| Objective: Colon cancer is among the three leading causes of cancer-related deaths worldwide,and even though several antibodies/molecules have been developed for treating colon cancer,surgical removal and chemo/radiation therapy remains standard of care.Moreover,colon cancer patients often develop metastatic disease or recur years after comprehensive therapeutic regimen of the primary tumor.The patients are asymptomatic because the disseminated cells appear to become dormant and are undetectable.The dormant cancer cells have been attributed to cancer stem cells.Colon cancer stem-like cells(colon CSCs)are believed to be resistant to chemo/radiation therapy,and a major cause of relapse.MSC,human mesenchymal stem cells,is one component of cancer stem cells microenvironment.Because of its characteristics of adult stem cells and participating in the tumor microenvironment,the study of MSC on tumor cells invasion and metastasis,and proliferation process attracts more attention.The interaction between BM-MSCs and tumor cells could be through many manners.BM-MSCs could secrete a variety of cytokines in the tumor milieu.MSC could also cause a direct effect through intercellular signaling via physical contacts with neighboring tumor cells,and then ultimately determine the fate of the tumor growth kinetics.In addition to this,some studies suggest that MSCs avail special delivery systems,which exosomes are required.Exosomes are small nanometer-sized(50-100nm)vesicles of endocytic origin that are released in the extracellular milieu by several cell types.It was recently reported that exosomes also contain messenger RNA(m RNA)and micro RNA(mi RNA)that are transferred to target cells,where they can be translated or mediate RNA silencing.Thus exosomal vesicles might function as a potent tool for intercellular communication and gene delivery also in metastasis.In this study,we will show BM-MSCs release exosomes and transfer information to colon cancer cells to maintain the stemness of colon cancer stem cells,which facilitate invasion and metastasis of colon cancer cells.Methods: 1.The effect of human BM-MSCs on colon cancer cells:(1)To isolate and analyze human BM-MSCs;(2)To detect the expression of E-cadherin,N-cadherin,vimentin,alpha-SMA in BM-MSCs by immunofluorescence;(3)To co-culture the BM-MSCs with colon cancer cell lines HCT-116?HT-29?SW-480;(4)To detect the expression of CD133 and Lgr5 after co-culture by FCM.(5)To analyze the cell cycle by PI staining.(6)To analyze the CSCs related m RNA(Oct-4,Lin-28,KLF,Bmi-1,CD44,Sox-2)by real time PCR.(7)To detect the invasion ability of colon cancer cells by Transwell assay.2.The effect of conditional medium from BM-MSCs on colon cancer:(1)To culture the colon cancer cell lines HCT-116?HT-29?SW-480 with conditional medium(CM)from BM-MSCs;(2)To detect the expression of CD133-APC and Lgr5-PE after culturing by CM from BM-MSCs;(3)To analyze the cell cycle by PI staining by FCM.(4)To observe the phenotype of colon cancer stem cell sphere.(5)To analyze the CSCs related m RNA by real time PCR.(6)To detect the invasion ability of colon cancer cells by Transwell assay.3.The effect of BM-MSCs exosomes from BM-MSCs on colon cancer cells:(1)To isolate the exosomes from BM-MSCs cells by ultracentrifuge technology,and detect the effect of exosomes from BM-MSCs on the there colon cancer cell lines HCT-116?HT-29?SW-480;(2)To analyze exosomes,the exosomes marker: CD81,CD63 and Rab5 a were detected by EM and western blot;(3)To detect the expression of CD133-APC and Lgr5-PE after exosomes treatment;(4)To analyze the cell cycle by PI staining of FCM;(5)To observe the phenotype of colon cancer stem cell sphere;(6)To detect the invasion ability of colon cancer cells by Transwell assay.4.The effect of exosomal micro RNA-3120-5p on colon cancer cells:(1)To analyze the exosomes isolated from BM-MSCs,co-culture cells,colon cancer cell by micro RNA biological array and select several micro RNA;(2)To transfect micro RNA into HCT-116?HT-29?SW-480,and detect the effect of micro RNA on the three cancer cells;(3)To observe the phenotype of colon cancer stem cell sphere;(4)To detect the expression of CD133-APC and Lgr5-PE after micro RNA transfection;(5)To analyze the cell cycle by PI staining of FCM after micro RNA transfection;(6)To detect the invasion ability of colon cancer cells treated by BM-MSCs exosomes by Transwell assay;5.Target genes of micro RNA-3120-5p in colon cancer cells:(1)To analyze the target gene of micro RNA-3120-5p by biological array and select several target genes;(2).To construct 3'UTR of target gene in vector,and transfect mi R-3120-5p into HCT-116 and SW-480,TK as control,and identify the target gene of micro RNA;(3)To observe the phenotype of colon cancer stem cell sphere;(4)To detect the expression of CD133-APC and Lgr5-PE after micro RNA transfection;(5)To analyze the cell cycle by PI staining of FCM after micro RNA transfection;(6)To detect the invasion ability of the three colon cancer cells after micro RNA transfection by Transwell assay.6.The localization of colon cancer stem like cells in tissue and the relationship with metastasis:(1)To detect the expression of CD133 and alpha-SMA in colon cancer tissue,and make sure the location of colon CSCs;(2)To detect the expression of CD133 and Lgr5 in 80 colon cancer specimens;(3)To analyze the relationship between CD133 and Lgr5 positive cells and metastasis of colon cancer samples.Results: 1.Human bone marrow derived mesenchymal stem cells can maintain the cancer stem like cells phenotype and promote invasion and metastasis:(1)Human bone marrow derived mesenchymal stem cells could express N-cadherin,did not expresss E-cadherin and Vimentin and express alpha-SMA;(2)After co-culture with human bone marrow derived mesenchymal stem cells,the cancer cell lines HCT-116,HT-29,SW-480,were able to form colon CSCs spheres;(3)After co-culture,expression of CD133 and Lgr5 were increased significantly in colon cancer cells;(4)After co-culture,the cell cycles of three colon cancer cells were significantly inhibited;(5)After co-culture,the invasion ability was enhanced;(6)After co-culture,the ability of cell proliferation was decreased significantly.2.Conditional medium from human bone marrow mesenchymal stem cells can maintain the phenotype of cancer stem cells like cells and promote invasion and metastasis:(1)HCT-116,HT-29 and SW-480 cells treated with conditional medium from human bone marrow derived mesenchymal stem cells were able to form colon CSCs sphere,respectively;(2)The expression of CD133 and Lgr5 increased significantly after cultured by conditional medium;(3)The cell cycle of three kinds of colon cancer cells were significantly reduced after cultured by conditional medium;(4)Invasive ability of colon cancer cells was obviously increased after cultured by conditional medium.3.Human bone marrow derived mesenchymal stem cells exosomes can promote the invasion of cancer stem cell like cell phenotype and metastasis:(1)Exosomes from human bone marrow derived mesenchymal stem cells promoted cancer cell lines HCT-116,HT-29,SW-480 to form colon CSCs spheres;(2)After BM-MSCs exosomes treatment,the expression of CD133 and Lgr5 were increased significantly;(3)After BM-MSCs exosomes treatment,the cell cycles of three colon cancer cells were significantly reduced;(4)After BM-MSCs exosomes treatment,the invasion ability was enhanced;(5)Deprived exosomes,the conditional medium could not promote invasion ability and the expression CD133 and lgr5;4.Exosomal mi R-3120-5p can promote cancer stem like cells phenotype and invasion:(1)Exosomal mi R-3120-5p promoted the three colon cancer cell lines HCT-116,HT-29,SW-480 to form colon CSCs spheres;(2)Exosomal mi R-3120-5p increased the expression of CD133 and Lgr5 significantly;(3)Exosomal mi R-3120-5p inhibited cell cycle of the three colon cancer cells significantly;(4)Exosomal mi R-3120-5p enhanced the invasion ability of the three colon cancer cells.5.mi R-3120-5p can promote cancer stem cell like cells phenotype and metastasis through down-regulating the expression of Axin2:(1)by bioinformatics analysis,many target genes of mi R-3120-5p were found.Through si RNA transfection,Axin2 was chosen to be the target gene to influence the colon CSCs formation;(2)the results of function assays were accompany with above results after sh Axin2 transfection.6.The localization of colon CSCs is in stromal tissues and promotes metastasis:(1)Immunofluorescence showed that CD133 was expressed adjacent to alpha-SMA positive cells;(2)Immunofluorescence staining showed the number of CD133 and Lgr5 positive cells in colon cancer tissues played vital roles on invasion and metastasis of colon cancer.Conclusions: 1.BM-MSCs can maintain stemness of colon cancer cells and promote the invasion and metastasis of colon cancer cells.2.BM-MSCs promote invasion and metastasis of colon cancer cells through exosomes.3.Exosomal micro RNA-3120-5p plays important roles on colon CSCs and invasion and metastasis of colon cancer cells.4.Colon CSCs are mainly located in the tumor stroma and adjacent to tumor derived MSC,the content of colon CSCs population is related with distant metastasis in cancer patients and the occurrence of adverse prognosis. |
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