The Role And Mechanism Of ENO1 On The Regulation Of Cytokines Secreted By Tumor Associated Macrophages In OSCC

Objective:Oral squamous cell carcinoma has been promoted by the World Health Organization as the sixth largest malignant tumor in the world.As a prone tumor,its aggressiveness and high metastasis rate lead to poor prognosis and even life-threatening.In recent years,the mortality of patients with oral squamous cell carcinoma that has invaded adjacent tissues and metastasized has not improved significantly^[1].Therefore,it is urgent to further study the mechanism of invasion and metastasis of oral squamous cell carcinoma in order to prolong the survival of patients.Tumor-associated macrophages,as one of the major immune cell subsets in the tumor microenvironment,are mainly polarized into two phenotypes in the tumor microenvironment,M1 and M2.Among them,M2 tumor-associated macrophages can promote tumor invasion and metastasis.In addition,tumor-associated macrophages can also affect tumor growth by secreting a variety of cytokines,such as IL-6,IL-10,and IL-12.Invasion and transfer^[2].Studies have shown that ENO1 is an alpha-enolase during glycolysis and a metalloenzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolysis pathway.It is a multifunctional glycolytic enzyme that participates in various important physiological processes,such as cellular stress,the occurrence and metastasis of cancer^[3].Studies have found that ENO1 is highly expressed in tissues and cells of oral squamous cell carcinoma,and can promote the invasion and metastasis of tumors through ATP produced by glycolysis and inhibit the further development of tumors.However,the specific mechanism of ENO1 in oral squamous cell carcinoma has not been fully elucidated[4].The purpose of this research project is to investigate the role of ENO1 produced by tumor cells on the regulation of cytokines secreted by tumor-associated macrophages,invasion and metastasis in oral squamous cell carcinoma,and to further clarify the mechanism of invasion and metastasis of oral squamous cell carcinoma,which is helpful Intervention in finding more effective targets is of great significance for improving the treatment effect of oral squamous cell carcinoma.Methods:CAL27 cells and RAW264.7 cells were used as experimental research objects.Real-time PCR and Western blot were used to detect the transfection efficiency of three ENO1 siRNAs in CAL27 cells.Transwell chamber experiments were used to examine the expression of ENO1 on CAL27 cells invasion and metastasis Impact;Real-time PCR to detect the effects of ENO1 on the production of cytokines IL-6,IL-10,IL-12,TNF-α,and TGF-βby TAMs;Transwell chamber experiments were used to detect CAL27 cells after adding IL-6R inhibitors Changes in cell invasion and metastasis.Results:1.Transfection efficiency of ENO1 siRNA on CAL27 cells Compared with normal CAL27 cell group and NC group,Real-time PCR and Western blot results showed that after transfection of three ENO1 siRNAs:ENO1siRNA transfected CAL27 cells 36h,at the genetic level,with control group and NC Compared with the control group,the expression of ENO1 of siRNA1（1070）,siRNA2（1197）,and siRNA3（1353）all decreased by about 60%;48 hours after transfection of ENO1 siRNA into CAL27 cells,the protein level was comparable to that of control group and NC group Compared to this,the ENO1 expression levels of siRNA1（1070）,siRNA2（1197）,and siRNA3（1353）decreased by approximately 40%and 30%.The transfection efficiency of siRNA1（1070）is more obvious.2.Effect of ENO1 on tumor cell invasion and metastasis Transwell cell experiment results show that:compared with the NC group,ENO1siRNA transfected CAL27 cells can significantly reduce the invasion and metastasis ability of CAL27 cells;compared with the control group,exogenous ENO1 protein can significantly enhance CAL27 cell invasion and Transfer ability.3.The effect of ENO1 on cytokines secreted by TAMs After the CAL27 cell supernatant of the same growth density was applied to RAW264.7 for 24h,it was induced into TAMs,and then the CAL27 cell supernatant of Control,ENO1,NC,and siRNA were applied to TAMs cells for 6h and 12h respectively.Real-time PCR detects the gene expression levels of IL-6,IL-10,IL-12,TNF-α,and TGF-βat 6h and 12h respectively.Compared with NC group,the expression of ENO1by siRNA can be significantly reduced IL-6 secretion;compared with the control group,exogenous ENO1 protein increased ENO1 expression can significantly increase IL-6secretion.4.Effects of cytokines secreted by TAMs on invasion and metastasis of tumor cells Transwell chamber experiment results show that:compared with the control group,IL-6R inhibitor can significantly inhibit the invasion and metastasis of CAL27 cells.Conclusion:1.ENO1 produced by tumor cells promotes invasion and metastasis of OSCC cells;2.The expression of ENO1 produced by tumor cells can promote the secretion of IL-6 by TAMs;3.The secretion of IL-6 by TAMs can promote the invasion and metastasis of oral tumor cells.