缺氧环境下GAPDH通过抑制铁死亡增强喉鳞状细胞癌恶性生物学行为和顺铂耐药性研究  

Study on the enhancement of malignant biological behavior and cisplatin resistance in laryngeal squamous cell carcinoma by GAPDH-mediated inhibition of ferroptosis under hypoxic conditions

作　　者：岑瑞祥[1] 刘丹[1] 彭聪 万浪[1] CEN Ruixiang;LIU Dan;PENG Cong;WAN Lang(Department of Otolaryngology,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Huangshi,Hubei,435000,China;Department of Otolaryngology,Guizhou Provincial People’s Hospital,Guiyang,Guizhou,550002,China)

机构地区：[1]黄石市中心医院(湖北理工学院附属医院)耳鼻咽喉科,湖北黄石435000 [2]贵州省人民医院耳鼻咽喉科,贵州贵阳550002

出　　处：《中国耳鼻咽喉头颈外科》2024年第10期613-619,共7页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：湖北省自然基金(2022CFB498);湖北省自然科学基金创新发展联合基金(2024AFD027);黄石市卫生健康科研项目面上项目(WJ2024014)。

摘　　要：目的探讨缺氧环境下磷酸甘油醛脱氢酶(GAPDH)调控喉鳞状细胞癌(简称喉癌)铁死亡、恶性生物学行为和顺铂耐药性中的作用。方法人喉癌细胞系Hep-2和TU212在体外进行培养,分别在缺氧(1%O_(2))和常氧(21%O2)条件下进行无血清培养。Hep-2细胞分为5个实验组:DMSO对照组、Erastin组、缺氧+Erastin组、缺氧+Erastin+NC-GAPDH组和缺氧+Erastin+si-GAPDH组,以研究细胞在不同实验条件下的反应。使用定量聚合酶链反应(qPCR)和蛋白免疫印迹法(WB)分析各组细胞系GAPDH的mRNA和蛋白表达水平。细胞计数试验(CCK-8)、集落形成实验和Transwell侵袭实验用于评估各组喉癌细胞活力、增殖能力和侵袭能力。CCK-8实验用来测定细胞对不同浓度顺铂(5、15、20μg/ml)的耐药性。试剂盒法评估细胞内丙二醛(MDA)和谷胱甘肽(GSH)的水平。WB检测谷胱甘肽过氧化物酶4(glutathioneperoxidase4,GPX4)和4-羟基壬烯醛(4-hydroxynonenal,4-HNE)蛋白表达水平。同时,使用Fe2+探针和活性氧簇(ROS)荧光探针检测细胞内Fe2+含量及ROS水平,透射电子显微镜用于观察线粒体形态变化,以评估铁死亡发生情况。结果与常氧条件相比,缺氧显著增强Hep-2和Tu212细胞中GAPDH mRNA和蛋白的表达(P均<0.05),在缺氧条件下,细胞的集落形成数量、迁移数量以及在15μg/ml和20μg/ml顺铂处理下的细胞活力显著上升,均高于常氧组(P均<0.05)。GAPDH被沉默后,缺氧引起的这些增强效果得到逆转。在常氧条件下,与DMSO对照组相比,Erastin处理显著降低Hep-2细胞的集落形成数量、迁移数量以及在15μg/ml和20μg/ml顺铂处理下的细胞活力(P均<0.05),但在缺氧条件下,Erastin的抑制作用有所减弱。此外,缺氧+Erastin+si-GAPDH组的集落形成数、迁移数以及在15μg/ml和20μg/ml顺铂处理下的细胞活力显著低于缺氧+Erastin+NC-GAPDH组(P均<0.05)。结论在缺氧环境下,沉默GAPDH能够逆转喉癌细胞铁死亡抑制过程,进OBJECTIVE To investigate the role of glyceraldehyde-3-phosphate dehydrogenase(GAPDH)in regulating ferroptosis,malignant biological behavior,and cisplatin resistance in laryngeal squamous cell carcinoma(LSCC)under hypoxic conditions.METHODS Human laryngeal cancer Hep-2 and TU212 cell lines were cultured in vitro under hypoxic(1%O2)and normoxic(21%O2)conditions without serum.Hep-2 cells were divided into five experimental groups:DMSO control group,Erastin treatment group,Hypoxia+Erastin group,Hypoxia+Erastin+NC-GAPDH group,and Hypoxia+Erastin+Silenced GAPDH group,to study the cellular response under various experimental conditions.Quantitative polymerase chain reaction(qPCR)and Western blot(WB)techniques were employed to analyze GAPDH mRNA and protein expression levels in each cell line.Cell Counting Kit-8(CCK-8),colony formation,and Transwell invasion assays were used to assess the vitality,proliferation,and invasion capabilities of each group of laryngeal cancer cells.Additionally,CCK-8 assays were used to determine cell resistance to various concentrations of cisplatin(5,15,20μg/ml).Kit methods were used to evaluate intracellular malondialdehyde(MDA)and glutathione(GSH)levels.WB was employed to detect the expression levels of glutathione peroxidase 4(GPX4)and 4-hydroxynonenal(4-HNE)proteins.Fe2+probes and reactive oxygen species(ROS)fluorescence probes were used to measure intracellular Fe2+content and ROS levels.Transmission electron microscopy was utilized to observe mitochondrial morphology changes to assess ferroptosis occurrence.RESULTS Compared to normoxic conditions,hypoxia significantly increased the expression of GAPDH mRNA and protein in Hep-2 and Tu212 cells(P<0.05).Under hypoxic conditions,the number of cell colonies,migration capacity,and cell viability at 15 and 20μg/ml cisplatin treatment were significantly higher than those under normoxic conditions(P<0.05).When GAPDH was silenced,these hypoxia-induced enhancements were reversed.Under normoxic conditions,compared to the DMSO control group,Erast

关 键 词：缺氧 喉肿瘤 癌 鳞状细胞 顺铂 磷酸甘油醛脱氢酶 铁死亡 

分 类 号：R739.65[医药卫生—肿瘤]