The Mechanism Of BGN And G9a Promote Tumor Invasion And Metastasis Of Gastric Cancer

Objection: Biglycan(BGN)highly expressed abnormally in gastric cancer,previous studies had shown that BGN expression level in gastric cancer was mainly related with lymph node metastasis,TNM staging and depth of tumor invasion.At the same time,BGN promoted tumor metastasis of gastric cancer through activating FAK signaling pathway and promoted the tube formation of endothelial cells,which suggested that BGN might be involved in the interaction of endothelial cells and tumor cells in gastric cancer.In this study,we invastigated whether BGN could affect the expression of VEGF mRNA and protein in endothelial cells,and clarified the effect of BGN on secreting VEGF in endothelial cells.Histone methyltransferase G9a is up-regulated in a wide variety of human tumors.This suggests that G9a plays an important role in the development of tumors.The aims of this study are to clarify G9a expression in tumor tissues and its adjacent normal tissues of gastric cancer.To investigate the effect of G9a on tumor invasion and metastasis of gastric cancer by up-regulating and down-regulating the expression of G9a in gastric cancer cells,and to validate it in animal model.Methods: The effect of BGN on the expression of VEGF in endothelial cells was detected by ELISA and q RT-PCR and the expression of VEGF in endothelial cells after treated with TLR2/4 monoclonal antibody,sh HIF1? or NF-k B inhibitor PDTC was detected by ELISA.The correlation between the upstream and downstream molecules in the signaling pathway and the binding sites were analyzed by double luciferase assay,Western-blot and CHIP.Endothelial cell tubule formation assay,CCK-8 test for cell proliferation and transwell chamber were used to detect endothelial cell formation,proliferation and migration of endothelial cells after TLR2/4 monoclonal antibody,sh HIF1? or PDTC treatment and then treated with BGN.Finally,the interaction between endothelial cells and gastric cancer cells was analyzed by transwell co-culture system.Immunohistochemistry(IHC)was used to detect the expression of G9a in gastric cancer tissues and its adjacent non-tumor tissues and to analyze the relationship between G9a and clinicopathological parameters.After overexpressing or knock-down G9a stable gastric cancer cell lines were constructed,the effect of G9a on tumor invasion and metastasis of gastric cancer cells was detected by transwell assay,ankylosis resistance assay,soft agar formation assay and adhesion assay,nude mice model of cell peritoneal transplantation used to validate the in vitro results.The expression of related pathway gene was detected by immunoblotting(WB),the interaction between the protein and the promoter region was detected by double luciferase reporter assay and chromatin immunoprecipitation(Ch IP)and Co-IP was used to detect the interaction between proteins.Results: This study showed that endothelial cells stimulated by BGN increased the interaction between NF-k B and the HIF-1? promoter,leading to enhanced promoter activity and increased HIF-1a mRNA levels,as well as augmented HIF-1 activity that resulted in VEGF expression.All of this was dependent on the interaction of BGN with its receptors,TLR2 and TLR4.Moreover,we found that BGN enhanced endothelial cell migration and proliferation,as well as tube formation,in a TLR signaling pathway-dependent manner.In addition,endothelial cell-derived VEGF in turn was found to act on GC cells and promoted their migration.In this study,we found that histone methyltransferase G9a highly expressed in GC tissues than its adjacent non-tumor tissues.G9a expression level was correlated with advanced stage and shorter overall survival.In vitro and in vivo experiments reveal that silencing G9a attenuate the peritoneal metastasis-relevant traits activities of GC cells,whereas ectopic overexpression of G9a contributes to promote tumor metastasis.Moreover,we demonstrate that G9a expression can be induced by Reg IV via p-ERK /p-SP1 pathway.SP1 directly binds to G9a promoter and promotes its expression then the upregulated G9a can form a transcriptional activator complex with P300 and GR to participate in ITGB3 expression induced by dexamethasone(DEX),which contributes to GC peritoneal metastasis.However,the process of G9a participates in ITGB3 expression induced by DEX was not dependent on the SET domain and methyltransferase activity of G9a.Conclusion: BGN secreted by gastric cancer cells acts on endothelial cells and induces VEGF secretion through TLR2(4)/ NF-k B / HIF-1a signaling pathway,the secretion of VEGF reaction in gastric cancer cells and caused migration of gastric cancer cells,thereby promoting tumor invasion and metastasis of gastric cancer.The expression of G9a in gastric cancer was significantly up-regulated,and the expression level of G9a was correlated with lymph node metastasis and TNM stage.Overexpression of G9a in gastric cancer cells promoted tumor invasion and metastasis behavior of cancer cells and vise versa.G9a was involved in the process of tumor invasion and metastasis of gastric cancer through the regulation of ITGB3.