NRAGE影响人食管癌细胞放射抗性的作用及其机制研究

The role and mechanism of NRAGE in radioresistance of human esophageal cancer cells

背景与目的:食管癌是全球威胁人类生命和健康的常见恶性肿瘤之一,中国每年食管癌发病人数占全球发病总人数的一半以上,以食管鳞癌最为常见。放疗是食管癌三大治疗手段之一,而放射抗性是导致其治疗失败的主要原因。神经营养因子受体相互作用MAGE类药物(neurotrophin receptor-interacting MAGE homolog,NRAGE)在放射抗性细胞株TE13R120中表达量明显高于亲本TE13细胞,且NRAGE亚细胞定位变化可能参与食管癌细胞放射抗性的形成。通过基因转染构建NRAGE稳定表达的食管癌细胞系,以进一步明确NRAGE基因与食管鳞癌细胞放射抗性的关系,分析该基因影响放射抗性的具体机制。方法:通过基因转染构建NRAGE稳定表达的食管癌细胞系。采用细胞克隆形成实验检测细胞放射敏感性,采用流式细胞术检测细胞周期及凋亡;细胞划痕、Transwell侵袭实验检测细胞迁移、侵袭能力,采用实时荧光定量聚合酶链反应(real-time fluorescence quantitive polymerase chain reaction,RTFQ-PCR)和蛋白质印迹法(Western blot)检测细胞中β-catenin表达情况。组间差异采用t检验或方差分析。结果:实验分为转染过表达组(Eca109/NRAGE组)和空白对照组(Eca109组)。Eca109/NRAGE细胞中NRAGE的表达量明显高于Eca109(t=29.65,P<0.05)。照射后Eca109/NRAGE细胞的放射抗性显著高于Eca109。流式细胞术检测结果显示,Eca109/NRAGE细胞中对射线抵抗性最强的S期细胞比例增加,对射线最敏感的G2/M期减少。Eca109/NRAGE细胞凋亡率较Eca109细胞降低(t=3.268,P<0.05)。Eca109/NRAGE细胞迁移和侵袭能力均高于Eca109。RTFQ-PCR和Western blot检测结果显示,β-catenin的mRNA表达及蛋白水平在Eca109/NRAGE细胞中明显高于Eca109(t=15.87,P<0.05)。结论:NRAGE参与Eca109细胞放射抗性的形成,改变细胞的细胞周期分布和凋亡情况,影响细胞迁移及侵袭能力并可能影响食管癌细胞的放射敏感性,该作用可能与激活Wnt/β-catenin信号转导通路有关。

Background and purpose:Esophageal cancer is one of the most common malignant tumors threatening human life and health worldwide.The annual incidence of esophageal cancer in China accounts for more than 50 percent of cases around the world,and squamous cell carcinoma is the most common type.Radiotherapy is one of the three main treatments for malignant tumors,but radioresistance could result in radiotherapy failure.Neurotrophin receptor-interacting MAGE homolog(NRAGE)expression in esophageal carcinoma resistance cell line TE13R120 is significantly higher than that in parental cell line TE13.Moreover,the change of NRAGE subcellular localization might be involved in the formation of radioresistance of esophageal cancer cells.In the present study,we established the esophageal cancer cell line Eca109 stably expressing NRAGE gene using gene transfection technology,then further analyzed the relationship between NRAGE gene and the radioresistance of esophageal squamous cancerous cells.Methods:The esophageal cancer cell line stably expressing NRAGE was constructed through gene transfection.Radiosensitivity of cells was detected using colony formation assay.Cell cycle and apoptosis were detected using flow cytometry,and cell migration and invasion capacities were detected using scratch-wound and Transwell assays.In addition,β-catenin expression in cells was detected using real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot,and t-test or analysis of variance was adopted for determining intergroup difference.Results:The experiments were divided into transfection over-expression group(Eca109/NRAGE group)and blank control group(Eca109 group).The expression level of NRAGE in Eca109/NRAGE cells was markedly higher than that in Eca109 cells(t=29.65,P<0.05).After radiation,the radioresistance of Eca109/NRAGE cells was remarkably higher than that in Eca109 cells.Flow cytometry indicated that the proportion of cells at S phase that had the highest resistance to X-rays in Eca109/NRAGE cells was increased,while those at G2/M phase with the highest sensitivity to X-rays was decreased.The apoptotic rate of Eca109/NRAGE cells was lower than that of Eca109 cells(t=3.268,P<0.05).Moreover,the Eca109/NRAGE cells showed higher migration and invasion capacities compared with Eca109 cells.Results of RTFQ-PCR and Western blot suggested that,theβ-catenin mRNA expression and protein levels in Eca109/NRAGE cells were notably higher than those in Eca109 cells(t=15.87,P<0.05).Conclusion:Over-expression of NRAGE is involved in the formation of radioresistance of Eca109 cells,which changes the cell cycle distribution and apoptosis,affects the cell migration and invasion capacities,and may influence the radiosensitivity of esophageal cancer cells.Such effect may be related to the activation of the Wnt/β-catenin signal transduction pathway.