摘要
目的探究CDGSH铁硫结构域1(CDGSH iron-sulfur domain 1,CISD1)对肺癌细胞铁死亡的影响及其机制。方法利用生物信息在线网站分析CISD1在健康人群和肺癌患者中的表达情况及其对预后的影响。人非小细胞肺癌细胞A549分为对照组、阴性对照组(LV-sh-NC)和CISD1敲低组(LV-sh-CISD1)。细胞克隆实验检测CISD1敲低组在细胞程序性死亡抑制剂作用下的细胞克隆形成能力;BODIPY(581/591)C11荧光探针检测细胞脂质过氧化物含量;FerroOrange荧光探针检测细胞亚铁离子水平;GSH/GSSG试剂盒检测细胞谷胱甘肽含量;免疫荧光检测铁死亡蛋白谷胱甘肽过氧化物酶4(GPX4)和酰基辅酶A合成酶长链家族成员4(ACSL4)含量;蛋白质免疫印迹实验检测铁代谢蛋白2(IRP2)和转铁蛋白受体(TFR)表达水平。结果生物信息学分析结果显示,与正常人群相比,CISD1在肺腺癌患者组织中高度表达(P<0.05),且这种高表达可能与较差的预后有关(P<0.05)。与阴性对照组相比,CISD1敲低组细胞克隆形成能力显著降低(P<0.05);与CISD1敲低组相比,CISD1敲低+铁死亡抑制剂(Fer-1)组细胞克隆形成能力显著升高(P<0.05)。与阴性对照组相比,CISD1敲低组细胞内脂质过氧化物水平、亚铁离子含量均显著升高(P<0.05),谷胱甘肽水平显著降低(P<0.05);铁死亡相关蛋白GPX4表达显著下调(P<0.05),ACSL4表达显著上调(P<0.05);铁代谢相关蛋白IRP2和TFR表达水平均显著上调(P<0.05)。结论肺癌细胞中CISD1高表达;敲低CISD1可以诱导肺癌细胞铁死亡,其作用机制与铁代谢密切相关。
Objective To investigate the effect of CDGSH iron-sulfur domain 1(CISD1)on ferroptosis in lung cancer cells and its underlying mechanism.Methods The expression of CISD1 in healthy individuals and lung cancer patients,and its effect on prognosis were analyzed using online bioinformatics databases.Human non-small cell lung cancer A549 cells were divided into control group,negative control group(LV-sh-NC),and CISD1 knockdown group(LV-sh-CISD1).Cell cloning experiments were conducted to assess the colony-forming ability of CISD1 knockdown cells in the presence of cellular programmed death inhibitor.BODIPY(581/591)C11 fluorescent probe was used to detect cellular lipid peroxide levels.FerroOrange fluorescent probe was utilized to measure intracellular ferrous ion levels.GSH/GSSG kits were employed to detect glutathione content in A549 cells.Immunofluorescence was conducted to assess the levels of ferroptosis-related proteins glutathione peroxidase 4(GPX4)and acyl-CoA synthetase long-chain family member 4(ACSL4).Western blotting was performed to detect the expression levels of iron regulatory protein 2(IRP2)and transferrin receptor(TFR).Results Bioinformatics analysis revealed that CISD1 expression was significantly higher in tissues from patients with lung adenocarcinoma than in healthy individuals(P<0.05),and the overexpression was associated with poor prognosis(P<0.05).Compared with negative control group,the cell colony formation ability was significantly reduced in CISD1 knockdown group(P<0.05).The cell colony formation ability was significantly increased in CISD1 knockdown+ferroptosis inhibitor ferrostatin-1(Fer-1)group compared to CISD1 knockdown group(P<0.05).Compared with negative control group,the levels of intracellular lipid peroxides and ferrous ions were significantly increased in CISD1 knockdown group(P<0.05),while the glutathione level was significantly decreased(P<0.05),the expression of ferroptosis-related protein GPX4 was significantly downregulated(P<0.05),the expression of ACSL4 was significantly upregulated(P<0.05),and the expression levels of iron metabolism-related proteins IRP2 and TFR were both significantly upregulated(P<0.05).Conclusion CISD1 is highly expressed in lung cancer cells,and CISD1 knockdown can induce the ferroptosis in lung cancer cells,which may be closely related to the iron metabolism.
作者
尚文丽
王君
田应选
霍树芬
SHANG Wenli;WANG Jun;TIAN Yingxuan;HUO Shufen(Department of Respiratory and Critical Care Medicine Ⅱ,Shaanxi Provincial People′s Hospital,Xi′an 710068,China)
出处
《山西医科大学学报》
2025年第5期476-483,共8页
Journal of Shanxi Medical University
基金
陕西省重点研发计划项目(2023-YBSF-064)
陕西省自然科学基础研究计划重点项目(2022JZ-59)。
作者简介
尚文丽,女,1984-11生,硕士,副主任医师,E-mail:zaixuyuan1115@126.com;通讯作者:霍树芬,E-mail:hazeline219@163.com。
