The Role And Mechanism Of Mig/CXCR3 Axis Which Induces Epithelial-mesenchymal Transition, Migration And Invasion In Oral Tongue Squamous Cell Carcinoma Cal-27 Cells

[Objective] Invasion and metastasis which caused the deaths of patients is a major characteristic of malignant tumor.Recent studies found that Chemokines and their receptors play a vital role in migration, invasion and metastasis of tumor cells. To investigate the role and mechanism of chemokine Mig/chemokine receptor CXCR3 axis which induces epithelial-mesenchymal transition, migration and invasion in oral tongue squamous cell carcinoma Cal-27 cells and provide new ideas for prevention and control of oral cancer metastasis. In our previous researches, Chemokines Mig/receptor CXCR3 was closely related to invasion and metastasis in oral tongue squamous cancer cells, but the exact molecular mechanisms are still unclear. Oral tongue squamous cancer cells was employed to study the role and mechanism of Mig/CXCR3 axis which induces epithelial-mesenchymal transition, migration and invasion, it may provide a novel strategy for prevention and control of the oral cancer.[Methods](1) Overexpression and knockdown CXCR3 cells were constructed by gene transfection and gene knockdown in Oral tongue squamous cell carcinoma Cal-27, then cultured conventionally.(2) Experimental group 1:Cal-27 cells which exposed to concentration gradients for mig(50 nM,100nM,150nM) and CXCR3 neutralized antibody are divided in four groups:control group,Mig group,CXCR3 neutralized antibody group and IgG group;Experimental Group 2:over-expression CXCR3 in Cal-27 cells which were treated by 100 nM mig were divided in four groups:GFP control group,GFP control group+Mig,over-expression CXCR3 group,over-expression CXCR3 group +Mig;Group3:knowdown CXCR3 in Cal-27 cells which were treated by 100 nM mig were divided in four groups:Scramble control group,Scramble control group +Mig,knockdown hCXCR3 group and knockdown hCXCR3 group+Mig.(3) CCK8 assay was used to evaluate the relative growth rate of Cal-27 cells.(4) Wound scratch assay was utilized to measure the migration ability of Cal-27 cells.(5) Transwell chamber system was used to test the invasion ability of Cal-27 cells.(6) Apoptosis rates of Cal-27 cells were analyzed with Annexin V-FITC/PI labeling through flow cytometry when cells were cultured in dishes which were coated by poly-HEMA.(7) The phalloidin labeled by Rhodamine staining assay was used to observe F-actin polymerized.(8) Fluorescence microscopy was used to observe the expression of EMT related markers E-cadherin and Vimentin.(9) The expressions of E-cadherin,Vimentin,Akt2,p-Akt2,Erkl/2,p-Erkl/2,eIF4E, p-eIF4E and Snail were analyzed by Western blot.(10) Statistical analysis was carried out with SPSS 17.0.[Results](1) the relative growth rate of Cal-27 was no statistically significant differences with or without mig induced and it was independent of CXCR3.(2) Cell scratch experiments showed that Mig promoted the migration of Cal-27 cells in concentration-dependent manners (p<0.05);The migration rate of Cal-27 cells treated by CXCR3 neutralized antibody were gradually decreased compared with Mig group (p<0.05);over-expression CXCR3 in Cal-27 cells enhance the migration ability which induced by mig and there was statistically significant differences between these two groups (p<0.05);Furthermore,the migration ability which induced by mig was weakened by knockdown CXCR3 in Cal-27 cells and there was statistically significant differences between these two groups (p<0.05).(3) Transwell invasion assay showed that Mig could promote the invasion of tumor cells compare with control group;after induced by CXCR3 neutralizing antibody,the number of invasion cells was reduced compare with Mig group (p<0.05);over-expression CXCR3 in Cal-27 cells enhanced invasive ability of Cal-27 cells which induced by Mig and there was statistically significant differences between GFP group and over-expression CXCR3 group (p<0.05);The invasion ability were weakened by knockdown CXCR3 in Cal-27 cells and there was statistically significant differences between scramble-shRNA group and shRNA-CXCR3 group (p<0.05).(4) apoptosis assay found that Mig inhibit anoikis of Cal-27 cells compare with control group(p<0.05);over-expression CXCR3 in Cal-27 cells enhanced the apoptotic resistance ability which induced by Mig and there was statistically significant differences between GFP group and over-expression CXCR3 group (p<0.05);The apoptotic resistance ability which induced by Mig were weakened by knockdown CXCR3 in Cal-27 cells and there was statistically significant differences between scramble-shRNA group and shRNA-CXCR3 group (p<0.05).(5) The phalloidin labeled by Rhodamine staining assay found that Mig could induce the polymerization of F-actin in tumor cells;After blocking CXCR3 with neutralized antibodies,the polymerization of F-actin was weakened;over-expression CXCR3 in Cal-27 cells enhanced F-actin polymerized ability which induced by mig and knockdown CXCR3 in Cal-27 cells declined F-actin polymerized ability which induced by mig.(6) The fluorescence intensity and Western blotting of E-cadherin was obviously decreased while Vimentin was increased after induced by Mig (p<0.05);over-expression CXCR3 in Cal-27 cells promoted effect which induced by mig (p<0.05);ablated CXCR3 with CXCR3 neutralizing antibody or knockdown CXCR3 inhibited effect which induced by mig (p<0.05).The relative expression quantity of Snail was up-regulated in Cal-27 cells induced by mig; over-expression CXCR3 in Cal-27 cells enhanced effect which induced by mig (p<0.05); blocked CXCR3 with CXCR3 neutralizing antibody or knockdown CXCR3 inhibited effect which induced by mig (p<0.05).(7) Western blotting found that the relative expression quantity of p-Akt2 and p-eIF4E was upregulated after induced by Mig compare with control group (p<0.05); the relative expression quantity of p-Akt2 and p-eIF4E was downregulated compare with Mig group after blocking CXCR3 by neutralized antibodies (p<0.05). however,no change of the relative expression quantity of p-Erkl/2 was detected after induced by mig or abated CXCR3 with CXCR3 neutralizing antibody (p>0.05). The p-Akt2 and p-eIF4E expression was significantly upregulated after over-expression CXCR3 in Cal-27 cells induced by mig and there was statistically significant differences between GFP group and over-expression CXCR3 group (p<0.05);The p-Akt2 and p-eIF4E expression was down-regulated after knockdown CXCR3 in Cal-27 cells induced by mig and there was statistically significant differences between scramble-shRNA group and shRNA-CXCR3 group (p<0.05).[Conclusion] Akt signaling pathway can be activated by Mig/CXCR3 biological axis,and contributed to induction of EMT, anoikis inhibition, Cytoskeleton rearrangement and promoted migration and invasion in oral tongue squamous cell carcinoma Cal-27 cells.