The expression of chemokine receptor 6 in esophageal cancer tissue and its effects on epithelial-to-mesenchymal transition

Abstract

Objective To explore the clinical significance of the expression of chemokine receptor 6 (CCR6) in esophageal cancer tissue, and evaluate its effects on epithelial-to-mesenchymal transition (EMT) in esophageal cancer cells. Methods Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to determine the expression of CCR6 and/or EMT markers in esophageal tissues/cells. Cell counting kit-8 (CCK-8) assay, wound healing assay and trans-well system were used to determine the influence of CCR6 on esophageal cancer cell proliferation, migration and invasion in response to CCL20. Results 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 (8.00±1.23, 4.57±1.12 vs. 1.00±0.32, t=11.000, P=0.000; t=6.100, P=0.001). The positive expression rates of CCR6 in the subgroup of esophageal cancer with lymph node metastasis and the subgroup with no lymph node metastasis were 87.1% and 63.8% respectively (P=0.020). The positive expression rates of CCR6 in the group with higher grade TNM and the group with lower grade were 87.9% and 62.5% respectively (P=0.038). Proliferation test revealed that CCL20 stimulus induced a significant decrease in the proliferation ability of esophageal cancer cell lines (0.35±0.02 vs. 0.49±0.03, t=7.003, P=0.002 and 0.38±0.033 vs. 0.48±0.04, t=3.719, P=0.021). Scratch tests showed that, 24 h after scratches in ECA-109 cell lines, the healing speed in CCL20 group was significantly greater than in the control group (1.34±0.02 vs. 1.00±0.04, t=4.680, P=0.009), whereas the healing speed in CCR6+ CCL20 group was significantly lower than in the CCL20 group (1.36±0.06 vs. 1.00±0.04, t=4.344, P=0.012). Invasion assay results suggested that the number of the cells permeabling through the polycarbonate membrane in CCL20 group was significantly more than that in the control group (401.33±20.00 vs. 239.33±21.18, t=5.560, P=0.005). In contrast, the cell number penetrating the polycarbonate membrane in CCR6+ CCL20 group was significantly reduced compared to CCL20 group in ECA-109 cell lines (311.33±12.12 vs. 401.33±20.00, t=3.849, P=0.018). Moreover, CCR6-CCL20 chemotactic experiments showed that, after CCL20 stimulus in ECA-109 cell lines, both the mRNA and protein expression of E-cadherin was significantly decreased compared to the control group (t=9.758, P=0.001), and that of Vimentin was significantly higher than that in the control group after stimulus (t=6.352, P=0.003). After treatment of ECA-109 cells with CCR6+ CCL20, the mRNA and protein expression of E-cadherin was significantly increased compared to the CCL20 group (t=7.707, P=0.002), and that of Vimentin were significantly lower than in the CCL20 group (t=6.095, P=0.004). Conclusion The high expression of CCR6 exsits in the esophageal cancer patients with lymph node metastasis. CCR6 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. Key words: Chemokine receptor 6; Esophageal cancer; Epithelial-to-mesenchymal transition; Lymph node metastasis