Fusobacterium Nucleatum Stimulates The Progression Of Oral Squamous Cell Carcinoma Through The Up-regulation Of CCL20

Background:Fusobacterium nucleatum(F.nucleatum)is a common oral symbiotic bacterium,which can cause a variety of infectious diseases in oropharynx and other parts outside of the oral cavity.At present,it has been found that F.nucleatum has a high abundance in oral squamous cell carcinoma(OSCC)and can induce the production of inflammatory cytokines to promote the proliferation and invasion of OSCC cells.However,there are few reports on its mechanism of action.Based on the above research background,this study will verify the abundance of F.nucleatum in OSCC,verify its promoting effect on OSCC by cell experiments and animal models,and explore the possible molecular mechanism.Methods:The abundance of F.nucleatum in OSCC and paracancerous tissue was analyzed by 16S rRNA sequencing and fluorescence in situ hybridization(FISH).Human normal oral keratinocytes(HOK),human OSCC cell lines CAL-27 and SCC9 cells were cultured and infected with F.nucleatum,respectively.The proliferation,migration and invasion of cells were detected by CCK-8,wound healing,transwell migration and invasion assay.Mouse OSCC model induced by 4-nitroquinolin-1 oxide(4NQO)was constructed.F.nucleatum was applied to the oral cavity of mice.Tumor number,volume and body weight were recorded.The promotion of F.nucleatum to OSCC was evaluated by hematoxylin-eosin(HE)staining and immunohistochemistry(IHC).The chemokines with the highest differential expression after infection of F.nucleatum with CAL-27 cells were screened out by transcriptomics.Quantitative realtime polymerase chain reaction(qRT-PCR),western blot and siRNA interference assay were used to verify the mechanism of the selected chemokines in the tumor-promoting process of F.nucleatum.Results:Compared with paracancerous tissues,the abundance of F.nucleatum was higher in OSCC tissues,suggesting that it may be related to OSCC.F.nucleatum could promote the proliferation of HOK,CAL-27 and SCC-9 cells.Regarding the ability of cell migration and invasion,F.nucleatum showed an inhibitory effect in HOK cells but a promoting effect in CAL-27 and SCC-9 cells.Compared with the control group,the volume and number of lesions in mice infected with F.nucleatum were larger,the pathological changes were more serious,and the Ki67 positive rate was higher.Transcriptomic analysis showed that CCL20 was the most differentially expressed chemokine after infection of F.nucleatum with CAL-27 cells,and qRT-PCR and western blot showed that the expression of CCL20 increased after infection of F.nucleatum with CAL-27 cells.SiRNA interference experiment showed that the migration and invasion ability of CAL-27 cells were significantly weakened after the reduction of CCL20 expression.Conclusion:This study confirmed that F.nucleatum was enriched in OSCC.F.nucleatum could promote the proliferation,migration and invasion of OSCC cells.F.nucleatum infection can promote the progression of OSCC in mice.F.nucleatum infection significantly increased the expression of CCL20 in CAL-27 cells,while inhibition of CCL20 significantly weakened the ability of F.nucleatum to promote the migration and invasion of CAL-27 cells.It is suggested that F.nucleatum can promote the progression of OSCC through the up-regulation of CCL20.