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

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 mL细胞悬液,建立裸鼠结肠癌移植瘤模型。4周后处死裸鼠,分离组织,剥取肿瘤称重。运用免疫组织化学染色检测裸鼠结肠肿瘤组织中Ki-67阳性细胞数。结果SMOX在8种结肠癌细胞株和结肠癌组织中的表达水平均高于正常结肠细胞株和癌旁组织,且与不良预后有关(均P<0.05)。与正常结肠细胞和癌旁组织比较,8种结肠癌细胞和结肠癌组织中的代谢产物亚精胺合成酶和ROS水平随SMOX的高表达同步升高(均P<0.05)。与shCtrl组比较,shSMOX组G_(0)/G_(1)期细胞数目增多,PI/Annexin V双阳性细胞数目增加,细胞增殖活性降低,侵袭数量减少,SMOX和TR1表达水平降低(均P<0.05)。与shSMOX组和shSMOX+pcDNA3.1组比较,shSMOX+TR1组TR1表达水平升高,S期细胞数目增多,PI/Annexin V双阳性细胞数目减少,细胞增殖活性升高(均P<0.05)。裸鼠结肠癌移植瘤模型显示,注射后14 d,与shCtrl组比较,shSMOX组和shSMOX+pcDNA3.1组肿瘤体积和重量均降低,结肠肿瘤组织中Ki-67阳性细胞数减少,而shSMOX+TR1组肿瘤体积、重量和结肠肿瘤组织中Ki-67阳性细胞数则较shSMOX+pcDNA3.1组增加(均P<0.05)。结论SMOX在结肠癌细胞和组织中表达上调,可能通过靶向提高TR1的表达促进细胞周期运转,抑制细胞凋亡,促进细胞增殖和侵袭能力及肿瘤生长,进而促进结肠癌恶性进展。

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 invasion ability of tumor cells.HCT116 cells in the shCtrl,shSMOX,shSMOX+pcD-NA3.1,and shSMOX+TR1 groups were diluted to 1×107 cells/mL and injected subcutaneously into the right armpit of nude mice to es-tablish a colon cancer transplantation tumor model.Four weeks later,the nude mice were killed,tissues were separated,and t umors were peeled and weighed.The number of Ki-67 positive cells in the colon tumor tissues of nude mice was detected by immunohistochemical staining.Results The expression of SMOX in the eight colon cancer cell lines and the colon cancer tissues was higher than that in the normal cells and the adjacent tissues,and was related to a poor prognosis(all P<0.05).Compared with the normal colon cells and the adjacent tissues,the levels of spermidine synthase and ROS increased synchronously with the high expression of SMOX in the colon cancer cell lines and the colon cancer tissues(all P<0.05).Compared with the shCtrl group,the number of G_(0)/G_(1)cells and the number of PI/Annexin V double-positive cells increased significantly in the shSMOX group,while cell proliferation activity,invasion and the expressions of SMOX and TR1 decreased(all P<0.05).Compared with the shSMOX and shSMOX+pcDNA3.1 groups,the expression of TR1,the number of S-phase cells and cell proliferation activity increased,while the number of PI/Annexin V double-positive cells decreased,in the shSMOX+TR1 group(all P<0.05).The nude mouse model showed that 14 days after injection,compared with the shCtrl group,the tumor volume and weight,and the number of Ki-67 positive cells in the colon tumor tissue decreased in the shSMOX and shSMOX+pcDNA3.1 groups.The tumor volume and weight,and the number of Ki-67 positive cells in the colon tumor tissue in the shSMOX+TR1 group were significantly increased compared with those in the shSMOX+pcDNA3.1 group(all P<0.05).Conclusions The expression of SMOX is up-regulated in colon cancer cells and tissues.SMOX may promote cell cycle progression,cell proliferation,invasion,and tumor growth,and inhibit cell apoptosis by targeting the expression of TR1,thereby promoting the malignant progression of colon cancer.