Fusobacterium Nucleatum Infected Tca8113 Tongue Carcinoma Cells To DNA Damage And Promoted Proliferation By Ku70/p53 Pathway

Objective:In recent years,studies have found that microbial infection is associate with many important tumors,such as Porphyromonas gingivalis and oral squamous cell carcinoma,Fusobacterium nucleatum and colon cancer,Helicobacter pylori and gastric cancer.Fusobacterium nucleatum(F.nucleatum)is an important pathogenic bacteria of periodontal disease,which is widely found in oral cavity and belongs to the orange complex,Fusobacterium genus and facultative anaerobes.In other organs,F.nucleatum is also more widespread than other bacteria and is closely linked to several diseases such as respiratory diseases,liver abscesses,and premature births.Cell DNA damage is an important mechanism by which bacteria destroy the genome stability and integrity and thus affect the genetic alteration of tumor cells,the most serious damage is DNA double-strand break(DSB),the major repair pathway of DSB in human and higher eukaryotes is Non-homologous end joining(NHEJ).Therefore,we explore whether F.nucleatum may induce DSB in oral squamous carcinoma cell and thus affect the stability and integrity of the genome,and the impact of cell proliferation and possible mechanisms.In this study,DNA damage model of F.nucleatum infected Tca8113 tongue squamous cell carcinoma was constructed by detecting the expression of γH2AX,then cell proliferation and cell cycle changes were observed,NHEJ major repair factor Ku70,tumor suppressor wild-type p53,tumor suppressor p27 and tumor promoter mutant p53 expression,we also explore the effect of F.nucleatum on the proliferation of oral tumor cells and the possible dependent pathway,that may provide a theoretical basis for further clarifying the mechanism of DNA damage and repair.Methods: In the first part,we selected Tca8113 tongue carcinoma cells to infect F.nucleatum ATCC 25586 and constructed a DNA damage model.Cells were stimulated by F.nucleatum with a multiplicity of infection of 500: 1,and samples were collected at 0h,4h,12 h,24h and 36 h respectively.Western blot was used to detect the expression ofγH2AX.The immunofluorescence was used to verify the injury model by detecting DNA double-strand break factor γH2AX.The cell proliferation was detected by CCK-8 assay,then the changes of cell cycle were also detected.In the second part,real-time PCR was used to detect the expression of Ku70 mRNA and p53 mRNA,and further detected the expression of tumor suppressor gene p27 and tumor suppressor gene mutant p53.The repair factor Ku70 overexpression of Tca8113 cells were using plasmid transfection,and then CCK-8 assay was used to detect the cell proliferation after co-culturing with F.nucleatum for 24 h,real-time PCR was used to detect the change of wild type p53 at last.Results: In the first part,we successfully constructed the Tca8113 cell DNA damage model by co-culturing with F.nucleatum with a multiplicity of infection of 500: 1.The expression of γH2AX at 0h,4h,12 h,24h and 36 h showed a time-dependent enhanced manner with a significant change at 24h(P<0.05).Choosing 24 h time-point to further verify the damage model by immunofluorescence,we found DNA damage factor γH2AX fluorescence intensity and the number of significantly increased compared with the control group.Meanwhile,within 36 hours after infection with F.nucleatum,the proliferation ability of infected cells was significantly increased compared with the control group at each time point(P <0.05),indicating that the cell proliferation ability was significantly enhanced under the damaged model.The results of cell cycle showed that the percent of cells in G1 phase decreased while the percent of S phase cell increased,indicating that F.nucleatum infection regulates cell cycle arrest in S phase(P <0.05).In the second part,within 36 hours after F.nucleatum infection,the expression of Ku70 mRNA and protein was down-regulated(P <0.05),while the damage factor was continuously increased,indicating that DNA damage of F.nucleatum infected Tca8113 cells may activate non-homologous recombination repair pathway and the damage was out of cell repair capacity.Within 36 hours after F.nucleatum infection,the expression of wild p53 which is a downstream of pathway,showed a continuous downward trend(P<0.05),so we further detected the expression of tumor suppressor gene p27 and tumor suppressor gene mutant p53,p27 showed a downward trend,while the mutant p53 showed an upward trend(P <0.05),and the repair factor continued to decrease,indicating that the ability of suppressing tumor cells may also down-regulated.Transfection of over-expressed DNA repair factor Ku70 was about 5-fold(P <0.05)compared with the control group.After 24 h infection of F.nucleatum,the proliferation of transfected cells was inhibited compared with non-transfected cells.Meanwhile,theexpression of tumor suppressor gene wild p53 mRNA increased compared with the control group(P <0.05).Conclusion: 1.Fusobacterium nucleatum infect Tca8113 tongue carcinoma cells DNA double-strand breaks and may initiate the pathway of non-homologous recombination repair.2.When repair capacity is not enough,Tca8113 tongue carcinoma cells are arrested in S phase,and the proliferation of Tca8113 tongue carcinoma promote via Ku70/ p53 pathway,meanwhile,it may also reduce tumor suppressor ability.