Bone Mesenchymal Stem Cell-derived Wnt3a Via-mediated Wnt Signal Pathway Promote Colorectal Carcinogenesis Induced By Chronic Fusobacterium Nucleatum Infection

Part Ⅰ The pathologic features of colorectal lesions in the model of chronic fusobacterium nucleatum infection-BMSC transplantation in APFCMin/+ miceObjective:To establish the model of chronic fusobacterium nucleatum infection-BMSC transplantation in APCMin/+ mice and evaluate the pathologic features of colorectal lesions.Methods:After breeding enough amount of APCMin/+ mice,We randomly divided into 4 groups:phosphate buffer saline transplantation alone(Control group),BMSC transplantation alone(BMSC group),chronic F.n infection group(F.n group),and BMSC transplantation with chronic F.n infection group(F.n +BMSC group).To establish the model of chronic F.n infection,APCMin/+mice were inoculated orally F.n(ATCC25586)for 8 weeks.Successfully isolated and identified bone marrow-derived mesenchymal stem cells(BMSC)/green fluorescent protein-BMSC were used to establish the model of chronic F.n infection-BMSC transplantation.The mice were sacrificed after 8 weeks and remained some mice to observe survival.The colorectal pathologic lesions were studied by HE stained paraffin sections and the nude mouse transplantation tumor experiment was to identify the role of F.n and BMSC in the mechanism of colorectal carcinogenesis.Results:(1)Among the breed mice,the APC mutation rate was from 12.5%to 37.5%,the average mutation rate only about 20%;(2)The F.n group could be observed weight loss,hematochezia,sporadic colon tumors.After BMSC transplantation 8 weeks,the F.n+BMSC group could be observed worse weight loss,rectocele,and hematochezia.The tumor numbers and size were significantly difference compared with F.n group(P<0.05);(3)The histopathology results showed that the incidences of colorectal high-grade intraepithelial neoplasia and cancer were significantly heightened in F.n+BMSC group(P<0.05);(4)The confocal results suggested that BMSC+F.n+SW480 group of BMSC was acted as cancer associated fibroblasts(CAFs)to further promote the growth of nude mice subcutaneous transplantation tumors.Conclusion:The model of chronic fusobacterium nucleatum infection in APCMin/+ mouse was established successfully by the method of F.n ATCC25586 oral inoculation.Bone marrow-derived mesenchymal stem cells significantly promoted the development of colorectal lesions and participated in the colorectal carcinogenesis induced by chronic fusobacterium nucleatum infection.Part Ⅱ The role of BMSC in the activation of Wnt pathway in fusobacterium nucleatum-induced colorectal carcinogenesis Objective:To investigate the role of BMSC in the activation of Wnt pathway in colorectal carcinogenesis induced by chronic fusobacterium nucleatum infection.Methods:To establish the model of chronic fusobacterium nucleatum infection-BMSC transplantation in APCMin/+mice in vivo.After 8 weeks,the colorectal specimens were harvested.The expression levels of β-catenin、TCF4、Axinland TGIF in cytoplasm and nucleus were detected by Real-time PCR,Western-blot and co-immunoprecipitation.The co-culture system in vitro of BMSC、SW480 and the fusobacterium nucleatum culture supernatant were established.Experiments were studied in vitro to analyse the effect of fusobacterium nucleatum infection to the activation of Wnt pathway in BMSC.Results:(1)After transplantation of BMSC,Real-time PCR and Western-blot results showed that the expression level of β-catenin、TCF4 in cytoplasm and nucleus in colorectal cancer of F.n+BMSC group was obviously heightened compared to that in F.n group;(2)Co-immunoprecipitation results indicated that the Axinl protein combined of TGIF and inhibited Axin-GSK3β-APC degradation compounds formation,thus β-catenin protein was accumulation(P<0.05);(3)After co-culture in vitro,Real-time PCR,Western-blot results showed that the expression of β-catenin and TCF4 in cytoplasm and nucleus in colon of F.n+BMSC group were obviously higher than those in other groups(P<0.05).(4)After prestimulation of F.n culture supernatant,BMSC could further enhance the capacity of colon cancer cell migration and invasion(P<0.05).Conclusion:BMSC participated in the activation of Wnt pathway in the colorectal lesions induced by chronic fusobacterium nucleatum infection,which indicated that the activation of Wnt pathway may be the pathogenesis of BMSC involved in the colorectal cancer induced by chronic F.n infection.Part Ⅲ The role of BMSC-derived Wnt3a in the activation of Wnt pathway in fusobacterium nucleatum-induced colorectal carcinogenesisObjective:To investigate the role of BMSC-derived Wnt3a in the activation of Wnt pathway in colorectal carcinogenesis induced by chronic fusobacterium nucleatum infection.Methods:We used Elisa kit to measure the level of Wnt3a secreted by BMSC after F.n stimulated.The expressions of Wnt3a and TGIF in colorectal specimens were detected by immunohistochemical.Western-blot and co-immunoprecipitation.We also built Wnt3a-RNAi virus and transfected into BMSC.Experiments were further studied in vitro and vivo to analyse the effect of fusobacterium nucleatum infection to the activation of Wnt pathway in BMSC.Results:(1)Real-time PCR results showed that Wnt3a gene expression increased most significantly among classic Wnt signaling pathways ligand;(2)Compared with other groups,the prestimulated by F.n culture supernatant of BMSC could secreted high level and specificity of Wnt3a proteins;(3)Western blot results indicated that the amount of Wnt3a and TGIF protein expression was positively related;(4)The F.n group could be observed better rectocele and hematochezia after Wnt3a-RNAi-BMSC transplantation compared with F.n+BMSC group;(5)Interference of BMSC-derived Wnt3a could significantly inhibit colon cancer cell migration,invasion,and nude mice tumor formation.Conclusion:BMSC participated in the activation of Wnt pathway in the colorectal lesions induced by chronic fusobacterium nucleatum infection.The secretion of Wnt3a may be the pathogenesis of BMSC involved in the colorectal cancer induced by chronic F.n infection.