Establishment Of An Esophageal Cancer Cell Line With Stably Expression Of NRAGE Gene And Its Related Mechanism On Radioresistance

Objective: Esophageal cancer is one of the most common malignance threatening human life and health worldwide.In China,it accounts for more than 50 percent of esophageal cancer 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.Our previous study found that NRAGE expression in esophageal carcinoma resistance cell line TE13R120 was 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 by gene transfection technology,and verified the efficiency of transfection by Real-Time PCR and Western Blot,then further analyzed the relationship between NRAGE and the radioresistance of esophageal squamous cancerous cells by detecting the cell cycle,apoptosis,and ?-catenin expression.It will provide the cell model and experimental basis for the future research of NRAGE on the treatment of esophageal cancer.Methods:1 Real-Time PCR and Western Blot were utilized to detect NRAGE expression in three esophageal cancer cell lines TE13,Eca109 and Kyse170.2 Eukaryotic Expression Vector(p3XFLAG-CMV-14-NRAGE)carrying NRAGE gene was transfected into Eca109 cells.The experiment was divided into two groups: NRAGE transfection group(Eca109/NRAGE group)and control group(Eca109 group).A stably transfected cell line(Eca109/NRAGE)was obtained by G418 screening.3 NRAGE gene expression in Eca109 group and Eca109/NRAGE group were verified by Real-Time PCR and Western Blot.4 Colony formation assay was utilized to observe the role of NRAGE gene on cellular radioresistance.5 Cell cycle distributions of cells in Eca109 group and Eca109/NRAGE group were detected by flow cytometry.6 Apoptosis of cells in Eca109 group and Eca109/NRAGE group were detected by flow cytometry.7 ?-catenin expression in Eca109 group and Eca109/NRAGE group were detected by Real-Time PCR and Western Blot.Results:1 Results of Real-Time PCR and Western Blot showed that NRAGE expression was lowest in Eca109 cells,so Eca109 cell line was selected for the following experiments.2 Results of Real-Time PCR and Western Blot showed that both of the m RNA and protein expression of NRAGE in Eca109/NRAGE group was higher than that in control group(P=0.000).It suggested that the exogenous NRAGE gene successfully integrated into the cell genome and Eca109 cells obtained stable NRAGE expression.3 Results of colony formation assay demonstrated that the D0,Dq,SF2 and N value in Eca109 and Eca109/NRAGE group were different(1.54,1.29,0.44 and 1.838 vs 1.76,1.41 Gy,0.51,and 1.802).The D0,Dq and SF2 values were all increased in Eca109/NRAGE group,and SF2 value was 1.159 times higher than that of control group,furthermore,the width of the cell survival curve also increased,suggesting that cells of Eca109/NRAGE group were more resistant to radiation than that in control group.4 Results of cell cycle analysis showed that the cell proportion in the radioresistant S-phase of Eca109/NRAGE group increased compared with that in control group,while that in radiosensitive G2/M phase decreased.5 Results of cell apoptosis assay showed that the apoptosis rate of cells in the Eca109/NRAGE group was lower than that in Eca109 group(P=0.0029).6 Results of Real-Time PCR and Western Blot revealed that the m RNA and protein level of ?-catenin was both significantly higher in ca109/NRAGE group compared with Eca109 group(P=0.000).Conclusions:1 An esophageal cancer cell line with stably expression of NRAGE gene could be established by liposome mediation.2 NRAGE maybe involved in the formation of radioresistance in esophageal cancer cells of Eca109/NRAGE group.3 NRAGE may change the radiosensitivity of esophageal cancer cells by influencing the cell cycle distribution and apoptosis.4 NRAGE may participate in the formation of radioresistance by activating Wnt/?-catenin signaling pathway.