| Objective: To study the effect of TIGAR in hypoxia-induced esophageal cancer radioresistance.Methods: 1)To detect the mitochondrial localization level of TIGAR in different esophageal squamous cell carcinoma cell lines(ECA109,KYSE410,KYSE450).The cells were lysed by the cell mitochondrial isolation kit,and the expression level of TIGAR in the mitochondria of each cell line was detected by Western-blot.The esophageal squamous carcinoma cells with the highest and lowest expression ofTIGAR in the mitochondria were screened for subsequent experiments.2)To examine the effect of hypoxia on the mitochondrial localization level of TIGAR.The cells were cultured in a normoxic or hypoxic cell culture incubator(1% oxygen concentration)for 20 hours,and the mitochondrial localization level of TIGAR in esophageal cancer cells under normoxia and hypoxic culture environment was detected by Western-blot.3)To study the effects of TIGAR in oxidative stress and autophagy in esophageal squamous cell carcinoma under hypoxic conditions.TIGAR was overexpressed or knocked down,and the effect of TIGAR in ROS level was detected by flow cytometry.Autophagy-related proteins(LC3-II,Beclin-1,P62)were detected by Western-blot.4)Colony formation assay was performed to study the effect of TIGAR in radiosensitivity of hypoxic esophageal cancer cells.5)To study the effects of TIGAR in DNA damage,oxidative stress and autophagy after irradiation of esophageal squamous cell carcinoma in hypoxic environment.The expression of r-h2 ax and autophagy-related proteins was detected by Western blot,ROS were detected by flow cytometry.Results:1.The mitochondrial localization levels of TIGAR in different esophageal squamous carcinoma cells(ECA109,KYSE410,KYSE450)were different.Among them,KYSE410 cells had the highest expression ofTIGAR and KYSE450 had the lowest expression of TIGAR.2.Under hypoxic culture conditions,the expression of TIGAR in mitochondria was elevated in esophageal squamous carcinoma cells.3.After knocking downthe TIGAR expression in KYSE410 cells,cell ROS level and autophagy flux was increased.After overexpressing TIGAR in KYSE450 cells,cell ROS level and autophagy flux was decreased.4.After knocking down the TIGAR expression in KYSE410 cells,the radiosensitivity of hypoxic esophageal cancer cells was increased.After overexpressing TIGAR in KYSE450 cells,the radiosensitivity of hypoxic esophageal cancer cells was decreased.5.After knocking down the TIGAR expression in KYSE410 cells,radiation-induced cell DNA damage,ROS level and autophagy flux was increased.After overexpressing TIGAR in KYSE450 cells,radiation-induced cell DNA damage,ROS level and autophagy flux was decreased.Conclusion: Hypoxia can induce TIGAR mitochondria localization in esophageal squamous cell carcinoma,which can reduce the oxidative stress and autophagy level of esophageal cancer cells,reduce the DNA damage of cells,and finally mediate the hypoxia-induced esophageal cancer radioresistance. |
