靶向抑制GOT1介导的铁死亡途径逆转食管鳞癌细胞顺铂耐药的机制

Targeted inhibition of GOT1-mediated iron death pathway reverses cisplatin resistance in esophageal squamous cell carcinoma cells

目的:探究靶向抑制谷氨酸草酰乙酸转氨酶1(GOT1)是否通过介导铁死亡途径影响食管鳞癌细胞顺铂(DDP)耐药性,并探究分子机制。方法:CCK-8法和细胞集落形成实验检测DDP敏感细胞株Eca-109与DDP耐药细胞株Eca-109/DDP的存活率、集落形成数目,RT-qPCR和Western blot检测Eca-109细胞和Eca-109/DDP细胞中GOT1表达水平差异;将Eca-109/DDP细胞分为对照组、siNC组、siGOT1组、siGOT1+铁死亡抑制剂Ferrostatin-1(Fer-1)组,CCK-8法检测不同浓度DDP处理下各组细胞存活率,细胞集落形成实验检测各组细胞集落形成数目,流式细胞术检测各组细胞凋亡率,DCFH-DA染色法检测各组细胞内活性氧(ROS)水平,比色法检测各组细胞中铁离子(Fe2+)浓度,Western blot检测各组细胞中铁死亡效应因子酰基辅酶A合成酶长链家族成员4 (ACSL4)、谷胱甘肽过氧化物酶4(GPX4)、溶质载体家族7成员11(SLC7A11)的蛋白表达水平。结果:与Eca-109细胞比较,不同浓度DDP处理下Eca-109/DDP细胞存活率增高(P<0.05),细胞集落形成数目增加(P<0.05),细胞中GOT1 mRNA与蛋白相对表达量上调(P<0.05)。与对照组比较,siGOT1组Eca-109/DDP细胞在不同浓度DDP处理下的存活率降低(P<0.05),细胞集落形成数目减少(P<0.05),细胞凋亡率增加(P<0.05),细胞中ROS水平和Fe2+浓度升高(P<0.05),ACSL4蛋白相对表达量上调(P<0.05),GPX4和SLC7A11蛋白相对表达量下调(P<0.05);与siGOT1组比较,siGOT1+Fer-1组Eca-109/DDP细胞在不同浓度DDP处理下的存活率升高(P<0.05),细胞集落形成数目增加(P<0.05),细胞凋亡率减少(P<0.05),同时,细胞中ROS水平和Fe2+浓度降低(P<0.05),ACSL4蛋白相对表达量下调而GPX4和SLC7A11蛋白相对表达量上调(P<0.05)。结论:靶向抑制GOT1能够提高食管鳞癌耐药细胞Eca-109/DDP对DDP的敏感性,促进该耐药细胞凋亡,这一作用与促进铁死亡途径有关。

Objective:To explore whether targeted inhibition of glutamic-oxaloacetic transaminase 1(GOT1)affects cisplatin(DDP)resistance in esophageal squamous cell carcinoma cells through mediating iron death pathway,and to explore the molecular mechanism.Methods:The survival rate and colony formation number of DDP sensitive cell line Eca-109 and DDP resistant cell line Eca-109/DDP were detected by CCK-8 method and cell colony formation assay.The expression levels of GOT1 in Eca-109 cells and Eca-109/DDP cells were detected by RT-qPCR and Western blot.Eca-109/DDP cells were divided into control group,siNC group,siGOT1 group and siGOT1+Ferrostatin-1(Fer-1)group.The cell survival rate of each group under different DDP concentrations was detected by CCK-8 method.The number of cell colony formation in each group was detected by cell colony formation experiment.The apoptosis rate in each group was detected by flow cytometry.The level of intracellular reactive oxygen species(ROS)in each group was detected by DCFH-DA staining,and the concentration of iron ion(Fe 2+)in each group was detected by colorimetry.The protein expression levels of iron death effector acyl coenzyme A synthetase long chain family member 4(ACSL4),glutathione peroxidase 4(GPX4)and solute carrier family member 711(SLC7A11)were detected by Western blot analysis.Results:Compared with Eca-109 cells,the survival rate of Eca-109/DDP cells under different concentrations of DDP was increased(P<0.05),the number of cell colony formation was increased(P<0.05),and the relative expression of GOT1 mRNA and protein in the cells was up-regulated(P<0.05).Compared with the control group,the survival rate of Eca-109/DDP cells in siGOT1 group under different concentrations of DDP was decreased(P<0.05),the number of cell colony formation was decreased(P<0.05),the apoptosis rate was increased(P<0.05),and the ROS level and Fe 2+concentration in cells were increased(P<0.05),the relative expression of ACSL4 protein was up-regulated(P<0.05),and the relative expression of GPX4 and SLC7A11 protein was down-regulated(P<0.05).Compared with siGOT1 group,the survival rate of Eca-109/DDP cells in siGOT1+Fer-1 group under different concentrations of DDP was increased(P<0.05),the number of cell colony formation was increased(P<0.05),and the apoptosis rate was decreased(P<0.05),ROS level and Fe 2+concentration were decreased(P<0.05),the relative expression of ACSL4 protein was down-regulated and the relative expression of GPX4 and SLC7A11 protein was up-regulated(P<0.05).Conclusion:Targeted inhibition of GOT1 can enhance the sensitivity of Eca-109/DDP to DDP and promote the apoptosis of the drug-resistant cells,which is related to the promotion of iron death pathway.