HES1通过铁死亡调控胃癌细胞对赫赛汀耐药的机制研究  

Mechanism of HES1 regulating herceptin resistance in gastric cancer cells by ferroptosis

作　　者：张金花 常晋霞[3] 汤建才[3] 刘文虎[1,2] ZHANG Jin-hua;CHANG Jin-xia;TANG Jian-cai;LIU Wen-hu(Department of Pharmacy,North Sichuan Medical College,Nanchong 637100,China;Innovation Centre for Science and Technology,Nanchong 637100,China;School of Basic Medical Sciences and Forensic Medical,North Sichuan Medical College,Nanchong 637100,China)

机构地区：[1]川北医学院药学院,四川南充637100 [2]川北医学院科技创新中心,四川南充637100 [3]川北医学院基础医学与法医学院,四川南充637100

出　　处：《药学学报》2023年第11期3354-3365,共12页Acta Pharmaceutica Sinica

基　　金：四川省应用基础科研项目(2019YJ0378);川北医学院重点发展项目(CBY22-ZDA01);南充市市校合作项目(19SXHZ0298)。

摘　　要：肿瘤耐药是导致化疗失败的主要原因,而基因突变或功能缺失是引起耐药的关键因素。前期研究显示,SPLIT多毛增强子1(hairy and enhancer of SPLIT1,HES1)在赫赛汀耐药胃癌细胞中表达上调,抑制其活性可逆转其耐药,其机制尚未明确。本研究以赫赛汀耐药胃癌细胞NCI N87R为对象,基于CRISPR/Cas9构建HES1敲除细胞(△HES1/NCI N87R),探究HES1在胃癌赫赛汀耐药中的潜在作用。采用定量蛋白质组学分析△HES1/NCI N87R细胞蛋白质表达谱;基于基因集富集分析(GeneSet Enrichment Analysis,GSEA)和Metascape数据库进行基因本体分析;利用GeneAnalytics进行通路富集分析,并通过免疫印迹和抑制剂对筛选分子及通路研究。结果显示,相比NCI N87R细胞,△HES1/NCI N87R对赫赛汀的抗药性降低;敲除HES1使NCI N87R细胞1263种基因表达改变,其中上调761种,下调502种,且铁死亡、脂肪酸β-氧化、自噬、谷胱甘肽代谢等多条通路显著变化。功能研究显示,△HES1/NCI N87R细胞铁离子和丙二醛浓度增加,而谷胱甘肽降低,进一步发现,铁死亡抑制剂Fer-1能够逆转△HES1/NCI N87R中pTP53、溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)的表达,且降低对赫赛汀的敏感性,提示HES1通过TP53/SLC7A11/GPX4通路参与调控NCI N87R细胞对赫赛汀耐药,靶向HES1介导的TP53/SLC7A11/GPX4信号轴可能是逆转胃癌赫赛汀耐药的潜在策略。Drug resistance of cancer cells is the main causes of chemotherapy failure,and gene mutation or function loss is key factor to induce drug resistance.Previous studies have shown that hairy and enhancer of split 1(HES1)is up-regulated in herceptin-resistant gastric cancer cells,and inhibition of its activity can reverse its resistance while the potential mechanism has not yet been elucidated.In this study,we employed CRISPR/Cas9 to establish HES1 knock-out cell line(△HES1/NCI N87R)to investigate the functions of HES1 in herceptin resistance of NCI N87R cells and its potential mechanisms.We investigated proteomics profiling of△HES1/NCI N87R cells based on quantitative proteomics.Gene ontology analysis was conducted by GeneSet Enrichment Analysis(GSEA)and Metascape database,and pathway enrichment analysis was done using GeneAnalytics database.The selected molecules were quantified by Western blot and some pathways were verified by using inhibitors.The results showed that the resistance to herceptin of△HES1/NCI N87R cells decreased compared to NCI N87R cells.Proteomic data demonstrated that the expression of 1263 genes changed significantly in△HES1/NCI N87R cells,among which 761 genes were up-regulated while 502 ones down-regulated comparing with NCI N87R cells.Pathway analysis showed that ferroptosis,fatty acidβ-oxidation,autophagy and glutathione metabolism,etc.exhibited notable changes in△HES1/NCI N87R cells.The functional studies showed that the levels of iron ion and malondialdehyde increased,and glutathione decreased in△HES1/NCI N87R cells.It was further found that Fer-1,a ferroptosis inhibitor,could reverse the expression of pTP53,solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)in△HES1/NCI N87R cell,and reduce the sensitivity of△HES1/NCI N87R cells to herceptin.It is suggested that HES1 regulated the resistance of NCI N87R cells to herceptin through TP53/SLC7A11/GPX4 signaling pathway,and targeting TP53/SLC7A11/GPX4 signal axis mediated by HES1 is a potential strate

关 键 词：SPLIT多毛增强子1 赫赛汀 抗药性 蛋白质组学 铁死亡 TP53/SLC7A11/GPX4 

分 类 号：R966[医药卫生—药理学]