神经营养因子受体亚细胞定位改变参与食管癌细胞上皮间质转化及放射抗性的形成

Effects of subcellular localization of neurotrophin receptor p75 interacting MAGE homoiogue on the epithelial to mesenchymal transition and radioresistance of esophageal cancer cells

目的探讨神经营养因子受体（NRAGE）亚细胞定位改变与人食管癌细胞上皮和间质转化及放射抗性形成的关系。方法应用转化生长因子（TGF—β1）诱导人食管癌细胞TE13，建立上皮间质转化（EMT）模型细胞。以TE13细胞、EMT模型细胞、放射抗拒TE13R120细胞为研究对象，通过Real—timePCR和Westernblot检测EMT标记物mRNA和蛋白表达，验证EMT模型的建立和TE13R120存在EMT样表型。Real-timePCR比较NRAGE在3组细胞中mRNA水平的表达情况，Westernblot检测3组细胞中NRAGE总蛋白及胞质、胞核蛋白的表达。结果TGF—β1诱导TE13细胞形态向间质型细胞转化，EMT标志物E—cadherin和Vimentin表达发生改变（t=13．56、-232．84，P〈0．05），成功建立EMT模型细胞。放射抗拒TE13R120细胞中EMT标记物表现出与EMT模型细胞相似的趋势（t=15．84、-54．54，P〈0．05）。NRAGE在EMT模型细胞、TE13R120细胞中的表达较TE13细胞明显上调，且均在细胞核内表达明显升高（t=-8．73、-5．62，P〈0．05），而在细胞质中表达差异无统计学意义。NRAGE在EMT模型细胞和TE13R120细胞胞质、胞核中的表达差异均无统计学意义。结论放射抗拒TE13R120细胞存在EMT样表型，且其放射抗性的形成可能因EMT的发生参与了NRAGE亚细胞定位的改变而导致。

Objective To investigate the role of NRAGE subcellular localization in the EMT and radioresistance of esophageal cancer cells. Methods EMT model cells were established by the treatment of TE13 cells with TGF-β1. To verify the establishment of EMT model and the phenotype of EMT-like TEI3R120 cells, EMT marker mRNA and protein were detected by Real-time PCR and Western blot, respectively. Real-time PCR was also used to detect the expression of NRAGE mRNA in three groups. Total NRAGE protein, cytoplasm protein and nuclear protein were measured by Western blot. Results It was found that TGF-~I could induce morphological alterations of TEl3 cells from epithelial to mesencbymal and change the expressions of EMT maker E-cadherin and vimentin （t = 13.56, -232.84, P 〈 0.05 ）, indicating the successful establishment of EMT model cells. Similar expression trends of EMT makers were observed in TE13R120 cells （t = 15.84, -54.54,P〈0.05）. NRAGE mRNA （t= -8.73, -5.62,P〈 0.05） and total protein in both EMT model cells and TEI3R120 ceils were higher than that in TEl3 cells, especially for the nuclear proteins. However, no differences in NRAGE cytoplasm protein expression were found among the three groups. In addition, there were also no difference of NRAGE mRNA （ t = - 0.88, P 〉0.05）, cytoplasm and nuclear protein between TE13R120 cells and EMT model cells. Conclusions The radioresistant cell line TE13R120 has the EMT-like phenotype that may cause cell radioresistance bychanging the subcelluar localization of NRAGE.