RUNX3调控胃癌细胞对赫赛汀耐药的非标记定量蛋白质组学研究  

Label-free quantitative proteomic study of RUNX3 regulating Herceptin resistance in gastric cancer cells

作　　者：常晋霞[1] 王仕宝[2] 袁江北 刘文虎[1] CHANG Jin-xia;WANG Shi-bao;YUAN Jiang-bei;LIU Wen-hu(School of Basic Medical Sciences,Department of Pharmacy,North Sichuan Medical College,Nanchong 637100,China;Department of Pharmacy,Hanzhong Vocation and Technology College,Hanzhong 723000,China;Peking University Shenzhen Hospital,Shenzhen Peking University-The Hong Kong University of Science and Technology Medical Center,Shenzhen 518036,China)

机构地区：[1]川北医学院基础医学院药学院,四川南充637100 [2]汉中职业技术学院药学院,陕西汉中723000 [3]北京大学深圳医院,深圳北京大学-香港科技大学医学中心,广东深圳518036

出　　处：《药学学报》2021年第7期1953-1964,共12页Acta Pharmaceutica Sinica

基　　金：四川省应用基础科研项目(2019YJ0378);南充市市校合作项目(18SXHZ0402,19SXHZ0298);川北医学院博士科研项目(CBY17-QD05).

摘　　要：肿瘤耐药是多机制、多因子参与的复杂生物学过程,而肿瘤细胞抗凋亡是导致其耐药的重要原因。前期研究显示,Runt相关转录因子3(Runt-related transcription factor 3,RUNX3)在胃癌赫赛汀耐药细胞中与DNA的结合活性显著增强,然而其活性变化是否与耐药有关,尚不明确。本实验以赫赛汀耐药细胞(NCI N87R)为对象,采用CRISPR/Cas9构建RUNX3敲除细胞株(△RUNX3/NCI N87R),探究RUNX3在赫赛汀耐药中的作用及其潜在机制。在此基础上,基于非标记定量蛋白质组学研究△RUNX3/NCI N87R细胞蛋白质表达谱;采用倍数变化及显著性水平筛选差异表达分子;利用GeneAnalytics数据库通路富集分析;使用DAVID Bioinformatics Resources数据库基因本体分析;基于STRING数据库构建蛋白-蛋白互作网络。结果表明,敲除RUNX3使NCI N87R细胞对赫赛汀的敏感性增加。蛋白质组数据显示,577种基因在△RUNX3/NCI N87R中的表达显著改变,其中上调191种、下调386种。根据通路富集率及显著性水平,自噬、细胞周期、凋亡、线粒体脂肪酸β氧化、神经源性位点notch同源蛋白1(neurogenic locus notch homolog protein 1,NOTCH1)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、Hedgehog及DNA损伤响应信号变化显著(P<0.05),表明敲除RUNX3干扰耐药细胞多条通路。免疫印迹证实,自噬相关蛋白(autophagy-related protein,ATG)13、7及BECN1在△RUNX3/NCI N87R中的表达显著增加,而细胞周期调节分子丝氨酸/苏氨酸蛋白激酶Chk2(serine/threonine-protein kinase Chk2,CHEK2)及凋亡调节分子Bcl-2(apoptosis regulator Bcl-2,BCL2)显著下调;与NCI N87R细胞相比,磷酸化丝氨酸/苏氨酸蛋白激酶AKT(p-AKT)在△RUNX3/NCI N87R中的表达显著降低(P<0.01),且赫赛汀能够降低p-AKT水平,表明敲除RUNX3改变了耐药细胞周期、增加赫赛汀对p-AKT的抑制作用,促进其自噬并诱导凋亡,提示RUNX3可能是逆转或降低胃癌赫赛汀耐药的潜在Resistance of tumor cells is a complex biological process involving multiple mechanisms and factors,in which anti-apoptosis is the most important cause of drug resistance.Previous studies have shown that the DNA binding activity of Runt related transcription factor 3(RUNX3)increased prominently in Herceptin resistant gastric cancer cells(NCI N87R)while the relevance of which to drug resistance has not yet been confirmed.In this study,we employed CRISPR/Cas9 to establish RUNX3 knock-out cell line(△RUNX3/NCI N87R)to investigate the functions of RUNX3 in Herceptin resistance of NCI N87R cells and its potential mechanisms.We investigated proteomics profiling of△RUNX3/NCI N87R cells based on label free quantitative proteomics.Differentially expressed proteins were screened out according to fold change and significance level between△RUNX3/NCI N87R and NCI N87R cells.Pathway enrichment analysis was done using GeneAnalytics database,and gene ontology analysis was conducted by DAVID Bioinformatics Resources database.Protein-protein interaction networks were constructed based on STRING database.The results showed that△RUNX3/NCI N87R cells increased the sensitivity to Herceptin.Proteomic data demonstrated that the expression of 577 genes changed significantly in△RUNX3/NCI N87R cells,among which 191 genes were up-regulated while 386 ones downregulated comparing with NCI N87R cells.Pathway analysis showed that autophagy,cell cycle,apoptosis,mitochondrial fatty acidβoxidation,neurogenic locus notch homolog protein 1(NOTCH1),mammalian target of rapamycin(mTOR),Hedgehog and DNA damage response pathways exhibited notable changes based on pathway enrichment ratio and significance level(P<0.05).These results indicated that RUNX3 knock-out altered multiple signaling pathways of NCI N87R cells.Western blotting manifested that the expression of autophagy regulatory molecules autophagy-related protein(ATG)13,7 and BECN1 increased remarkably while cell cycle molecules serine/threonine-protein kinase Chk2(CHEK2)and apoptosis r

关 键 词：RUNT相关转录因子3 赫赛汀 耐药性 非标记定量蛋白质组学 自噬 凋亡 

分 类 号：R966[医药卫生—药理学]