The Regulatory Mechanism Of Subcellular Localization Of NRAGE In Radioresistance Of Esophageal Carcinoma Cell

Objective: Esophageal carcinoma is one of the most common cancers in the digestive tract, which seriously affects people's health, especially in China for its high incidence. Radiotherapy is one of the main treatments of esophageal cancer. However, radiation resistance is an important reason to restrict the efficacy of radiotherapy. Therefore, to explore the reason of radiation resistance of esophageal cancer cells will provide clues and evidence to improve the local control rate and the prognosis of esophageal carcinoma. In our previous studies, we found that the expression of NRAGE in the radiation resistant cell line TE13R120 was significantly higher than that of the parental TE13 cells, suggesting that the gene may be involved in the formation of the radiation resistance. It is proposed to define the role of NRAGE in esophageal radiation resistance by our experiments, and then explore the possible mechanism from the perspective of cell biology.Methods:1 MTT assay was used to compare the growth rate and radiation sensitivity between TE13 and TE13R120 cells under different doses of irradiation.2 We used small interfering RNA transfection to knockdown the expression of NRAGE in TE13R120(si-NRG) and non-targeting si RNA as the negative control(NC).3 Realtime PCR and Western Blot were used to test the expression of NRAGE between TE13 and TE13R120, si-NRG and NC.4 The distribution of cell cycle of TE13 and TE13R120, NC and si-NRG was detected by flow cytometry.5 The intracellular location of NRAGE in TE13 and TE13R120 cells was tested by immunofluorescence.Results:1 The results of MTT assay showed that the cell growth rate decreased with the increase of irradiation dose, but the survival rate of TE13R120 cells was significantly higher than that of TE13, indicating that overexpression of NRAGE in esophageal cancer cells could confer resistance to IR treatment.2 Western Blot and Realtime PCR verified that the expression of NRAGE in TE13R120 and NC cells was significantly higher than that in TE13 and si-NRG-TE13R120 cells, respectively.3 TE13R120 cells transfected with NRAGE-targeting si RNA showed a clear reduction of the colony formation capacity compared with control si RNA.4 Flow cytometry indicated that high expression of NRAGE in TE13R120 cells arrested cell cycle at S phase, which is the most radioresistant cell stage in cell cycle and a lower ratio in the most radiosensitive cell stage G2/M. However, si-NRG showed a reduction of S phase and an increase of G2/M phase compared with NC.5 Immunofluorescence analysis manifested that NRAGE displayed an enhancement of nuclear distribution in TE13R120 compared with TE13.Conclusions:1 NRAGE is likely participated in the formation of ESCC radioresistance.2 NRAGE could change the radiosensitivity of esophageal cancer cells by affecting cell cycle distribution.3 Subcellular localization of NRAGE may be involved in the radiosensitivity of esophageal cancer cells.