| Objective:The relationship between HNSCC and microorganisms and the possible pathogenic mechanisms were initially investigated by bioinformatics analysis of TCGA database.To evaluate the association between Fusobacterium nucleatum and laryngeal and hypopharyngeal cancer,we investigated the possibility of Fusobacterium nucleatum as a potential biomarker for laryngeal and hypopharyngeal cancer by measuring the relative quantitative levels of Fusobacterium nucleatum in the tissues and feces using real-time PCR(q PCR),gene sequencing,and fluorescence in situ hybridization.Methods:Expression profiling data related to head and neck squamous cell carcinoma were downloaded from TCGA database,including gene expression files and data annotation files,the gene expression data were collated into gene expression matrices,and significance levels were calculated using a linear model based on empirical Bayes distribution and corrected using benjamini Hochberg method.The screening criteria for significant difference genes were: |fold change|≥2.0 and P<0.05.To understand the biological functions represented by the genes with significant differences between normal tissues and HNSCC and where they are located in the biological processes,go analysis and KEGG pathway analysis of the up-regulated genes and down regulated genes in HNSCC were performed,and the significant enrichment of inflammatory and immune related biological processes and pathways in HNSCC was initially defined,These data suggest that HNSCC may be associated with microbial infection and that several significantly different genes are involved in pathways related to Fusobacterium nucleatum pathogenesis,so we chose to initially explore the association between Fusobacterium nucleatum and laryngeal and hypopharyngeal cancer.Tissue samples and stool samples of laryngeal and hypopharyngeal cancers in this study were obtained from patients who visited the otolaryngology department of the second hospital of Jilin University between November 2017 and August 2020.The tissue samples were obtained from 51 patients(30 patients with laryngeal cancer and 21 patients with hypopharyngeal cancer,including both cancerous and paracancerous tissues)in the larynx and hypopharynx cancer group and 20 controls(obtained from the oropharyngeal mucosa after UPPP for snoring).Faecal samples: 25 from laryngeal and hypopharyngeal cancer group and 21 from normal controls.The relative quantification levels of Fusobacterium nucleatum in tissue and fecal samples were determined by real-time PCR,respectively,and the results were statistically analyzed by means of the statistical analysis software graphpad prism 9,and differences were considered statistically significant when p<0.05.Result:A total of 545 HNSCC related gene expression profiling data were retrieved in the TCGA database,including 501 HNSCC expression data and 44 normal tissue expression data,and differential analysis revealed that a total of 9031 genes were differentially expressed between the two groups.Of these,5619 were upregulated and 3412 were downregulated in the HNSCC group.Go analysis and KEGG analysis of the differential genes revealed that the enriched biological processes included: complement activation(classical pathway),humoral immune response mediated by circulating immunoglobulins,immune response mediated by immunoglobulins,humoral immune response,lymphocyte mediated immunity,etc.The significantly enriched pathways included: ECM-receptor interaction Neutrophil and extracellular trap formation,human papillomavirus infection,cytokine cytokine receptor interactions,NK cell – mediated cytotoxicity,JAK-STAT signaling,transcriptional dysregulation in cancer,etc.Initially,we identified the enrichment of inflammatory and immune related biological processes and pathways in HNSCC tissues,suggesting that HNSCC may be associated with microbial infection.Differential gene screening of head and neck squamous carcinoma revealed that multiple E-cadherin family molecules(CDH)and Wnt family molecules were up-regulated in expression among them.This is consistent with Fusobacterium nucleatum secreting the virulence factor Fada adhesin acting on the E-cadherin family and thereby causingβ-Catenin activation and Wnt pathway activation promote intestinal tumorigenesis by the same mechanisms,suggesting that Fusobacterium nucleatum may be associated with HNSCC,so we chose to further explore the association between Fusobacterium nucleatum and laryngeal and hypopharyngeal cancer.The relative quantification of Fusobacterium nucleatum in the tissues of the experimental and control groups by qPCR showed that the relative quantification of Fusobacterium nucleatum in laryngeal and hypopharyngeal cancer tissues was 22.96±27.88(mean ± SD),and the relative quantification of Fusobacterium nucleatum in adjacent tissues was 1.662±2.902(mean±SD),the relative quantification levels of Fusobacterium nucleatum were higher in laryngeal and hypopharyngeal cancer tissues than in adjacent and normal tissues,and the differences were statistically significant(P<0.05).The relative quantification of Fusobacterium nucleatum within laryngeal cancer tissues was 23.44 ±28.62(mean±SD),and the relative quantification of Fusobacterium nucleatum in the adjacent laryngeal tissue was 1.620±2.54(mean±SD),the relative quantification level of Fusobacterium nucleatum in laryngeal cancer tissues was higher than that in adjacent tissues and normal tissues,and the difference was statistically significant(P<0.05).The relative quantification of Fusobacterium nucleatum within hypopharyngeal cancer tissues was 22.28±27.46(mean±SD),and the relative quantification of Fusobacterium nucleatum in the parapharyngeal tissues was 1.722±3.418(mean±SD),the relative quantification levels of Fusobacterium nucleatum in hypopharyngeal cancer tissues were higher than those in adjacent noncancerous and normal tissues,and the differences were statistically significant(P<0.05).There was no significant difference in the relative quantification level of Fusobacterium nucleatum between laryngeal and hypopharyngeal cancer tissues(P>0.05).Correlation analysis of clinical factors between patients with laryngeal and hypopharyngeal cancer revealed that the relative quantification levels of Fusobacterium nucleatum in cancer tissues and the associations between gender,age,smoking history,alcohol consumption history,lymph node metastasis and TNM stage were not statistically significant(P>0.05).The relative quantifications of Fusobacterium nucleatum in stool samples of laryngeal and hypopharyngeal cancers were 1.278e-5±4.024e-5(mean±SD)、1.533e-6 ± 2.496e-6(mean ± SD),and the relative quantification of Fusobacterium nucleatum in controls was 5.961e-7±9.796e-7(mean±SD),while there was no significant difference in the relative quantification level of Fusobacterium nucleatum in fecal samples between laryngeal cancer,hypopharyngeal cancer and normal subjects(P>0.05).Conclusion:1.After filtering out differential genes by bioinformatics analysis of TCGA database,go analysis and KEGG analysis of differential genes revealed that inflammation and immune related pathways and biological processes were significantly enriched in HNSCC,suggesting that HNSCC might be associated with microbial infection.2.The abundance of Fusobacterium nucleatum was higher in laryngeal and hypopharyngeal cancer tissues than in paired adjacent cancerous and normal tissues.3.There was no correlation between the abundance of Fusobacterium nucleatum and gender,age,tobacco or alcohol history,lymph node metastasis or TNM stage in laryngeal and hypopharyngeal cancer tissues.4.There was no statistical difference in the abundance of Fusobacterium nucleatum in the fecal samples of patients with laryngeal and hypopharyngeal cancer compared with that in the normal group. |
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