes and 640 downregulated genes,were identified between the HCC and control samples.RESULTS The results of the hierarchical clustering analysis demonstrated that 95%of the DEGs were sample specific,and that 5 of these were potential key genes(CASP9,SARDH,GNMT,CBS and DDX39).Furthermore,cysteine proteinase 9(CASP9),sarcosine dehydrogenase(SARDH),glycine N-methyltransferase(GNMT),cystathionine-?-synthase(CBS)and ATP independent RNA helicase(DDX39)were included in the obtained network module The main biological processes involved in hepatocellular carcinoma were cell apoptosis,programmed necrotic cell death,cell migration,cell metabolism and cell motility.SARDH,GNMT and CBS are involved in the majority of GO terms and KEGG pathways associated with hepatocellular carcinoma.CONCLUSIONS The combination data mining method increases the probability of identifying the biological processes and functional candidate genes that could represent the high-throughput data and have a great effect on the studied disease.A number of studies have revealed that applying in silico bioinformatic approaches in mining data from high-throughput microarray profiles is reliable and effective in predicting disease-causing biomarkers and has high accuracy.In conclusion,the present study identified critical node proteins exhibiting close interactions with other proteins in the network module,including CASP9,SARDH,GNMT,CBS and DDX39.These proteins may be involved in the tumorigenesis of HCC via modulating the cell apoptosis and amino acid metabolic process,and may be used as molecular biomarkers for the early diagnosis of HCC.The results of the present study assist in further understanding of the tumorigenesis of HCC,and provide potential targets for developing effective therapeutic treatment strategies for this disease.Bioinformatic tools allow us to identify key genes and pathways that are closely related to the development fo hepatocellular carcinoma.Amino-acid metabolism may be the crucial pathway to affect signal transduction in hepatocellular carcinogenesis.The differently expressed genes and disturbed biological functions uncovered in present study may play important roles in the development of HCC and can contribute to the understanding on molecular mechanisms of HCC.Further these DEGs we obtained can be acted as potential biomarkers for diagnosis and therapy of HCC.Part? Genetic association of aminoacid metabolism pathway polymorphisms with susceptibility to high incidence family of liver cancerBACKGROUND As HCC is the third most common cause of cancer-related death worldwide,its overall incidence remains alarmingly high in China and is steadily rising across most of the developed and developing world.Over the past 15 years,the incidence of HCC has more than doubled and it increases with advancing age.Liver cancer,one of the most deadly cancers,is the second-most common cancer in China.Approximately 383,000 people die from liver cancer every year in China,which accounts for 51%of the deaths from liver cancer worldwide.Chronic infection with hepatitis B virus is the leading cause of HCC,closely followed by infection with hepatitis C virus.Aflatoxin exposure,cigarette smoking,heavy alcohol consumption,low vegetable intake,inorganic arsenic ingestion,radioactive thorium dioxide exposure,iron overload and the use of oral contraceptives and anabolic steroids have been documented as HCC risk factors.Other agents such as genetic susceptibility or genomic polymorphisms may also play important roles in the development of liver cancer.Familial aggregation of HCC exists and a major susceptibility gene of HCC has been hypothesized.Patients of some genetic diseases are at an increased risk of HCC.By recognizing the risk factors for HCC,high-risk groups can be identified and followed up with screening strategies.In fact,the management of high-risk patients with screening and surveillance has the real potential to detect HCC early and improve patient outcomes.When HCC is detected earlier,patients are candidates to receive curative treatments.Great effort aimed at primary and Secondary prevention of this cancer,such as universal hepatitis B vaccination in children,chemoprevention in selected population,and early detection in at-risk population,has been undertaken.These strategies might be further emphasized in the future for the effective prevention of liver cancer in China.OBJECTIVE We have screened and analyzed aminoacid metabolic pathways have been previously reported to associated with the development of hepatocellular carcinoma through bioinformatics.Which mining single nucleotide polymorphism of the key gene SARDH,CBS,GNMT,CASP9 in this pathway.The aminoacid metabolism pathway is important in the control of vital processes in the carcinogenesis of hepatocellular carcinoma(HCC),including cell apoptosis,programmed nectonic cell death,cell migration and cell metabolism.In the current study,we aim to assess if genetic variants in the genes of the aminoacid metabolism pathway are associated with the carcinomagenesis and development of HCC.MATERIAL AND METHODS We utilized the International Hap Map Project database and db SNP database to search for candidate variants in the promoter region,all exons including intron–exon boundaries,the 5'-untranslated region(5'-UTR)and the 3'-untranslated region(3'-UTR).We identified 10potential functional polymorphisms.We selected tagging SNPs from 5-kb flanking and within the gene regions of four genes by using the tagger algorithm.6 tagging SNPs were identified with a cut-off value of r~2<0.8 and a minor allele frequency greater than 0.1 in the Chinese population,based on data from the Hap Map Project.We genotyped 10 single nucleotide polymorphisms(SNP)in four core genes(SARDH,CBS,GNMT,CASP9)by using DNA from blood samples of79 high incidence family of liver cancer(20 HCC patients with surgical resection and 59 family members)and 40 control family members from Fusui county in Guangxi.We screened SNPs in-7.2kb promoter region of SARDH gene and constructed a refined linkage disquilibrium(LD)block map:six SNPs(rs2502740?rs129932?rs129886?rs2118852?rs227836 and rs234706).Genomic DNA was extracted from blood samples.Genotyping was performed with Sequenom Mass ARRAY i PLEX platform by use of allele-specific MALDI-TOF mass spectrometry assay.Polymerase chain reaction(PCR)and extension primers for these 36 SNPs were designed using the Mass ARRAY Assay Design 3.0 software.Data analysis,with the exception of haplotype construction and haplotype frequency estimation,was performed with SPSS software version 19.All tests were two-sided and a p<0.05 was considered statistically significant.Odds ratios(ORs)and 95%confident intervals(CIs)were estimated for the multivariate survival analyses by Cox proportional hazards regression models,adjusting for age,gender,family history,HBs Ag and AFP.RESULTS The SARDH rs2502740 allele were G and A.There frequencies in the HCC high incidence family patients were 97.5%and 2.5%,in the healthy control family were 75%and 25%,respectively.It was found that the frequency of the SARDH rs2502740 G allele genotype in HCC high incidence family patient cases was observably higher than that of normal subjects(p=0.001).In comparison to the A allele,the G allele genotype improved the susceptibility to HCC(OR=13,95%CI:1.68?110.82).The CASP9 rs2118852 allele were G and T.There frequencies in the HCC high incidence family patients were 77.5%and2 2.5%,in the healthy control family were 91.03%and 8.97%,respectively.Our finding showd that the frequency of the The CASP9 rs2118852 G allele genotype in HCC high incidence family patient cases was observably lower than that of normal subjects(p=0.042).The G allele have a reduced risk of HCC(adjusted odds ratio OR=0.34,95%confidence individual CI:0.12-0.99)when T allele were blended and they were considered as the reference.The SARDH rs2502740 genotypes were GG,GA and AA.There frequencies in the HCC high incidence family patients group were 95%,5%and 0%,in the healthy control family group were 52.5%,45%and 2.5%,respectively.Allele and genotype associations of SARDH rs2502740 polymorphism With HCC suscep-tibility were observed in comparisons between the HCC high Incidence family A group and the healthy control samples C group(P<0.0001).Risk of HCC development in this high incidence family was significantly increased in carrier of the GG genotype polymorphism when compared with hetrozygote G/A genotype(OR=16.29,95%CI:1.98-133.94).The SARDH rs129886 genotypes were CC,CT and TT.There frequencies in the HCC high incidence family patients group were 50%,25%and 25%,in the healthy control family group were 25.64%,53.85%and 22.51%,respectively.Allele and genotype associations of SARDH rs129886 polymorphism With HCC susceptibility were observed in comparisons between the HCC high Incidence family A group and the healthy control samples C group(P<0.03).Risk of HCC development in this high incidence family was significantly increased in carrier of the GG genotype polymorphism when compared with hetrozygote G/A genotype(OR=4.3,95%CI:1.13?15.59).When stratifying by sex,smoking,dring,HBVcarrier status,AFP level.We found that the GG genotype of the SARDH rs2502740 were associated with a significant in creased risk of HCC among no-drinking individual in comparison to the GA and AA genotype(OR=10.93,95%CI:1.08?18.69).Multivariate regession analyses showed that HCC patients of high incidence family carrying the SARDH rs129886 CC genotype had significantly increased risk of HCC with adjusted odd ratio OR=4.5.In addition cases carrying the CC genotype had a 4.5 fold compared the CT and TT genotypes increased in the risk for HCC(OR=4.5,95%CI:1.08?18.69).CONCLUSIONS Our association studies have found that rs2502740 and rs129886 of SARDH gene are associated with susceptibility to HCC.These SNPs may have a causal role in tumorigenesis or be in LD with the real causal polymorphism,so we continue to carry out the functional studies on these SNP.By in vivo and in vitro functional assays including IHC,Western-blot and quantitation m RNA,we demonstrated in protein and RNA level that rs2502740and rs129886 was a functional regulatory polymorphism in the SARDH splicing region.This polymorphism could regulate the expression of SARDH gene in an allele-specific manner and the G allele was related with incresed expression of SARDH gene.