精胺氧化酶靶向调控硫氧还蛋白还原酶1促进结肠癌恶性进展  

Spermine oxidase targets thioredoxin reductase 1 to promote malignant progression of colon cancer

作　　者：王慧 刘超 章春雪[2] 姚懿芹 陈龙 祁悦欣[5] 费禹翔 赵树立 胡容 杜前明[2,5] Wang Hui;Liu Chao;Zhang Chunxue;Yao Yiqin;Chen Long;Qi Yuexin;Fei Yuxiang;Zhao Shuli;Hu Rong;Du Qianming(College of Nursing and Health Management&College of Life Science and Chemistry,Wuhan Donghu University,Wuhan 430212,China;School of Basic Medicine&Clinical Pharmacy,China Pharmaceutical University,Nanjing 210009,China;Department of Pharmacy,Nanjing First Hospital,Nanjing Medical University,Nanjing 210006,China;Schoolof Pharmacy,China Pharmaceutical University,Nanjing 210009,China;General Clinical Research Center,Nanjing First Hospital,Nanjing Medical University,Nanjing 210006,China)

机构地区：[1]武汉东湖学院护理与健康管理学院(生命科学与化学学院),湖北武汉430212 [2]中国药科大学基础医学与临床药学学院,江苏南京210009 [3]南京医科大学附属南京医院南京市第一医院药学部,江苏南京210006 [4]中国药科大学药学院,江苏南京210009 [5]南京医科大学附属南京医院南京市第一医院临床医学研究中心实验室,江苏南京210006

出　　处：《实用肿瘤杂志》2024年第4期317-332,共16页Journal of Practical Oncology

基　　金：国家自然科学基金项目(82172558,82273971,82204394);江苏省重点研发计划(临床前沿技术)(BE2019617);南京市卫生科技发展专项资金项目杰出青年基金项目(JQX20008)。

摘　　要：目的探讨精胺氧化酶(spermine oxidase,SMOX)靶向调控硫氧还蛋白还原酶1(thioredoxin reductase 1,TR1)对结肠癌恶性进展的影响。方法收集2018年9月至2019年6月就诊于南京市第一医院的30例结肠癌患者的结肠癌组织及其癌旁组织。采用免疫组织化学染色检测组织中的SMOX表达。采用Western blot实验检测结肠癌细胞株(HCT116、SW480、DLD-1、SW620、Caco-2、HCT-15、T84和LS123)和正常结肠细胞株NCM460的SMOX表达。利用基因表达谱互作分析(Gene Expression Profiling Interactive Analysis,GEPIA)网站查询SMOX在结肠癌组织与癌旁组织中的表达情况和SMOX表达水平与患者总生存的关系。采用Western blot实验检测上述细胞和组织中代谢产物亚精胺合成酶的水平。利用2',7'-二氯二氢荧光素二乙酸酯(2',7'-dichlorodihydrofluorescein diacetate,DCFH-DA)结合流式细胞术和组织免疫荧光实验分别检测结肠细胞和组织中代谢副产物活性氧(reactive oxygen species,ROS)水平。选取SMOX表达水平最高的结肠癌细胞株HCT116和SW480为实验对象,构建慢病毒载体,用PLKO.1-puro-shCtrl、PLKO.1-puro-shSMOX、PLKO.1-puro-shSMOX+pcDNA3.1和PLKO.1-puro-shSMOX+pcDNA3.1-TR1分别转染细胞,分别为shCtrl组、shSMOX组、shSMOX+pcDNA3.1组和shSMOX+TR1组。Western blot实验检测HCT116和SW480细胞shCtrl组和shSMOX组SMOX与TR1的表达情况,以及HCT116细胞shSMOX+pcDNA3.1组和shSMOX+TR1组的TR1表达水平。利用细胞计数试剂盒(Cell Counting Kit-8,CCK-8)检测细胞增殖活力。碘化丙啶(propidium iodide,PI)单染结合流式细胞术检测肿瘤细胞周期变化。PI/异硫氰酸荧光素-膜连蛋白(PI/fluorescein isothiocyanate-Annexin V,PI/FITC-Annexin V)双染结合流式细胞术检测细胞凋亡/坏死情况。Transwell体外侵袭实验分析细胞侵袭能力。shCtrl组、shSMOX组、shSMOX+pcDNA3.1组和shSMOX+TR1组的HCT116细胞稀释至浓度为1×107个/mL的细胞悬液分别向裸鼠右侧腋窝皮下注射0.2 Objective To investigate the effect of spermine oxidase(SMOX)on the malignant progression of colon cancer by targeting thioredoxin reductase 1(TR1).Methods Thirty cases of colon cancer tissues and the adjacent tissues were derived from colon cancer patients who were admitted to Nanjing First Hospital from September 2018 to June 2019.Immunohistochemical staining was used to detect the expression of SMOX in the tissues.Western blot was used to detect the expression of SMOX in colon cancer cells,including HCT116,SW480,DLD-1,SW620,Caco-2,HCT-15,T84,and S123,and normal colon NCM460 cells.The Gene Expression Profiling Interactive Analysis(GEPIA)website was used to investigate the expression of SMOX in the colon cancer tissues and adjacent tissues,and the correlation between the SMOX expression and the overall survival of the patients.Western blot was used to detect the level of spermidine synthase in the cells and tissues.2',7'-Dichlorodihydrofluorescein diacetate(DCFH-DA)combined with flow cytometry(FCM)and immunofluorescence was used to detect the level of reactive oxygen species(ROS)in the colon cells and tissues.Two cell lines with the highest SMOX expression,HCT116 and SW480,were used for further experiments.HCT116 and SW480 were transfected with PLKO.1-puro-shCtrl,PLKO.1-puro-shSMOX,PLKO.1-puro-shSMOX+pcDNA3.1,or PLKO.1-puro-shSMOX+pcDNA3.1-TR1,and labeled as shCtrl group,shSMOX group,shSMOX+pcDNA3.1 group,and shSMOX+TR1 group,respectively.Western blot was used to detect the expressions of SMOX and TR1 in HCT116 and SW480 cells in the shCtrl and shSMOX groups,and the expression of TR1 in HCT116 cells in the shSMOX+pcDNA3.1 and shSMOX+TR1 groups.Cell Counting Kit-8(CCK-8)assay was used to detect cell pro-liferation activity.Propidium iodide(PI)single staining combined with FCM was used to detect tumor cell cycle changes.PI/fluorescein isothiocyanate-Annexin V(PI/FITC-Annexin V)double staining combined with FCM was used to detect cell apoptosis/necrosis.Tran-swell invasion experiment was used to analyze the invasio

关 键 词：结肠癌 精胺氧化酶 硫氧还蛋白还原酶1 恶性进展 

分 类 号：R735.35[医药卫生—肿瘤]