| Background:Gastric cancer is still an extremely severe health issue with high mortality due to lacking of effective biomarkers.Therefore,screening of the potential biomarker of gastric cancer is still hot research area in cancer-biology.In this study,we aimed to investigate the alterations of salivary protein glycosylation among HV,AG and GC,as well as assess the possibility of salivary glycopatterns as potential biomarkers for the diagnosis of GC.Method:Totally,201 individual samples were enrolled and random assigned as two parts,test group and validation group.Firstly,the salivary glycopatterns of 124 samples(HV:n=30,AG:n=30,GC:n=64)were detected by lectin microarrays,and validated the results by saliva microarrays and lectin blotting analysis.Secondly,the diagnostic model of GC(Model GC)and AG(Model AG)were constructed based on 15 candidate lectins(e.g.,PSA,PHA-E and ECA)that exhibited significantly alterations of salivary glycopattern by logistic stepwise regression.And the two diagnostic models were assessed in the validation group including HV(n=30)and patients with GC(n=23)and AG(n=24).Finally,the glycoproteins from human saliva were selectively isolated by AAL-magnetic particle conjugates.Half of isolated glycoprotein were dealt with NaClO to release O-glycan.And others were subjected to reduction,alkylation,and trypsin digestion transform into polypeptides for further releasing glycans.The collected polypeptides were dealt with PNGase F to release N-glycan.The released N-glycan and O-glycan were purified and performed to identify the entire glycan structural using MALDI-TOF/TOF-MS.Result:Totally,there were 15 lectins reveal significant alterations in salivary glycopatterns between HV,AG and GC(including different stages).The Fuca-1,6GlcNAc(core fucose)binder PSA,and the bisecting GlcNAc and the biantennary complex-type N-glycan with outer Gal binder PHA-E exhibited significantly decreased NFI in all patients with AG or GC compared with the HV(all p<0.05).However,the Galβ-1,4GlcNAc and Galβ1-3GlcNAc binder ECA,the high-Mannose,Manα1-3Man,and Manα1-6Man binder HHL,the Galβ1-3GalNAcα-Ser/Thr(T antigen)binder PNA,the Galα1-3(Fucαl-2)Gal(blood group B antigen)binder EEL,the T antigen,and GalNAc binder MPL,and the Galβ1-3GalNAc,αGalNAc,and αGal binder GSL-I exhibited significantly increased NFIs in all patients with AG or GC compared with the HV(all p<0.01).As shown in Fig.2B,the β-D-G1cNA,(GlcNAcβ1-4)n,Galβ1-4GlcNAc binder DSA,the(GlcNAc)n and high mannose-type N-glycan binder LEL exhibited significantly increased NFIs in AG compared with HV and GC(all p<0.01),however,the Fucal-2Galβl-4GlcNAc and Fucal-3(Galβ1-4)GlcNAc binder LTL exhibited significantly decreased NFIs in AG compared with HV and GC(all p<0.01).The Fucal-6 GlcNAc(core fucose)and Fucal-3(Gaβ1-4)GlcNAc binder AAL exhibited significantly decreased NFIs in GC compared with HV and AG(all p≤0.001),however,the GalNAcα-Ser/Thr(Tn antigen)and GalNAc binder VVA,α-Gal,α-GaINAc,Galα-1,3Gal,and the Galα-1,6Glc binder BS-I,and the a-or p-linked terminal GalNAc,(GalNAc)n,and GalNAcal-3Gal binder SB A exhibited significantly increased NFIs in GC compared with HV and AG(all p<0.01).The principal component analysis(PCA)and hierarchical cluster analysis(HCA)were performed to show the whole level of glycans expressed in salivary proteins among HV,AG and GC.The results showed that the salivary glycopattens were significant difference among HV,AG and GC.So Model GC and Model AG were constructed by logistic stepwise regression to distinguish GC from HV&AG and AG from HV&GC,respectively.The Receiver operating characteristics(ROC)curves indicated that the Model GC(cutoff value:0.91,AUC:0.89,sensitivity:0.96,and specificity:0.80)had high diagnostic accuracy for distinguishing GC from HV and AG(Fig.4D and 4E).22 cases of 23 GC and 19 cases of 24 AG as well as 24 cases of 30 HV were correctly classified by Model GC.While the ROC curves indicated that the Model AG(cutoff value:0.93,AUC:0.83,specificity:0.72,and sensitivity:0.92)also had high diagnostic accuracy for distinguishing AG from HV and GC(Fig.4F and 4E).22 cases of 24 AG and 13 cases of 23 GC as well as 25 case of 30 HV were correctly classified by Model AG.This study provides pivotal information to distinguish HV,AG,and GC based on precise alterations in salivary glycopatterns,which have great potential to be biomarkers for diagnosis of GC.The results of MALDI-TOF/TOF-MS showed that the entire fucosylated glycan structural were significantly different among HV,AG and GC,and the types of fucosylated O-glycan exhibited decreased trends during the development of gastric cancer.Interestingly,the glycan in m/z 1776.629 was the highest abundance peaks in HV and AG,respectively,but became a quite lower abundance peak in GC.Conversely,the same glycans of m/z 1730.636 and 1752.610 were the highest abundance intensity peaks in GC,but became the moderate intensity peaks in HV and AG. |
