The Expression Of Chemokine Receptor CCR6 In Esophageal Cancer And Its Effects On Epithelial-to-mesenchymal Transition

Objective:To explore the clinical significance of the expression of chemokine receptor CCR6 in esophageal cancer,and evaluate its effects on epithelial-to-mesenchymal transition(EMT)in esophageal cancer cells.Methods:Tissue microarray and immunohistochemical techniques were used to detect the expression of CCR6、E-cadherin and Vimentin in 99 cases of human esophageal cancer tissues and 11 cases of normal esophageal epithelial tissues;qRT-PCR was performed to measure CCR6 expression in human esophageal cancer cell lines ECA-109,TE-1,and normal esophageal squamous epithelial cell line HEEC;CCK-8,Wound Healing and Transwell assays were used to examine the influence of CCR6 on esophageal cancer cellproliferation,migrationandinvasion;Evaluateitseffectson epithelial-to-mesenchymal transition(EMT)in esophageal cancer cells by qRT-PCR and Western blot.Results:71.9%cases of esophageal cancer tissues with a significantly high expression in CCR6 were detected by immunohistochemical,but a low or undetectable expression of CCR6 was detected in normal esophageal tissues;Furthermore,the positive proportion of CCR6 in the subgroup with lymph node metastasis was significantly higher than the subgroup with no lymph node metastasis(χ~2=5.431,P=0.020).The positive proportion of CCR6 in the group with higher grade TNM was also significantly higher than the group with lower grade(χ~2=8.425,P=0.038).The results of qRT-PCR suggested that CCR6 was highly expressed in esophageal cancer cell lines ECA-109 and TE-1,whereas kept in a low expression in normal HEEC cell lines.Proliferation test revealed that CCL20stimulus induced a significant decrease in the proliferation ability of esophageal cancer cell lines(P<0.05).Scratch test showed that,after 24 hours scratches in ECA-109 cell lines,the healing speed of CCL20 group was significantly higher than the control group(P<0.01),whereas the healing speed ofαCCR6+CCL20 group was significantly lower than the CCL20 group(P<0.05).Invasion assay was also performed,the results suggested that the number of the cells permeabling through the polycarbonate membrane in CCL20 group was much higher than the control group(P<0.01).In contrast,the cell number penetrating the polycarbonate membrane inαCCR6+CCL20 group was significantly reduced compared to CCL20 group in ECA-109 cell lines(P<0.05).Moreover,CCR6-CCL20chemotactic experiments showed that,2 hours after CCL20 stimulus in ECA-109 cell lines,both the mRNA and protein expression of E-cadherin significantly decreased compared to the control group,while the expression of Vimentin was significantly higher than the control group until 6 hours after stimulus.ECA-109 cell lines were also treated withαCCR6+CCL20,2 hours after treatment,the mRNA and protein expression of E-cadherin significantly increased compared to the CCL20 group,while the expression of Vimentin were much lower than the CCL20 group.In TE-1 cell lines,there were no significant differences between CCL20 andαCCR6+CCL20 group in E-cadherin and Vimentin expression.Conclusions:The expression of CCR6 was significantly associated with lymph node metastasis and tumor TNM stages.CCR6 were highly expressed in esophageal cancer cell lines ECA-109、TE-1 and esophageal cancer tissues.Abnormal expression of CCR6 in esophageal cancer and the chemotactic effects of CCR6-CCL20,play a role in the regulation of tumor cell proliferation,invasion and migration.CCR6 may participate in regulating the occurrence of EMT in esophageal cancer cells.