端粒酶TERT通过向线粒体转位抵抗胃癌细胞铁死亡的发生机制

The mechanism of telomerase TERT resisting ferroptosis in gastric cancer cells by translocating to mitochondria

目的:探究胃癌细胞铁死亡发生时,端粒酶TERT如何抵抗铁死亡的发生。方法:提取胃癌细胞BGC823的线粒体及细胞总蛋白,通过蛋白质印迹实验检测10μmol/L erastin处理细胞后,铁死亡相关蛋白SLC7A11、谷胱甘肽过氧化物酶4(GPX4)及端粒酶蛋白TERT表达量的变化以及线粒体中TERT含量变化;通过免疫荧光实验检测TERT是否定位于线粒体;使用5μmol/L bosutinib预处理细胞后再加入10μmol/L erastin处理细胞,通过免疫荧光实验检测TERT与线粒体定位情况;通过活性氧簇(ROS)检测试剂盒、丙二醛(MDA)检测试剂盒、Fe^(2+)检测试剂盒分别检测ROS、MDA、Fe^(2+)含量变化;通过CCK-8及划痕实验检测细胞生长、增殖及迁移能力。结果:蛋白质印迹实验结果显示,GPX4(t=15.30,P<0.001)、SLC7A11(t=5.228,P<0.01)、TERT(t=3.682,P<0.05)水平明显降低,线粒体中TERT含量降低(t=6.736,P<0.001)。免疫荧光实验结果显示,TERT定位于线粒体,联合用药组细胞免疫荧光实验结果显示TERT更多的定位在细胞核。联合用药组与单独用药组相比,铁死亡指标ROS(t=4.109,P<0.05)、MDA(t=6.491,P<0.01)、Fe^(2+)(F=9.703,P<0.01)含量均升高,CCK-8(F=4.706,P<0.05)及划痕实验(t=4.631,P<0.05)结果显示细胞增殖、迁移能力进一步降低。结论:铁死亡发生时,TERT由细胞核向线粒体进行转位,使用bosutinib抑制TERT向线粒体转位后,铁死亡相关指标增强,抑制TERT转位可以增强铁死亡的发生,抑制胃癌细胞生长、增殖。

Objective:To explore how telomerase TERT resists ferroptosis in gastric cancer cells.Methods:Mitochondria and total cell proteins of gastric cancer cell BGC823 were extracted.Changes in the expression levels of ferroptosis related proteins SLC7A11,glutathione peroxidase 4(GPX4),and telomerase protein TERT after treatment with 10μmol/L erastin in cells were detected by Western blotting.Whether TERT was localized in mitochondria was detected through immunofluorescence assay.TERT and mitochondrial localization were detected by immunofluorescence assay after 5μmol/L bosutinib pretreatment and 10μmol/L erastin treatment of cells.Changes in ROS,MDA,and Fe^(2+)contents were detected by reactive oxygen species(ROS)detection kit,malondialdehyde(MDA)detection kit,and Fe^(2+)detection kit,respectively.Cell growth,proliferation,and migration were measured by CCK-8 and wound healing.Results:Western blotting showed a significant decrease in GPX4(t=15.30,P<0.001),SLC7A11(t=5.228,P<0.01),and TERT(t=3.682,P<0.05)protein levels,and reduction in the content of TERT in mitochondria(t=6.736,P<0.001).The immunofluorescence experiment results showed that TERT was localized in mitochondria,and the combined use of the drug group showed that TERT was more localized in the nucleus.Compared with the monotherapy group,the combination therapy group showed an increase in iron death markers ROS(t=4.109,P<0.05),MDA(t=6.491,P<0.01),and Fe^(2+)(F=9.703,P<0.01)content.The CCK-8 and wound healing results showed a further decrease in cell proliferation and migration ability.Conclusion:When ferroptosis occurs,TERT translocates from the nucleus to mitochondria.Inhibiting TERT translocation to mitochondria with bosutinib enhances ferroptosis related indicators,indicating that inhibiting TERT translocation can enhance ferroptosis and inhibit the growth and proliferation of gastric cancer cells.