Spermine oxidase targets thioredoxin reductase 1 to promote malignant progression of colon cancer
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 tumors 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 G0/G1 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.
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