耐阿霉素食管癌细胞株EC9706/ADM的EMT-MET现象及机制

EMT-MET phenomenon and mechanism of adriamycin resistant esophageal cancer cell line EC9706/ADM

目的:建立耐阿霉素食管癌细胞株EC9706/ADM,探讨耐药细胞的EMT-MET现象及机制。方法:采用中等浓度间歇法建立耐阿霉素食管癌细胞株EC9706/ADM,用MTT法测定细胞的耐药指数及多药耐药性,Transwell小室实验检测耐药细胞的体外侵袭能力,实时荧光定量PCR和Western blot检测细胞在不同黏附时间EMT相关分子标志物E-cadherin、N-cadherin、Vimentin、Fibronectin和基质金属蛋白酶MMP2、MMP7、MMP9等的表达。结果:成功建立耐阿霉素食管癌细胞株EC9706/ADM,细胞的耐药指数为32.2,对顺铂、5-氟尿嘧啶、环磷酰胺产生交叉耐药性。与EC9706亲本细胞相比,EC9706/ADM细胞体外侵袭能力明显增强。黏附实验中,在黏附早期,耐药细胞E-cadherin表达降低而N-cadherin表达显著增高,黏附后期,E-cadherin表达升高而N-cadherin表达显著降低,基质金属酶在细胞黏附过程中表达均升高。结论:食管癌耐阿霉素细胞EC9706/ADM侵袭能力增强,可能与基质金属酶表达量增高和细胞发生EMT-MET转换相关。

Objective:To investigate the EMT-MET phenomenon and mechanism of drug-resistant cell by establish adriamycin resistant esophageal cancer cell line.Methods:To establish a human multidrug-resistant esophageal tumor cell line(EC9706/ADM) by middle doses of intermittent induction.The multidrug resistance of EC9706/ADM was detected by MTT assay.Matrigel invasion assays were used to study the invasion of cells.The mRNA and protein that the EC9706 and EC9706/ADM cells adhered at different times expression levels of the selected genes(E-cadherin,N-cadherin,Vimentin,Fibronectin,MMP2,MMP7,MMP9) showing statistically significant changes in the microarray analysis were validated with real-time quantitative RT-PCR and Western blot.Results:EC9706/ADM had obvious cross-resistance to other chemotherapy drugs(cisplatinum,5-FU,cyclophosphamide).The drug resistant index of EC9706/ADM cell kept on 32.2 times.In vitro invasion images of Transwell assay show increased number of EC9706/ADM cells.EC9706/ADM cells expressed increased levels of mRNA and protein for N-cadherin but decreased the level of E-cadherin at early stage when the cells adhered.The results were adverse at late stage when the cells adhered.While EC9706/ADM cells expressed increased levels of mRNA and protein for MMP2,MMP7 and MMP9 all the time.Conclusion:EC9706/ADM cells possessed highly invasive and metastatic capabilities with epithelial-mesenchymal transition phenotype.