关键词: 神经营养因子受体基因, 稳定转染, 食管鳞状细胞癌, 放射抗性

Abstract: 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 G 2 /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, the β-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  ca109 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.

Key words: Neurotrophin receptor-interacting MAGE homolog gene, Stable transfection, Esophageal squamous cell carcinoma, Radioresistance