TNF-α Promotes The Expression Of Angiogenesis Factors In OSCC Cells Via TNFR2/Akt/mTOR Axis

Background and PurposesOral squamous cell carcinoma is a lethal cancer,which develops at the lips,tongue,salivary glands,gingiva,floor of the mouth,oropharynx,buccal surfaces and other intra-oral locations.It is a kind of characteristic locally aggressive tumor.Due to the specificity of the maxillofacial position,OSCC causes oral function and facial appearance impairment,and seriously impairs patients’ qualities of lives.OSCC is highly aggressive with rapid local tumor growth and exhibits early metastasis to the regional lymph nodes.For this reason,the prognosis remains poor despite aggressive treatment regimens encompassing radiation therapy,chemotherapy and surgery.Although the progress has been made in the treatment of OSCC in recent years,the overall prognosis is still poor due to its high metastasis rate and high risk of recurrence.Therefore,more accurate treatments for OSCC are urgently needed to be developed.Thus,it is important to explore the molecular mechanism of the occurrence and development of OSCC.To support the high proliferative rate of cancer cells,tumors need to rapidly develop a new vascular network,and it is also an essential step for tumor invasion,lymphatic metastasis and distant metastasis.Angiogenesis is regulated by a variety of biologically active molecules and cells,during which cell signaling pathways are involved.Moreover,pathological angiogenesis in tumor tissue could be affected by tumor cells,especially by biologically active molecules derived from tumor cells.Periodontitis is a bacterial infection disease with destruction of periodontal tissue,in which inflammation and corresponding host immune response play an important role.It is characterized by both dysbiosis of oral microbiome and proinflammatory events involving both cells and mediators from immunity,in which inflammatory cytokines such as TNF-α have been shown to be increased.The key factor TNF-α and other proinflammatory factors could enter the blood circulation,and increase the level of systemic inflammation.Thus Periodontitis is associated with a variety of systemic diseases.Existing studies have shown that periodontitis is closely related to cardiovascular diseases,respiratory diseases,premature and low birth weight infants,cancer and other systemic diseases.Recently,scientists found that the inflammatory microenvironment could regulate the biological functions of tumor cells.Moreover,when produced in the tumor microenvironment,inflammation can act as an endogenous tumor promoter.However,previous studies evaluating the correlation between periodontitis and OSCC are limited to a small number of clinical studies,and have reported contradictory findings.Besides,the specific molecular mechanism still needs further exploration.Therefore,this study aims to explore the role of the TNF-α in the process of angiogenesis function in OSCC cells.Moreover,we aim to illustrate the role of TNFR2,one of the key receptors of TNF-α in the cells,on the above process.We hope to provide new ideas for studying the molecular mechanism of OSCC,and provide a promising molecular target for the treatment of OSCC.Materials and Methods1.Study whether TNF-α promote the expression of angiogenesis factor in OSCC cellsOSCC cell line Ca127 was cultured,and was stimulated with different concentrations of TNF-α.CCK-8 was used to detect cell proliferation.RT-PCR and Western blot were used to detect the expression of VEGF and HIF-1a on the mRNA levels and protein levels.2.Study whether TNFR2 is expresses on the OSCC cellsRT-PCR and immunofluorescence were used to detect the expression of TNFR1 and TNFR2 on the mRNA levels and protein levels.3.Study the role of TNFR2 on the process of TNF-α promoting angiogenesis function in OSCC cells.TNFR2 neutralizing antibody was used to inhibit the function of TNFR2.RT-PCR and Western blot were used to detect the expression of VEGF and HIF-1a on the mRNA levels and protein levels.Western blot was used to detect the activation of Akt/mTOR signaling pathway on protein levels.The supernatants of OSCC cells were collected to obtain the CM.ELISA was used to detect the expression of VEGF,FGF-2 and TGF-β.4.Detection of the effect of OSCC cells’ CMs on HUVECs angiogenesis and migration in vitro.HUVECs were cultured and pre-treated with CM collected previously.The effect of OSCC cells’ CMs on HUVECs angiogenesis was determined by tube experiment in vitro.The transwell chamber was used for the cell migration experiment to detect the effect of OSCC cells’ CMs on HUVECs migration.Results1.TNF-α could promote the expressions of VEGF and HIF-la in OSCC cells on mRNA levels and protein levels.2.TNFR2 is expressed in OSCC cells.Moreover,TNF-α could promote the expressions of TNFR2 on mRNA levels and protein levels.3.TNFR2 antibody inhibited TNF-α-augmented angiogenesis factors expression in OSCC cells.Moreover,the Akt/mTOR signaling pathway could participate in the process mentioned above.4.TNF-α-CM could promote HUVECs angiogenesis and migration.TNFR2 neutralizing antibody-CM could inhibit the function of TNF-α-CM mentioned above.ConclusionsTNF-α could promote angiogenesis related function in OSCC via TNFR2/Akt/mTOR axis.