赫赛汀获得性耐药胃癌细胞非标定量蛋白质组学研究  

Label-free quantitative proteomic analysis of acquired herceptin resistance in gastric cancer cells

作　　者：刘文虎[1,2] 汪宜 李生茂 张建武[1] 常晋霞[3] LIU Wen-hu1,3, WANG Yi3, LI Sheng-mao1, ZHANG Jian-wu1, CHANG Jin-xia2(1. Department of Pharmacology, 2. School of Basic.Medical Sciences, North Sichuan Medical College, Nanchong 637007, China;3. State Key Laboratory of Proteomics, National Center for Protein Sciences, Beijing 102206, China)

机构地区：[1]川北医学院药学院,北京102206 [2]国家蛋白质科学中心,蛋白质组学国家重点实验室,北京102206 [3]川北医学院基础医学院,四川南充637007

出　　处：《药学学报》2018年第4期553-560,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(31270822);四川省教育厅基金项目(17ZB0170)

摘　　要：基于非标定量(label-free quantitative)蛋白质组学对HER2表达阳性胃癌细胞(N87)和赫赛汀获得性耐药细胞(N87/R)进行蛋白质组学研究,发现耐药细胞蛋白质组的变化。提取的蛋白质样品经还原烷基化、FASP酶解;肽段经自制反相柱(small manual reversed phase,s RP)分离、LC-MS/MS分析;获取的数据通过Protein Database 2.1软件搜库鉴定。采用基于强度定量法(intensity based quantification,IBQ)进行蛋白质定量,寻找差异表达蛋白质。基于Web Gestalt数据库对差异蛋白质进行基因本体分析(gene ontology,GO)、基因–疾病网络构建及通路富集分析。共鉴定蛋白质8 509个,对其中7 163个蛋白质进行生物信息学分析,与母本组相比,耐药组中110个蛋白质显著上调,70个下调。GO富集显示,差异蛋白质在细胞成分、生物过程、分子功能方面明显不同;基因–疾病网络分析表明,差异蛋白质与肿瘤转移、肿瘤侵袭及炎症等相关;Wikipathway富集表明,IL-2、MAPK/ERK、m TOR、aurora A、Ret激酶、NF-κB、免疫调控及代谢通路在耐药细胞中有显著变化;Western blot证实,ERK1/2在耐药组中表达显著增加;MAPK/ERK通路抑制剂SCH772984能够选择性降低耐药细胞活力。结果表明,MAPK/ERK通路激活是赫赛汀获得性耐药的重要机制。本研究为胃癌赫赛汀耐药机制研究提供了理论基础。This study was designed to explore proteins differentially expressed in HER2 positive gastric cancer N87 cells and N87/R ceils with an acquired resistance to herceptin based on label-free quantitative proteomics. The extracted proteins were reduced and alkylated, then digested using filter aided sample preparation （FASP）; peptides were separated via small manual reversed phase column, analyzed by LC-MS/MS, and identified with protein database 2.1 search engine. Proteins were quantified by intensity based quantification （IBQ） to search for differential proteins by comparison with relatively quantified proteins. The enrichment and network construction in gene ontology （GO） terms, genes-disease and Wikipathway of differential proteins were established through Web Gestalt. A total of 8 509 proteins were detected, among them, 7 163 proteins were further analyzed by bioinformatics, of which 110 proteins were up-regulated and 70 were down-regulated in N87/R cells. The differential proteins showed a significant difference in cellular component, biological process and molecular function in GO terms, respectively. Genes-disease network analysis indicated the association of these differential proteins with neoplasm metastasis, neoplasm invasiveness and inflammation, etc. Wikipathway enrichment analysis revealed the relevance of several signaling pathways to herceptin resistance, which included IL-2, MAPK/ERK, mTOR, aurora A, Ret, NF-κB, immune-regulatory and metabolic pathway. Western blot showed a significant increase of ERK1/2 activities in N87/R cells compared with N87 cells. Correspondingly, SCH772984, a MAPK/ERK inhibitor, preferentially reduced the viability of N87/R cells. Taken together, our data suggested that the MAPK/ERK signaling pathway is one of the key pathways that mediate herceptin resistance. This study provides the basic information for exploring the mechanisms of acquired resistance to herceptin in gastric cancer cells.

关 键 词：赫赛汀 获得性耐药 蛋白质组学 MAPK/ERK信号通路 

分 类 号：R966[医药卫生—药理学]