The Relationship Between Neoglycoproteins And Oral Microbiota In Gastric Cancer Patients

Background:Gastric cancer(GC)is a malignant tumor worldwide with a very high mortality rate,which poses a serious threat to human health.As the early clinical symptoms of GC patients are not obvious,most patients are diagnosed at the advanced stages,these due to the high mortality of GC.Therefore,it is still urgent demands to identify reliable biomarkers and establish efficient methods for early diagnosis of GC.Microbiota in the oral cavity plays an important role in maintaining oral health and even the whole body’s health.And the composition and distribution of oral microbiota also be regulated by distal diseases,which secrete cytokines and other substances to affect the oral microenvironment through blood circulation,endocrine and lymphatic systems.Thus,discovery of the altered oral microbial components while GC occurred is also very meaningful.Our previous study has shown the significantly altered salivary glycopatterns in GC patients.But it is unclear whether the altered salivary glycopatterns could cause the changes in species abundance of oral microbiota.Hence,this study first researched the oral microbiome of GC patients and healthy volunteers(HV),which provide a novel insight for early diagnosis,risk assessment,and efficacy assessment of GC.Then the neoglycoproteins which corresponding to the altered salivary glycopatterns in GC patients were synthesised.And the effects of neoglycoproteins on the up-regulated oral bacteria of GC patients were investigated,as well as the underlying mechanisms.Then to reveal the relationship between salivary glycopatterns and the abundance of oral microbiota in GC patient.And to elucidate the correlation between salivary glycopatterns and oral microbiota in the occurrence and development of human distal diseases.Methods:The study is divided into four parts:1.The species and abundance of oral microbiota were identified by 16 S rDNA sequencing.The α-diversity and β-diversity between HV and GC patients were analyzed and compared.Furthermore,PCR and qPCR were used to verify the results.2.The neoglycoproteins corresponding to the altered salivary glycopatterns in GC patients were synthesized.The number of monosaccharides attached to each BSA were calculated based on the analysis of LC-Q-TOF-MS and orcinol-sulfuric acid reaction.3.The effect of neoglycoprotein on the proliferation and adhesion functions of Aggregatibacter segnis(A.segnis)were analyzed.The outer membrane and total protein glycopatterns of A.segnis were analyzed by lectin microarrays.4.The toxicity of A.segnis and Fuc-BSA treated A.segnis to oral cells and that induced innate immune responses were preliminary studied.Results:1.The 24 oral microbiota samples sequencing results showed that no significant bacterial diversity(α-diversity)difference between HV and GC patients,but the β-diversity were significant differences.The results showed that one family,five genera and seven species were significant differences.Carnobacteriaceae,Granulicatella,Bergeyella,TM7[g-6],Streptococcus salivarius and Oral＿taxon＿870 showed significantly decreased in GC patients,while Alloprevotella,Megasphaera,A.segnis,Megasphaera micronuciformis,oraltaxon＿392,Oral＿taxon＿308,and Oral＿taxon＿396 significantly increased in GC patients.2.The results of LC-Q-TOF-MS showed that the neoglycoproteins were averagely composed of one BSA and 8.9 mannose,9.0 galactose or 8.5 fucose,while lichenol-sulfuric acid reaction results showed that each BSA was binded to 14.4 mannoose,9.4 galactose or 8.5 fucose.3.At the concentrations of 30 μg/mL and 100 μg/mL,Fuc-BSA significantly reduced the adhesion of A.segnis to oral cell lines(CAL-27 and HOEC).However,Gal-BSA,Man-BSA as well as three monosaccharides did not affect the adhesion ability of A.segnis at the same concentrations.4.Lectin microarrays results showed that the NFIs of three lectins(PHA-E,LEL and MAL-I)were very significantly increased in outer membrane glycopatterns of A.segnis,while the NFIs of 9 lectins(Jacalin,GSL-II,EEL,et al)were significantly decreased.And the results of total protein glycopatterns showed that only four lectins(PHA-E,PNA,LEL and GSL-I)were significantly increased.5.Three LPS-related glycosyltransferases including NCTC10977＿00605(Rfaq＿1),NCTC10977＿00729,and NCTC10977＿00649(Waa A)in A.segnis were significantly decreased in the Fuc-BSA treated A.segnis.6.The CCK8 experiments showed that the IC50 of A.segnis infected HOEC and CAL-27 were 10.67 and 9.72 MOI,respectively,while 100 μg/mL Fuc-BSA treated A.segnis infected HOEC and CAL-27 were 16.4 and 12.3 MOI.And Fuc-BSA did not affect the proliferation of HOEC and CAL-27.This results indicated that Fuc-BSA reduced the cytotoxicity of A.segnis.7.A.segnis induced the expression of IL-1β and IL-18 in HOEC,activation of cellular inflammatory signaling pathways.Treatment with Fuc-BSA could weaken this immunoreaction.TLR4,CD14,and MD2 as the main receptor complex for extracellular bacterial stimulation.NF-κB,P38 and JNK pathway were all involoved in the activation of inflammatory signaling pathways induced by A.segnis.Inflammasomes(NLRP1,NLRP3,and AIM2)with ASC,were involved in A.segnis induced inflammatory response,activating Caspase-1 to processes IL-1β and IL-18 for their maturation,releasing mature IL-1β and IL-18 and induces pyroptosis.Conclusion:1.Oral microbiota has the potential to distinguish GC patients from HV.2.Fuc-BSA could significantly reduce the adhesion ability of A.signis to oral cells HOEC and CAL-27.3.Treatment with Fuc-BSA change LPS glycopatterns changed,which through reducing three LPS-related glycosyltransferases NCTC10977＿00605(Rfaq＿1),NCTC10977＿00729 and NCTC10977＿00649(WaaA).4.Treatment with Fuc-BSA evidently attenuate the cytotoxicity and trigger inflammatory ability of A.segnis.5.TLR4,MD2 and CD14 as the primary co-receptor complex for extracellular stimulation of A.segnis,then activate downstream NF-κB,P38 and JNK signaling pathways,leading to transcription of IL-1β and IL-18.On the other hand,inflammasomes(NLRP1,NLRP3,and AIM2)with ASC are also involved in A.segnis induced inflammatory response,activating Caspase-1 to processes IL-1β and IL-18 for their maturation.