摘要
目的观察趋化因子受体6（CCR6）在食管癌组织的表达及其对食管癌细胞上皮-间充质转化（EMT）的影响。方法应用免疫组织化学技术检测99例食管鳞状细胞癌和11例食管正常组织中CCR6、E-钙黏蛋白（E-cadherin）和波形蛋白（Vimentin）的表达，分析其与临床病理特征的相关性。应用实时定量反转录聚合酶链反应（RT-qPCR）检测人食管癌细胞株ECA-109、TE-1及正常食管细胞株HEEC中CCR6的表达。应用细胞计数试剂盒（CCK-8）、划痕和Transwell实验检测CCR6对食管癌细胞增殖、迁移和侵袭能力的影响。应用RT-qPCR和Western blot实验测定CCR6-CCL20趋化作用对食管癌细胞EMT的影响。结果CCR6在食管癌细胞株中高表达与正常食管细胞株中低表达，差异有统计学意义（8.00±1.23、4.57±1.12比1.00±0.32，t＝11.000，P＝0.000；t＝6.100，P＝0.001）。CCR6在淋巴结转移组和未转移组的阳性表达率分别为87.1%和63.8% （P＝0.020）；在TNM分期级别高组和级别低组的阳性表达率分别为87.9%和62.5% （P＝0.038）。食管癌细胞经CCL20刺激后，增殖能力显著降低，差异有统计学意义（0.35±0.02比0.49±0.03，t＝7.003，P＝0.002；0.38±0.033比0.48±0.04，t＝3.719，P＝0.021）。ECA-109划痕24 h后，CCL20组的愈合速度显著升高，差异有统计学意义（1.34±0.02比1.00±0.04，t＝4.680，P＝0.009），封闭CCR6，愈合速度明显降低（1.36±0.06比1.00±0.04，t＝4.344，P＝0.012）。ECA-109细胞Transwell实验结果显示：CCL20组细胞穿膜数量显著增高，差异有统计学意义（401.33±20.00比239.33±21.18，t＝5.560，P＝0.005）；封闭CCR6后，穿膜细胞数量显著降低（311.33±12.12比401.33±20.00，t＝3.849，P＝0.018）。CCL20刺激后，ECA-109中E-cadherin的mRNA和蛋白表达显著降低（t＝9.758，P＝0.001）、Vimentin的表达显著升高（t＝6.352，P＝0.003）；封闭CCR6，E-cadherin的mRNA和蛋白表达显著升高（t＝7.707，P＝0.002）、Vimentin表达显著降低（t＝6.095，P＝0.004）。结论CCR6在食管癌淋巴结转移患者中呈高表达，CCR6及CCR6-CCL20趋化轴参与调控肿瘤细胞的增殖、侵袭和迁移，可能参与调节食管癌细胞EMT的发生。
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.
MethodsImmunohistochemistry, 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.ResultsCCR6 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）.ConclusionThe 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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2018年第1期141-144,共4页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（31570877、31570908）
国家科技支撑计划（2015BA112812）
国家自然科学基金海外及港澳学者合作研究基金（31729001）
常州市科技局应用基础项目（CJ20160021）
