Construction Of Spontaneous Gastric Cancer Mouse Model By MUC6 Knock-out And Molecular Mechanisms Of CEACAM6 On Prompting Gastric Cancer Progression

Part I Construction of spontaneous gastric cancer mouse model by knock-out MUC6Objection:MUC6 is one of secreted mucoproteins and is normally expressed in mucous of stomach.MUC6 plays important role to lubricate and protect mucous of stomach.However,the abnormally expression of MUC6 contribute to occurrence and progression of tumor and is observed in a wide variety of human tumors.Thus,to observe the pathologic changes of stomach,MUC6 was knocked out in C57BL/6J mice.The spontaneous gastric cancer mouse model was expected to be obtained in C57BL/6J mice.Methods:To build MUC6 knockout mouse model,CRISPR/Cas9 was used to knockout MUC6 in zygote of C57BL/6J mouse.The heterozygote MUC6 knockout mice were selected.MUC6^-/-mice were randomly divided into 7 groups with each group containing 6mice.MUC6^-/-and MUC6^+/+mice were sacrificed every 10 weeks.The stomachs were collected and paraffin embedded to examine the pathologic changed of stomach.HE staining was performed to evaluate the severity of changes and process of tumor formation.Results:We successfully obtained MUC6^-/-mouse and genotype of mouse was verified by sequencing.Hair color,diet,weight,reproductive capacity,and lifetime presented on difference.In mice stomach tissues,MUC6^-/-mice showed no significant change compared to MUC6^+/+mice.Mucosa edema was found in MUC6^-/-mice at 10W and 20W.Early tumor formation with 16.7%incidence was observed at 30W.Carcinoma in situ formed at 40W with tumor incidence 50%.The tumor incidence was increased by 66.7%at 50W and 100%at 60W.HE staining showed that inflamed cells were observed at 5W mice.With time going,the inflammation became more severity.Significant lymphoid follicles were detected at40W mice.Acid secreted gland showed significant atrophy with chief cells and parietal cells loss at 20W mice.The intestinal type cells were increased in MUC6^-/-mice.Slight atypical dysplasia was shown at 10W and moderate atypical dysplasia was detected at 20W.Early gastric cancer formed at 30W and 40W.Intra-mucosal carcinoma was observed at 50W and invasive carcinoma infiltrating submucosa was found at 60W.Conclusion:Knockout MUC6 resulted in intestinal gastric cancer through the following sequence inflammation,proliferation,intestinal metaplasia,and dysplasia.Part II Study of luteolin suppressed gastric cancer progression by inhibiting Notch1 signaling pathwayObjection: Luteolin is a flavonoid belonging to Chinese herbs,presenting in much green fruits and vegetables.Many studies showed that luteolin suppressed tumor progression in a variety of tumors.However,the effects of luteolin in gastric cancer were unclear.Thus,we focus on the function and molecular mechanisms of luteolin in gastric cancer.Methods: Different concentration of luteolin was used to treat gastric cancer cells.Then to examine cell proliferation,cell migration and invasion,cell apoptosis,and cytoskeleton.And cell signaling after luteolin treatment was examined.Furthermore,the effect of luteolin in vivo was examined after subcutaneous tumor formation in BABL/C nude mice.Results: Luteolin effectively suppressed cell proliferation,colony formation,cell migration and invasion,and angiogenesis.Furthermore,cytoskeleton of gastric cancer cell was shrunk and epithelial-mesenchymal transition(EMT)was inhibited by luteolin.Results of study showed that Notch1 cell signal was suppressed by luteolin treatment.Downregulated Notch1 expression could observe similar results as well as luteolin treatment.However,overexpression of Notch1 reversed the effects induced by luteolin.Moreover,the interaction between active molecular NICD of Notch1 and ?-catenin was inhibited by luteolin treatment,then the following signaling pathway was suppressed.Luteolin suppressed growth of grafted subcutaneous tumor in vivo.Conclusion: Luteolin suppressed gastric cancer progression by inhibiting EMT and angiogenesis through downregulating Notch signaling pathway.Part III Molecular mechanisms of CEACAM6 promoting gastric cancer progression by interacting with ITGA5Objection: CEACAM6 was overexpressed in gastric cancer tissues and associated with lymph node metastasis.Overexpression of CEACAM6 promoted gastric cancer cells invasion and migration,and angiogenesis.However,CEACAM6 was absence of intracellular domain and transmembrane domain.The molecular mechanisms of CEACAM6 promoting gastric cancer metastasis remain unknown and should be further explored.Methods: Co-immunofluorence and Co-IP assays were used to examine the interaction between CEACAM6 and ITGA5.With up-and down-regulating CEACAM6 in gastric cancer cells,ITGA5 expression was further altered by vectors.Then,transwell,tube formation,vascular mimicry tube formation and western blot assays were performed to examine the alteration of biological behaviors and cell signaling.Results: Our results showed that CEACAM6 and ITGA5 co-localized on the membrane of cell and interact with each other.We silenced ITGA5 expression in CEACAM6 overexpressed cells and found that phosphorylation of AKT/FAK and VEGF level were decreased,EMT,cell migration,and tube formation were also inhibited.However,overexpressed ITGA5 in CEACAM6 silencing cells upregulated phosphorylation of AKT/FAK and secretion of VEGF,promoted gastric cancer EMT and cell migration,elevated tube formation ability.Conclusion: CEACAM6 promotes gastric cancer progression by interacting with ITGA5,then activating FAK/AKT signaling and inducing EMT and angiogenesis.