Effect Of Chemerin Expression And Neutrophil Infiltration On Angiogenesis Of Oral Squamous Cell Carcinoma

Object:To investigate the effect of Chemerin expression on angiogenesis in oral squamous cell carcinoma(OSCC).The effect of neutrophil infiltration on tumor angiogenesis and the role of Chemerin in OSCC were investigated.To explore the possible molecular mechanism of neutrophils affecting tumor angiogenesis through Chemerin.Methods:immunohistochemistry technique(IHC)was applied to assess Chemerin expression and relationship between microvessel density(MVD)in 74 OSCC cases while immunohistochemistry technique(IHC)Coculture and angiogenesis experiments explored the effect of tumor tissue origin Chemerin on the angiogenesis ability of human umbilical vein endothelial cell(HUVECs).Immunohistochemical double staining was used to analyze the correlation between neutrophil infiltration and MVD in OSCC tissues and the relationship between the two and clinicopathological features of OSCC patients.Meanwhile,Kaplan-Meier method was used to analyze postoperative survival of OSCC patients with complete follow-up data.Cox regression model was used to analyze the independent prognostic factors affecting postoperative survival of OSCC patients.Angiogenesis assay to explore the effect of neutrophils on the angiogenesis of HUVECs via Chemerin.Western blot to explore the possible molecular mechanism of neutrophils promoting angiogenesis through Chemerin.The effect of Chemerin expression and neutrophil infiltration on tumor angiogenesis was investigated in nude mouse transplantation.Results:Immunohistochemical results showed that MVD was higher in OSCCs with high Chemerin expression.The MVD of OSCCs with low Chemerin expression was lower.In vitro experiments showed that Chemerin promoted the angiogenesis of HUVECs,and the angiogenesis ability of HUVECs was enhanced after the addition of R-Chemerin(P<0.05).In the co-culture system of tumor cells and HUVECs,the angiogenesis ability of HUVECs co-cultured with Chemerin overexpression group was stronger,and the total number of nodes,total number of branches,total crossover number and grid index of angiogenesis were significantly higher than those of control group(P<0.05).Immunohistochemical double staining results showed that the number of neutrophils was positively correlated with MVD,with higher MVD in tumor tissues with abundant neutrophils infiltration,and lower MVD in areas with less neutrophils infiltration.In OSCC,neutrophil infiltration was associated with high MVD and TNM staging(χ2=6.624,P=0.036),lymph node metastasis(χ~2=13.353,P=0.001),large tumor(χ2=6.791,P=0.034),and tumor recurrence(χ~2=11.517,P=0.002).survival analysis showed that the overall survival(OS)and disease free survival(DFS)of OSCC patients with abundant neutrophil infiltration and high MVD were shorter than those with low neutrophil infiltration and low MVD(P<0.05).In OSCC patients with tumor size≥4cm,patients with abundant neutrophilic infiltration and high MVD group had shorter OS and DFS than patients with low neutrophilic and MVD(P<0.05).The ROC operating curve showed that the AUC of neutrophil+MVD(AUC=0.813)was higher than that of neutrophil+MVD(AUC=0.635)and MVD(AUC=0.799),and the combination of the two was a better indicator to evaluate the survival of OSCC patients.Cox regression models showed that neutrophil number and MVD were independent prognostic factors for survival in OSCC patients(P<0.005).The results of in vitro tube formation experiment showed that in the co-culture system of tumor cells,neutrophils and endothelial cells,the angiogenesis ability of HUVECs in the overexpressing Chemerin tumor cells group(Cal27-Chemerin/SCC9-Chemerin)was stronger than that in the control group(P<0.05).On the contrary,the angiogenesis of SCC15-Chemerin tumor cells group was weaker than that of the control group(P<0.05),suggesting that neutrophils could promote tumor angiogenesis through Chemerin.Western-blot results showed that Chemerin may promote angiogenesis by up-regulating the expression of VEGF(VEGF-A,MMP-9,MMP-2,and S100A9)in neutrophil through MEK/ERK pathway.Tumor formation in nude mice showed that the tumors of nude mice in the Chemerin overexpression group(Cal 27-Chemerin/SCC9-Chemerin)were larger and heavier than those in the control group(P<0.05),while the tumors of nude mice in the knockdown group(SCC15-Chemerin)were smaller and lighter than those in the control group(P<0.05).Immunohistochemical results showed that the Chemerin overexpressed nude mice had abundant neutrophil infiltration and high MVD.Conclusion:1.The expression of Chemerin in OSCC could promote angiogenesis.There is a positive correlation between neutrophil infiltration and MVD in OSCC,and the two are related to the poor prognosis of OSCC patients.The combined detection of neutrophil infiltration and MVD can better evaluate the survival time of patients.Neutrophils can activate MEK/ERK signaling pathway through Chemerin to promote angiogenesis and participate in the progression and metastasis of OSCC.