Erastin对骨肉瘤细胞生长及增殖的影响  

Effects of erastin on the growth and proliferation of osteosarcoma cells

作　　者：郝名英 刘思琪 黄欣宇 耿鑫 HAO Mingying;LIU Siqi;HUANG Xinyu;GENG Xin(Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院生物化学与分子生物学系,天津300070

出　　处：《天津医科大学学报》2024年第3期193-199,共7页Journal of Tianjin Medical University

基　　金：天津市教委科研计划(2021ZD037)。

摘　　要：目的:探究erastin对骨肉瘤(OS)细胞生长、增殖的影响及其分子机制。方法:使用STRING数据库构建OS易突变蛋白、铁死亡相关蛋白和端粒相关蛋白的相互作用网络。检测2.5μmol/L erastin处理OS细胞后铁死亡相关指标的变化、端粒替代延长(ALT)相关表型的变化、端粒损伤情况、端粒保护蛋白的变化和对OS细胞生长增殖、迁移的影响。结果:STRING蛋白相互作用网络分析显示,铁死亡相关蛋白、OS中突变蛋白和端粒相关蛋白相互作用网络包括RPA1、BARD1、UBE3A、AURKA、MDM2、SIRT1、CREBBP、IGFBP5、DDX5及EP300。2.5μmol/L erastin处理OS细胞后,Western印迹显示谷胱甘肽过氧化物酶(GPX)4、SLC家族氨基酸转运蛋白(SLC7A11)表达量明显降低(t=13.24、4.399,均P<0.05),毛细血管扩张性共济失调和Rad3相关蛋白(ATR)表达量明显降低(t=10.68,P<0.001),端粒重复结合因子(TRF)1、2表达量明显升高(t=8.006、10.79,均P<0.01);丙二醛(MDA)检测显示脂质过氧化物含量升高(t=2.951,P<0.05);活性氧簇(ROS)检测试剂盒显示,ROS水平明显增加(q=11.03,P<0.001);ALT相关表型结果显示,c-circle水平明显降低(t=16.20,P<0.0001),早幼粒细胞白血病核小体(PML)与端粒的定位明显降低(t=6.018,P<0.01);免疫荧光结果显示,53BP1、γ-H2AX与端粒的定位明显增加(q=4.391、4.653,均P<0.05);CCK8、克隆形成实验结果显示,细胞生长增殖水平降低(t=10.86、4.972,均P<0.01);划痕实验结果表明,细胞的迁移水平降低(t=2.953,P<0.05)。结论:伴随铁死亡的发生,erastin可以减弱ALT并抑制OS细胞的生长、增殖和迁移。Objective:To explore the effect of erastin on the growth and proliferation of osteosarcoma(OS)cells and its molecular mechanism.Methods:The interaction network of mutant protein with ferroptosis associated protein and telomere related protein in OS was constructed using STRING database.The effects of 2.5μmol/L erastin treatment on ferroptosis associated protein,alternative lengthening of telomeres(ALT)related phenotype,telomere damage,telomere protective proteins,and growth,proliferation and migration of OS cells were detected.Results:The STRING protein interaction network analysis showed that ferroptosis related proteins,mutant proteins in OS,and telomere related protein interaction networks included RPA1,BARD1,UBE3A,AURKA,MDM2,SIRT1,CREBBP,IGFBP5,DDX5 and EP300.After 2.5μmol/L erastin treatment,Western blotting showed that glutathione peroxidase 4(GPX4)and SLC family amino acid transporter(SLC7A11)protein expression decreased significantly(t=13.24,4.399,both P<0.05).Ataxia telangiectasia and Rad3-related protein(ATR)expression was significantly decreased(t=10.68,P<0.001).The expression levels of shelterin telomere repeat binding 1 and 2(TRF1 and TRF2)proteins were significantly increased(t=8.006,10.79,both P<0.01).Malondialdehyde(MDA)detection results showed that lipid peroxide content increased(t=2.951,P<0.05),and reactive oxygen species(ROS)detection kit results showed that ROS level increased significantly(q=11.03,P<0.001).The results of ALT related phenotype showed that c-circle level was significantly decreased(t=16.20,P<0.0001),promyelocytic leukemia(PML)and telomere localization were significantly decreased(t=6.018,P<0.01).Immunofluorescence results showed that the localization of 53BP1,γ-H2AX and telomeres increased significantly(q=4.391,4.653,both P<0.05).CCK8 and the results of clonal formation experiment showed that the cell growth and proliferation level decreased(t=10.86,4.972,both P<0.01),the scratch test results showed that the migration level of the cells was reduced(t=2.953,P<0.05).Conclusio

关 键 词：骨肉瘤 铁死亡 端粒替代延长 

分 类 号：R34[医药卫生—基础医学]