Oral bacterial infections substantially affect the development of various periodontal diseases and oral cancers. However, the molecular mechanisms underlying the association between Fusobacterium nucleatum (F. nucleatum ), a major periodontitis (PT)-associated pathogen, and these diseases require extensive research. Previously, our RNAsequencing analysis identified a few hundred differentially expressed genes in patients with PT and peri-implantitis (PI) than in healthy controls. Thus, in the present study using oral squamous cell carcinoma (OSCC) cells, we aimed to evaluate the effect of F. nucleatum infection on genes that are differentially regulated in patients with PT and PI. Human oral squamous cell carcinoma cell lines OSC-2O, HSC-4, and HN22 were used. These cells were infected with F. nucleatum at a multiplicity of infection of 100 for 3 hours at 37℃ in 5% CO2. Gene expression was then measured using reverse-transcription polymerase chain reaction. Among 18 genes tested, the expression of CSF3, an inflammation-related cytokine, was increased by F. nucleatum infection. Additionally, F. nucleatum infection increased the phosphorylation of AKT, p38 MAPK, and JNK in OSC-20 cells. Treatment with p38 MAPK (SB202190) and JNK (SP600125) inhibitors reduced the enhanced CSF3 expression induced by F. nucleatum infection. Overall, this study demonstrated that F. nucleatum promotes CSF3 expression in OSCC cells through p38 MAPK and JNK signaling pathways, suggesting that p38 MAPK and JNK inhibitors may help treat F. nucleatum-related periodontal diseases by suppressing CSF3 expression.

Introduction
Materials and Methods
    1. Cell culture
    2. F usobacterium nucleatum culture and infection intoOSCC cells
    3. RNA extraction and reverse transcriptionquantitativepolymerase chain reaction
    4. Western blot analysis
    5. Inhibitor treatment
    6. Statistical analysis
Results
    1. F usobacterium nucleatum infection increases CSF3expression in OSCC cells
    2. F usobacterium nucleatum infection inducesphosphorylation of AKT, p38 MAPK, and JNK inOSC-20 cells
    3. The p38 MAPK and JNK signaling pathwaysregulate F. nucleatum -induced CSF3 expression inOSC-20 cells
Discussion
Funding
Conflicts of Interest
Supplementary Data
References

• Ahyoung Jo(Department of Oral Biochemistry, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea)
• Jung-Min Oh(Department of Oral Biochemistry, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea, Department of Life Science in Dentistry, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea) Corresponding author