Metagenomic Analysis Of The Taxonomic And Functional Diversity Of The Oral Mcirobiota In Salivary Adenoid Cystic Carcinoma

Background: More and more experiments have proved that oral microorganisms are closely related to human diseases.Besides the common periodontal diseases and caries,oral microorganisms are also closely related to cancer and other systemic diseases.A large number of studies have confirmed that microbiology is associated with diabetes mellitus,cardiovascular disease,preterm birth and low birth weight infants.At the same time,a large number of studies have found that oral microorganisms are related to microbiome and tumor occurrence,microbiome shifts in cancer patients,the feasibility of identifying early diagnostic markers from the microbiome,and the effects of tumor treatments on the microbiome.Currently,the research on oral microorganisms and tumors includes oral squamous cell carcinoma,gastric cancer and pancreatic cancer.Salivary adenoid cystic carcinoma(SACC)is one of the most common malignant tumors in salivary glands.The biological characteristics of SACC include slow growth,perineural invasion and distant lung metastasis.Because the boundary between SACC tumor tissue and surrounding healthy tissue is not clear,it is difficult to define the extent of resection during surgical treatment.The treatment of SACC includes surgery,radiotherapy and chemotherapy,but the effect is not satisfactory,and the recurrence rate is high.Therefore,early detection,early diagnosis and early treatment are the key points of SACC diagnosis and treatment.Objective: In this study,next-generation genome sequencing technology was used to comprehensively and systematically analysis the changes of oral microbial community structure and functional metabolism in SACC patients.To observe whether there is an imbalance of salvia microflora in SACC patients,and the key microbes associated with SACC patients,studying the correlation of SACC from the microbes function,revealing the role of oral microorganisms in the occurrence and development of SACC.From the composition and function of oral microorganisms,provide a preliminary research basis for understanding the pathogenesis of SACC,and is helpful to predict and evaluate the risk of SACC and to study new diagnostic and therapeutic methods of SACC.Methods: Thirteen patients with SACC confirmed by histopathology were selected,and ten healthy control subjects with sex and age matched with SACC.Genomic DNA was extracted from saliva samples of SACC patients and healthy controls.Illumina high-throughput sequencing and bioinformatics analysis were carried out.Metagenomics reads were compared and annotated with NR database by DIAMOND software,and the differences of annotated species were analyzed.KOBAS 2.0 software was used to annotate KEGG metabolic pathway and analyze the difference.Result:1 A total of 104.64 Gb of data was obtained from 23 saliva samples,with an average of 4.55 Gb for each sample.A total of 698 M high quality Reads were obtained,and the average high quality Reads with 30.35 M for each individual.Attributed to 123 phylum,237 classes,516 orders,938 families,2539 genus and 11300 species classifications.2 PCA analysis showed that there was a significant separation trend between the two groups,indicating that there was a significant difference in the bacterial community composition of oral saliva samples between SACC patients and the healthy control group.3 Through Venn diagram analysis,it was found that there were 10435 species in the healthy control group and 10664 species in the SACC group.There were 9799 common species in both groups,636 unique to the healthy control group,and 865 unique to the SACC group.There were a total of 2047,137 gene sets in the two groups,2777,647 in the SACC group,730,510 unique to the SACC group,2651,166 in the healthy control group,and 604,029 unique to the healthy control group.The SACC group had a higher number of species and genes than the healthy control group.4 In 23 salivary samples,there were 123 phyla at phylum level.The dominant phylum(relative abundance>1%)in all samples were Firmicutes? Proteobacteria? Bacteroidetes? Actinobacteria?Fusobacteria and Spirochaetes,and the highest content was Firmicutes.At the genus level,Streptococcus? Prevotella? Neisseria? Actinomyces? Veillonella?Haemophilus ? Rothia ? Porphyromonas ? Capnocytophaga ? Alloprevotella? Fusobacterium? Treponema all had high abundance in each sample(relative abundance > 1%).5 The difference between the SACC group and the healthy control group was analyzed.The phyla with statistical difference between the two groups were Bacteroides and spirochetes(P<0.05).There were 31 genera(p< 0.05)with statistical difference.The abundance of 15 genera in SACC group were higher than that in healthy control group,including Streptococcus,Rothia,Lautropia,Mycobacterium,Carnobacterium,Granulicatella,Kingella,Cardiobacterium,Morococcus,Gammaproteobacteria,Abiotrophia,Actinobaculum,Bordetella,Desulfobulbus,Curtobacterium.The remaining 16 genera: Prevotella,Alloprevotella,Treponema,Oribacterium,Bacteroides,Clostridium,Propionibacterium,Isoptericola,Butyrivibrio,Ruminococcus,Parabacteroides,Coprococcus,Ruminococcaceae,Arcanobacterium,Pedobacter were higher in healthy control group than in SACC group.6 In 23 saliva samples,the dominant of metabolic pathways including: Carbohydrate metabolism,Global and overview maps,Amino acid metabolism,Nucleotide metabolism,Replication and repair,Energy metabolism,Metabolism of cofactors and vitamins,Membrane transport,Translation.7 In the comparison and analysis of the metabolic function of oral microflora between SACC group and healthy control group,the functional metabolism of oral microflora in SACC group changed significantly compared with healthy control group.Statistically significant metabolic pathways have 21 species(P<0.05),there are 13 kinds of metabolism in SACC group is higher than the healthy controls,including: Biosynthesis of amino acids,Histidine metabolism,Tyrosine metabolism,Valine,leucine and isoleucine biosynthesis,Glutathione metabolism,Arginine and proline metabolism,ABC transporters,Two-component system,2-Oxocarboxylic acid metabolism,Glycerophospholipid metabolism,Glycerolipid metabolism,C5-Branched dibasic acid metabolism,Phosphotransferase system(PTS).Eight functional metabolisms were higher in the healthy group than in the SACC group,including: Aminoacyl-tRNA biosynthesis,Carbon fixation pathways in prokaryotes,RNA degradation,Carbon fixation in photosynthetic organisms,Terpenoid backbone biosynthesis,Vancomycin resistance,Drug metabolism-other enzymes,Streptomycin biosynthesis.In SACC group,functional related to amino acid metabolism and lipid metabolism were significantly increased,biosynthesis function genes of antibiotics were decreased,and the oral microbial gene expression in SACC patients might be weakened.8 LEfSe analysis revealed that Streptococcus,Lautropia and Rothia significant enrichment in SACC group.Meanwhile,the metabolic functions that significant enrichment in SACC group are ABC transporters,Valine,leucine and isoleucine biosynthesis,Glutathione metabolism,Phosphotransferase system(PTS),2-Oxocarboxylic acid metabolism,Two-component system?Conclusion: The structure and functional structure of saliva microflora in SACC patients have changed significantly.This experiment comprehensively analyzed the structure and function of oral microorganisms in SACC patients.It preliminarily discussed the changes of oral microorganisms in SACC patients,and provided a new theoretical basis for the pathogenesis,treatment and diagnosis of SACC.