PA-MSHA通过调控Wnt/β-catenin信号通路抑制结肠癌耐西妥昔单抗耐药结肠癌SW480细胞的增殖、迁移和侵袭  

PA-MSHA inhibits the proliferation,migration and invasion of cetuximab-resistant colon cancer SW480 cells through Wnt/β-catenin signaling pathway

作　　者：张欢欢 单武林 ZHANG Huanhuan;SHAN Wulin(Department of Cancer Epigenetics Program,The First Affiliated Hospital of USTC,Division of Life Sciences and Medicine,University of Science and Technology of China,Hefei 230031,Anhui Province,China;Department of Laboratory Diagnostics,The First Affiliated Hospital of USTC,Division of Life Sciences and Medicine,University of Science and Technology of China,Hefei 230031,Anhui Province,China)

机构地区：[1]中国科学技术大学附属第一医院肿瘤表观遗传学研究室,安徽合肥230031 [2]中国科学技术大学附属第一医院检验科,安徽合肥230031

出　　处：《肿瘤》2022年第4期254-264,共11页Tumor

基　　金：安徽省自然科学基金项目(1908085QH375)

摘　　要：目的:研究铜绿假单胞菌-甘露糖敏感血凝素(pseudomonas aeruginosa mannose-sensitive hemagglutinin,PA-MSHA)对西妥昔单抗耐药结肠癌SW480细胞(SW480-R)增殖、迁移和侵袭能力的影响,并探讨可能的作用机制。方法:用不同浓度的西妥昔单抗处理亲本SW480细胞和耐药SW480-R细胞,采用CCK-8法检测亲本和耐药细胞对西妥昔单抗的敏感性。用不同浓度的PA-MSHA处理SW480-R细胞,采用CCK-8法检测PAMSHA对SW480-R细胞增殖能力的影响,实时荧光定量PCR法检测对SW480-R细胞中微RNA(micro,miR)-7-5p表达的影响,划痕愈合实验和Transwell小室法检测对SW480-R细胞迁移和侵袭能力的影响。采用脂质体转染的方法将miR-7-5p-模拟物(mimics)转入SW480-R细胞再用PA-MSHA处理后,采用划痕愈合实验和Transwell小室实验检测miR-7-5p过表达对PA-MSHA抑制SW480-R细胞迁移和侵袭能力的影响,最后用蛋白印迹法检测对Wnt/β-连环蛋白(β-catenin)信号通路中β-catenin和E-钙黏蛋白(E-cadherin)表达水平的影响。结果:结肠癌SW480-R细胞对西妥昔单抗的敏感性低于SW480细胞(P<0.05),成功建立对西妥昔单抗耐药的结肠癌细胞。与对照组相比,PA-MSHA可以明显抑制SW480-R细胞的增殖、迁移和侵袭能力(P均<0.01),且呈剂量依赖性。PA-MSHA刺激后,miR-7-5p的表达水平上调(P<0.01);miR-7-5p过表达明显增强PA-MSHA抑制SW480-R细胞迁移和侵袭的能力(P<0.05)。蛋白质印迹法检测结果显示,PA-MSHA刺激后,Wnt/β-catenin信号通路中β-catenin的表达水平下调,E-cadherin的表达水平上调(P均<0.01),提示Wnt/β-catenin信号通路的活性被抑制;miR-7-5p过表达进一步降低β-catenin的表达水平,上调E-cadherin蛋白的表达水平(P均<0.05)。结论:PA-MSHA可能通过调控miR-7-5p/Wnt/β-catenin通路抑制西妥昔单抗耐药结肠癌细胞增殖、迁移和侵袭。Objective:The study aims to investigate the effect of pseudomonas aeruginosa mannose sensitive hemagglutinin(PA-MSHA)on the proliferation,migration and invasion of cetuximab-resistant colon cancer cells(SW480-R),and further elucidate the underlying mechanism.Methods:The parental sensitive colon cancer cells SW480 and cetuximab-resistant daughter cells SW480-R were treated with different concentrations of cetuximab,and the cell sensitivity to cetuximab was evaluated by CCK-8 assay.SW480-R cells were treated with different concentrations of PA-MSHA,and the effect of PA-MSHA on the proliferation,expression level of micro-RNA(miR)-7-5p,migration and invasion of SW480-R cells was examined by CCK-8 assay,real-time fluorescence quantitative PCR,wound healing assay and Transwell assay,respectively.SW480-R cells were transfected with miR-7-5p-mimics followed by PA-MSHA treatment,and the inhibitory effect of PA-MSHA on the migration and invasion of SW480-R cells was assessed with wound healing assay and Transwell assay,respectively,while the expression of Wnt/β-catenin pathway-related proteins,includingβ-catenin and E-cadherin,was detected by Western blotting.Results:The sensitivity of SW480 cells to cetuximab was significantly higher than that of SW480-R cells(P<0.05),indicating the successful establishment of the cetuximab-resistant colon cancer cell line.Compared with the control group,PA-MSHA could significantly inhibit the proliferation,migration and invasion of SW480-R cells in a dose-dependent manner(P<0.01).The real-time fluorescence quantitative PCR result showed that PA-MSHA could up-regulate the expression of miR-7-5p in a dose-dependent manner(P<0.01),and miR-7-5p overexpression could obviously enhance the inhibitory effect of PA-MSHA on the migration and invasion of SW480-R cells(P<0.05).In addition,the activity of Wnt/β-catenin pathway was remarkably down-regulated in PA-MSHA-treated cells as indicated by the decrease inβ-catenin expression(P<0.01)and increase in E-cadherin expression(P<0.01)after PA-MSHA

关 键 词：结肠肿瘤 PA-MSHA 西妥昔单抗 细胞增殖 细胞运动 

分 类 号：R735.35[医药卫生—肿瘤]