Analysis Of Serum Distinctive Glycans Profile And Differential Proteins In Gastric Cancer And The Association Between Rage Polymorphism And Risk Of Gastric Cancer

CHAPTER IAnalysis of serum distinctive glycans profile in patients with gastric cancer by using lectin microarray technologyObjective To investigate the distinctive glycan affinity profiling of serum glycoproteins between patients with gastric cancer and control.Methods Gastric cancer patients and health control were involved in this experiment. Experimental process included following steps:Pooled serum were obtained from equal volume sample mixed. The high abundance proteins depleted serum proteins were collected by removing albumin and IgG. After desalting and ultrafiltrating, the high abundance proteins depleted serum were labeled with fluorescence dye Cy5. Cy5 labeled proteins were hybridized with lectin microarray which contained 50 lectins. Affinity signals were obtained by scanning lectin microarray. Affinity ability to glycan of LEL, PHA-L, RCA-I, STL were identified using lectin blot. Data were analyzed by independent sample t test and nonparametric statistics according to the homogeneity of variance.Results The glycan affinity to 13 lectins with statistically significant differences were found in gastric cancer group compare with health control.11 lectins including CAL, DSA, EEL, LEL, PWA, PHA-L, RCA-I, RCA-II, STL, UEA and VAL showed the high affinity ability in gastric cancer (P<0.05). It implied the increase of T/Tn antigen, Tri/tetra-antennary, galactosyl(a-1,3)galactose, N-acetylglucosamine, (GlcNacβ4) n,β-1,6 branching structure, Terminal Ga1β 1-4 GlcNAcβ1, N-acetylgalactosamine, oligomers of N-acetylglucosamine, a-linked fucose residues, P-D-galactosyl residues in gastric cancer. Two lectins including CFL and GNL had the low affinity ability in gastric cancer (P<0.05). It suggested the decrease of N-acetyl-D-galatosamine, (a-1,3) mannose residues in gastric cancer. Lectin blot results of LEL, PHA-L RCA-I, STL confirmed the accuracy of lectin microarraysConclusions Serum glycan of glycoproteins were altered in gastric cancer compare with health control. This may imply it is closely related to the occurrence and development of gastric cancer.CHAPTER IITandem Mass Tags(TMT) based quantitative proteomics serum analysis in Gastric cancerObjective Using TMT quantitative proteomics technique and bioinformatics method to identify potential serum diagnostic biomarkers for Gastric cancer.Methods Gastric cancer patients and health control were involved in this experiment. Mixed serum were pooled with 10 individual sample. The high abundance proteins depleted serum proteins were collected by removing albumin and IgG. After desalting and ultrafiltrating, the trypsin digested proteins were labeled by TMT. The peptide mixture was fractionated by high pH separation using a Aquity UPLC system connected to a reverse phase column. The fractions were separated by nano LC and analyzed by on-line electrospray tandem mass spectrometry. The differential protein were screened out by using the cut off value of 1.2 fold change for up or down regulation. The GO classification was further analyzed by UniProtKB/Swiss-Prot database.Results A total of 594 serum distinct proteins were identified between gastric cancer group and health control.48 proteins were up-regulated and 57 proteins were down-regulated using the cut off value of 1.2 fold change. Using bioinformatics analysis of differentially expressed proteins, we found that most of differentially expressed proteins localized in extracellular exosome, extracellular region, extracellular space, plasma membrane; biological process involved in the antigen binding, calcium ion binding, protein homodimerization activity, zinc ion binding; molecular function involved in innate immune response, blood coagulation, cellular protein metabolic process.Conclusions These results showed that we have created the differential expressed protein database of Gastric cancer using TMT technology. These proteins as potential molecular markers would help us to reveal the potential molecular mechanism of Gastric cancer.CHAPTER ⅢRelevance between RAGE polymorphismand and sRAGE or the risk of gastric cancerObjective To investigate the relevance between RAGE gene rs2070600, rs 184003, rs 1800624 and rs 1800625 polymorphisms with serum sRAGE level and the risk of gastric cancer in Guangxi Chinese population.Methods 200 gastric cancer patients and 207 health controls were involved in our study. The polymerase chain reaction-restriction fragment length polymorphism strategy was used to detect RAGE gene rs2070600, rs184003, rs 1800624 and rs1800625 polymorphisms. Genotype results were verified by DNA sequencing method. The estimated odds ratios (ORs) and 95% confidence intervals (CIs) were obtained by using a binary logistic regression model after adjusting potential confounding variables such as gender, age, ethnicity, body mass index, smoking and alcohol consumption. Serum sRAGE level were detected by ELISA.Results1. The genotype distributions of rs2070600 and rs184003 were found to be consistent with the Hardy-Weinberg equilibrium, that means the samples were representative and comparable (P>0.05).2. There were three genotypes (GG, AG and AA) in rs2070600 site. Analysis for the rs2070600 polymorphism indicated that these subjects carrying the AG genotype had a moderately increased risk for gastric cancer compare to GG genotype, with adjusted ORs of 1.62 (95% CI=1.03-2.58; P=0.038). Subjects carrying combined genotype AG+AA had a moderately increased risk for gastric cancer compare to GG genotype, with adjusted ORs of 1.83 (95% CI = 1.01-2.39; P= 0.044).3. There were three genotypes (GG, GT and TT) in rs 184003 site. Analysis for the rs 184003 polymorphism indicated that these subjects carrying the GT genotype had a moderately decreased risk for gastric cancer compare to GG genotype, with adjusted ORs of 0.62 (95% CI= 0.39-0.99; P=0.048).4. There were three genotypes (TT, AT and AA) in rs 1800624 site. Binary logistic regression analysis for the rs 1800624 polymorphism revealed no differences in genotype and allele frequencies distribution between gastric cancer patients and health controls. The rs 1800624 polymorphisms were not associated with gastric cancer risk.5. There were three genotypes (CC, CT and TT) in rs 1800625 site. Binary logistic regression analysis for the rs 1800625 polymorphism revealed no differences in genotype and allele frequencies distribution between gastric cancer patients and health controls. The RAGE rs 1800625 polymorphisms were not associated with gastric cancer risk.6. Analysis for the RAGE gene rs2070600 polymorphism in nonsmoking population indicated that these subjects carrying the AG genotype had a moderately increased risk for gastric cancer compare to GG genotype, with adjusted ORs of 1.71 (95% CI=1.01-2.91; P=0.043). Analysis for the RAGE gene rs2070600 polymorphism in nondrinking population indicated that these subjects carrying the AG genotype had a moderately increased risk for gastric cancer compare to GG genotype, with adjusted ORs of 1.75 (95% CI=1.03-2.97; P=0.037).7. Subjects carrying the rs2070600 site AG genotype had a moderately decreased serum sRAGE level compared with those carrying GG genotype(P= 0.010). Subjects carrying the rs 184003 site TT genotype had a moderately increased serum sRAGE level compared with those carrying GG genotype (P= 0.014).Conclusions Our findings suggest that variants in RAGE gene rs2070600 site had decreased serum sRAGE level and associated with increased risk Gastric cancer in the Guangxi population.variants in RAGE gene rs 184003 site had increased serum sRAGE level and associated with decreased risk Gastric cancer in the Guangxi population.