Label-free定量蛋白质组学揭示GATA6调控胃癌细胞对曲妥珠单抗耐药的信号通路  

Label-free Quantitative Proteomics for Investigation of Signaling Pathways of GATA6 Regulating Trastuzumab Resistance in Gastric Cancer Cells

作　　者：刘文虎 袁江北[4] 常晋霞 LIU Wen-Hu;YUAN Jiang-Bei;CHANG Jin-Xia(Research Center of Molecular Metabolomics,Xiangya Hospital Central South University,Changsha 410008,China;Department of Pharmacy,North Sichuan Medical College,Nanchong 637100,China;School of Basic Medical Sciences,North Sichuan Medical College,Nanchong 637100,China;School of Pharmaceutical Sciences and Innovative Drug Research Center,Chongqing University,Chongqing 401331,China)

机构地区：[1]中南大学湘雅医院分子代谢组学研究中心,长沙410008 [2]川北医学院药学院,南充637100 [3]川北医学院基础医学院,南充637100 [4]重庆大学药学院,重庆401331

出　　处：《分析化学》2020年第2期187-196,共10页Chinese Journal of Analytical Chemistry

基　　金：四川省科技厅应用基础科研项目(No.2019YJ0378);四川省教育厅项目(No.17ZB0170);南充市市校合作项目(No.18SXHZ0402);川北医学院博士科研项目(No.CBY17-QD05)资助~~

摘　　要：肿瘤细胞对曲妥珠单抗耐药是导致其化疗失败的主要原因。前期研究表明,在曲妥珠单抗耐药胃癌细胞(NCI N87/R)中,转录因子GATA6与DNA的结合活性显著增强,这与其耐药是否有关尚不清楚。本研究以NCI N87/R为研究对象,采用Crispr/Cas9技术构建GATA6敲除细胞系(NCI N87R/GATA6)。在此基础上,基于Label-free定量蛋白质组学技术并结合生物信息学分析GATA6调控曲妥珠单抗耐药的信号通路。蛋白样品经还原烷基化,FASP酶切,肽段经液相色谱分离,LC-MS/MS定量分析,通过差异倍数及t检验筛选差异表达蛋白;采用WebGestalt数据库进行基因本体分析,利用GeneAnalytics数据库进行通路富集分析。结果表明,敲除GATA6增强了曲妥珠单抗对NCI N87/R的抑制作用,降低其侵袭。采用质谱定量检测出5792种蛋白质,其中305种在NCI N87R/GATA6细胞中表达上调,182种表达下调。通路富集分析显示,线粒体转运、细胞凋亡、DNA损伤、葡萄糖代谢、丙酮酸代谢及TCA循环、Wnt/β-catenin降解通路在NCI N87R/GATA6细胞中显著改变。蛋白免疫印迹实验(Western blot)表明,线粒体动力蛋白OPA1和DNM1L、凋亡蛋白Caspase-9、TCA循环代谢酶SUCLG2和MDH1、糖原代谢酶PYGL在NCI N87R/GATA6中显著改变,表明GATA6敲除导致NCI N87/R细胞线粒体功能障碍、能量代谢异常,进而诱导其凋亡,提示抑制GATA6转录活性可能是逆转胃癌曲妥珠单抗耐药的有效途径。Trastuzumab resistance is one of the principal causes of failure in tumor chemotherapy.Previous studies show that the DNA-binding activity of GATA6,a transcription factor,has a remarkable enhancement in trastuzumab resistant gastric cancer cells(NCI N87/R),while its correlation to resistance remains unclear.In this study,Crispr/Cas9 was employed to establish a GATA6 knock-out cell line(NCI N87R/GATA6).The signaling pathways regulated by GATA6 and related to trastuzumab resistance were investigated based on label-free quantitative proteomics combined with bioinformatics.The extracted proteins were alkylated,and digested using filter aided sample preparation(FASP),and the peptides were separated via high performance liquid chromatography and thereafter quantified by LC-MS/MS.Differentially expressed proteins were screened by fold change and student's t-test between NCI N87/R and NCI N87R/GATA6 cells.WebGestalt website was adopted for Gene ontology analysis,and GeneAnalytics was utilized for pathway enrichment analysis.The results demonstrated that GATA6 knock-out enhanced the antiproliferative effect of trastuzumab on NCI N87/R cells and suppressed their invasion ability.A total of 5792 proteins were quantified by LC-MS/MS,among which 305 proteins were up-regulated in NCI N87R/GATA6 cells while 182 ones down-regulated.Pathway enrichment analysis revealed that mitochondrial transport,apoptosis,DNA damage,glucose metabolism,pyruvate metabolism and TCA cycle and Wnt/β-catenin degradation pathways exhibited significant changes.Western blot manifested that the expression of mitochondrial dyneins OPA1 and DNM1L,apoptosis protein caspase-9,TCA metabolic enzymes SUCLG2 and MDH1,and glycogen metabolic enzyme PYGL changed significantly in NCI N87R/GATA6 cells,manifesting that GATA6 knock-out gave rise to mitochondrial dysfunction and abnormal energy metabolism,and therefore inducing the apoptosis of NCI N87R/GATA6 cells.The result implicated that inhibiting the transcriptional activity of GATA6 could be an effective strateg

关 键 词：曲妥珠单抗 抗药性 Label-free定量蛋白质组学 GATA结合蛋白-6 信号通路 

分 类 号：R735.2[医药卫生—肿瘤]