B7-H3 Is Highly Expressed In Gastric Cancer And Modulates Cancer Cell Migration And Invasion

Objective:1.To detect the expression of B7-H3 in gastric cancer and to analyze the relationship between the expression of B7-H3 and clinicopathological features of patients;2.To explore the effect of b7-h3 on the biological function of gastric cancer.3.To investigate the potential mechanisms on the cell migration and invasion by B7-H3 and CXCR-4 in gastric cell lines.Methods:1.B7-H3 expression was determined by immunohistochemistry(IHC)in 120 gastric cancer tissues.We analyzed the relationship between the expression of B7-H3 and clinicopathological features of patients;2.Lentiviral gene transfer vectors encoding green fluorescent protein(GFP)and either a human B7-H3(LV-B7-H3 virus)or a non-targeting control sh RNA(LV-NC virus)were used and The infection rate and infection efficiency were verified by fluorescence microscope observation,flow cytometry,FCM,western blot and real-time PCR.3.The effects of B7-H3 on proliferation,migration and invasion and metastasis ability of gastric cancer cell were detected by CCK-8,Wound healing assay and Migration and invasion assays.4.Gastric cancer cells were injected into the tail veins of 4-week-old female BALB/c nude mice.Mice were imaged using an in vivo imaging instrument at four weeks to detect GFP+ cells in organs.After five weeks,all mice were euthanized.Tissues were removed and fixed in 10% formalin for H&E staining,and metastasized colonies were counted using a dissecting microscope.5.B7-H3 and CXCR-4 expression was determined by immunohistochemistry(IHC)in 120 gastric patients' tissues and statistical analysis the relevance of B7-H3 and tumor metastasis related protein expression CXCR-4.6.B7-H3 silencing downregulated the metastasis associated molecule,CXCR-4,in N87 cells,as confirmed by flow cytometry,western blotting,and real-time PCR.Fluorescence imaging demonstrated whether B7-H3 localizated with CXCR-4 in the cell membrane or cytoplasm.Co-immunoprecipitation with each specific antibody showed an interaction between B7-H3 and CXCR-4.Expression of related molecular was evaluated by western blot.Results:1.B7-H3 expression was determined by performing immunohistochemistry(IHC)in 120 gastric patients' tissues.B7-H3 levels in cancer tissues were positively correlated with tumor infiltration depth.2.We used flow cytometry,western blotting,and real-time PCR to analyze B7-H3 expression in both LV-NC-and LV-B7-H3-infected N87 cells.LV-B7-H3-infected cells exhibited lower B7-H3 membrane,plasma protein and m RNA levels than LV-NC infected cells.3.CCK-8 assay showed no difference between LV-NC-and LV-B7-H3-infected N87 cell proliferation rates.However,wound healing assay resulted that wound healing was reduced in B7-H3-silenced N87 cells compared to controls.Similarly,LV-B7-H3-infected N87 cells migrated less in a transwell assay after 24 h compared to controls.Mice injected with LV-B7-H3-infected N87 cells had more lung metastases than mice injected with LV-NC-infected cells.4.B7-H3 expression was correlated with CXCR-4.B7-H3 silencing downregulated the metastasisassociated molecule,CXCR-4,in N87 cells,as confirmed by flow cytometry,western blotting,and real-time PCR.Fluorescence imaging demonstrated a high degree of B7-H3 localization with CXCR-4 in the cell membrane and cytoplasm.Co-immunoprecipitation with each specific antibody showed an interaction between B7-H3 and CXCR-4.5.B7-H3 silencing in N87 cells reduced AKT,ERK,Jak2 and Stat3 phosphorylation as shown by western blotting.Conclusions:1.Shallow depth of tumor tissue showed low B7-H3 expression and deep depth of tumor tissue showed high B7-H3 expression.2.B7-H3 can modulate gastric cancer cell migration and invasion.3.B7-H3 can targeting CXCR-4 and promote the invasion and metastasis ability of gastric cancer cells by phosphorylating Jak2,Stat3,AKT and ERK pathways.