The Role And Mechanism Of CXCL9/CXCR3 Axis Regulating Oral Squamous Cell Carcinoma Metastasis In Vivo

Objective:Oral cancer is the sixth most common tumor in the world and 90%is squamous cell carcinoma.Oral cancer mainly metastasize to cervical lymph nodes in early stage.Recent studies have found that the CXCL9/CXCR3 axis can regulate epithelial-mesenchymal transition(EMT)in tongue squamous cell carcinoma and induce the invasion and metastasis of tongue squamous cell carcinoma,but it has not been confirmed in vivo.In this study,a nude mice animal model was used to explore the role and mechanism of CXCL9/CXCR3 axis in the induction of epithelial-mesenchymal transition of tongue squamous carcinoma cells and regulating squamous cell carcinoma cells metastasize to cervical lymph node,so that provide a theoretical basis for the treatment of oral cancer.Methods:Gene transfection and gene knockdown methods were used to construct the oral tongue squamous cell carcinoma Ca127 cells with high expression and low expression of chemokine receptor CXCR3,then cultured conventionally.Then we establish animal models of tongue squamous carcinoma with nude mice.Suspension contain 2 × 106 TSCC cells were injected into the floor of mouth(FOM)of nude mice.After the tumor formation,we began to intervene its growth with drugs.The mice were randomly divided into several groups.Group ?:Transplanted tumors were conventionally intervened CXCL9(100nM)or saline,then divided into:Cal27 group,Cal27+NS group,Ca127+CXCL9 group.Group ?:Nude mice with Cal27 cells which overexpressed CXCR3 were treated with 100 nM CXCL9 and divided into Cal27-GFP control group,Cal2 7-GFP+CXCL9 group,Cal27-GFP-CXCR3 group and Cal27-GFP-CXCR3+CXCL9 group.Group?:Nude mice with Cal27 cells which knockdown CXCR3 were treated with 100nM CXCL9 and divided into Cal27-shRNA-Scramble group,Cal27-shRNA-Scramble+CXCL9 group,Cal27-shRNA-CXCR3 group and Cal27-shRNA-CXCR3+CXCL9 group.Each group has 24 mice.On the 10th day after the tumor formation,the nude mice were sacrificed and the fresh tumor tissues and cervical lymph nodes were harvested for HE staining.The protein expressions of E-cadherin,Vimentin,CXCR3,p-Akt2 and p-eIF4E in the tumor tissues were detected by immunohistochemistry(IHC).The results were analyzed with SPSS21.0 statistical software.Results:(1)The results of HE staining showed that CXCL9 promoted cervical lymph node metastasis in xenograft tumors of tongue squamous carcinoma in nude mice.Compared with Cal27 group and Cal27+NS group,the difference was statistically significant(P<0.05).The tumor metastasis rates were 29.2%in Cal27 group,33.3%in Cal27+NS group and 66.7%in Cal27+CXCL9 group.The upregulating of CXCR3 expression contribute to the promotion of CXCL9 on oral squamous cell carcinoma metastasis in vivo,which had statistical significance compared with Cal27-GFP group(P<0.05).The tumor metastasis rates were 33.3%in Cal27-GFP group,58.3%in Cal27-GFP+CXCL9 group,66.7%in Cal27-GFP-CXCR3 group and 91.7%Cal27-GFP-CXCR3+CXCL9 group.Decreasing the expression of CXCR3 inhibited the promotion of CXCL9 on oral squamous cell carcinoma metastasis in vivo,which was significantly different from that of Cal27-shRNA-Scramble group(P<0.05).The tumor metastasis rates were 29.2%in Cal27-shRNA-Scramble group,62.5%in Cal27-shRNA-Scramble+CXCL9 group,8.3%in Cal27-shRNA-CXCR3 group,20.8%in Cal27-shRNA-CXCR3+CXCL9 group.(2)Immunohistochemical detection of each group of transplanted tumors found that after CXCL9 treatment,the expression of E-cadherin protein in tongue squamous cell carcinoma in nude mice significantly decreased compared with Cal27 group and Cal27+NS group,The expression of Vimentin protein was enhanced(P<0.05).Overexpressed CXCR3 promoted the above-mentioned effect of CXCL9,while the effct of CXCL9 was inhibited when expression of CXCR3 were decreased(P<0.05).(3)Immunohistochemical detection of each group of xenografts found that,after CXCL9 treatment,the expression of Akt2 and eIF4E phosphorylation of tongue squamous carcinoma cells significantly increased,compared with the Cal27 group,expression levels of p-Akt2 and p-eIF4E were statistically significant(P<0.05).Overexpression of CXCR3 promoted CXCL9-induced phosphorylation of Akt2 and eIF4E.Compared with Cal27-GFP group,expression levels of p-Akt2 and p-eIF4E were up-regulated,with statistical significance(P<0.05).Decrease the expression of CXCR3 can inhibit CXCL9-induced phosphorylation of Akt2 and eIF4E,and expression levels of p-Akt2 and p-eIF4E decreased compared with Ca127-shRNA-Scramble group(P<0.05).Conclusion:CXCL9/CXCR3 axis can activate Akt signaling pathway,inducing epithelial-mesenchymal transition in tumor cells of tongue squamous carcinoma in nude mice and mediating the metastasis of cancer cells.