Study On The Expression,Role And Molecular Mechanism Of VEGFR3 In Gastric Cancer

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Analysis of the expression and clinicopathological characteristics of VEGFR3 in gastric cancerObjective:To detect the expression of VEGFR3 in early and advanced gastric cancer,and to further analyze the clinicopathological relationship between VEGFR3 expression and gastric cancer.Methods:The expression of VEGFR3 in gastric cancer tissues was analyzed from the TCGA database.Immunohistochemistry was used to detect VEGFR3 protein expression in 50 patients with early gastric cancer,48 patients with advanced gastric cancer and their paired normal gastric mucosa tissues.The clinicopathological relationship between VEGFR3 protein expression and patients with early and advanced gastric cancer was analyzed by statistical methodsResults:1.Database analysis resultsExpression of VEGFR3 was up-regulated in gastric cancer.Patients in the high VEGFR3 expression group had a poorer prognosis than those in the low VEGFR3 expression group.Compared with the control group,VEGFR3 expression in normal gastric mucosa increased with the increase of clinical and pathological staging and the number of lymph node metastasis.2.Immunohistochemical resultsThe positive expression rates of VEGFR3 in early and advanced gastric cancer tissues(60%and 93.75%)were significantly higher than that in normal gastric mucosa tissues(10%).The expression of VEGFR3 in early gastric cancer was related to the depth of tumor invasion and lymph node metastasis,but not to gender,age,tumor size or tumor differentiation.The expression level of VEGFR3 in advanced gastric cancer was only closely related to lymph node metastasis in patients with gastric cancer,and was not related to gender,age,tumor size,depth of tumor invasion and degree of differentiation.Patients with high expression of VEGFR3 had a poor prognosis.Conclusion:VEGFR3 expression is up-regulated in both early and advanced gastric cancer,and is associated with lymph node metastasis and poor prognosis in patients with gastric cancer.Part ? Effect of VEGFR3 on the biological function of gastric cancer cellsObjective:To observe the effects of VEGFR3 on the proliferation,migration,invasion and apoptosis of gastric cancer cells in vitro and in vivo.Methods:The effects of VEGFR3 on the proliferation,migration,invasion and apoptosis of gastric cancer cells were studied in vitro and in vivo.Methods:1.In vitro experimentsSmall interfering RNA(siRNA)and overexpressed plasmids of VEGFR3 were transfected into gastric cancer cell lines,respectively.Quantitative real-time PCR and Western blot were used to detect the expression effect of VEGFR3 in the transfected gastric cancer cell lines.The proliferation ability of gastric cancer cell lines transfected with VEGFR3 was detected by Edu and plate cloning.The scratch test and Transwell assay were used to detect the changes in the migration and invasion ability of gastric cancer cells transfected with VEGFR3.Flow cytometry was used to detect the apoptosis of gastric cancer cells transfected with VEGFR3 and its influence on the cell cycle time phase distribution.2.In vivo experimentsA stable gastric cancer cell line(MKN45)with knockdown VEGFR3 expression on lentivirus was constructed and the gastric cancer cells were amplified.Gastric cancer cells infected with lentivirus were injected into nude mice by subcutaneous and caudal intravenous injection.The influence of VEGFR3 on the proliferation,invasion and migration of gastric cancer cells in vivo was analyzed by observing the subcutaneous tumor formation and tumor metastasis to distant organs in nude mice after 5-8 weeks.Results:1.Rt-qpcr and Western blot resultsAfter MKN45 and BGC823 gastric cancer cells were transfected with small interfering RNA(siRNA),VEGFR3 mRNA and protein expression levels were significantly lower than those of the control group.However,mRNA and protein expression levels of VEGFR3 were significantly higher after transfection of MGC803 gastric cancer cells than those of the control group.2.Effects of VEGFR3 on the biological function of gastric cancer cellsIn vitro cytological experiments showed that knockdown of VEGFR3(siRNA transfected with VEGFR3)significantly inhibited the proliferation,migration and invasion ability of gastric cancer cells and the transformation of cell cycle from G1 phase to S phase,promoting the apoptosis of gastric cancer cells.While VEGFR3 overexpression(i.e.,transfected overexpression plasmid)promoted the proliferation,migration and invasion ability of gastric cancer cells,promoted the transformation of cell cycle from G1 phase to S phase,and inhibited the apoptosis of gastric cancer cells.In vivo animal experiments showed that after VEGFR3 knockdown,the growth rate of the subcutaneous tumor in nude mice was significantly reduced,the size and weight of the subcutaneous tumor were significantly smaller than those in the control group,and the enveloped invasion was also reduced.In the tail vein model,knockdown VEGFR3 expression was associated with a significant reduction in the number and size of metastatic tumors in the lung.Conclusion:The expression of VEGFR3 was positively correlated with the proliferation,invasion and migration of gastric cancer cells in vivo and in vitro.The expression of VEGFR3 was negatively correlated with the apoptosis of gastric cancer cells in vitro.The effects on the cell cycle of gc focus on the Gl-GS phase.Part ? Molecular mechanism of VEGFR3 effect on gastric cancer cell functionObjective:To investigate the effect of VEGFR3 on the proliferation,invasion and apoptosis of gastric cancer cells.Methods:Wesetern blot was used to detect the expression of related genes in the PI3K/AKT pathway,the epithelium mesenchymal transformation pathway and the endogenous apoptosis pathway in the gastric cancer cell lines transfected with VEGFR3.Results:Knockdown and/or overexpression of VEGFR3 in gastric cancer cells had little effect on the protein expression of PI3K/AKT,but positively correlated with the protein expression level of phosphorylated PI3K/AKT.After VEGFR3 depletion,n-cadherin and Vimentin expression were down-regulated and E-cadherin and Vimentin expression were up-regulated.However,after VEGFR3 overexpression,e-cadherin expression was down-regulated and N-cadherin and Vimentin expression were up-regulated.After VEGFR3 knockdown,BLC2 protein expression was significantly decreased,while that of BAX,Parp,Caspase3 protein was significantly increased.After VEGFR3 overexpression,BAX,Parp,Caspase3 protein expression was significantly decreased,while that of BCL2 protein was significantly increased.Conclusion:VEGFR3 affects the biological function of gastric cancer cells by affecting the phosphorylation of PI3K/AKT signaling pathway.The migration and invasion of gastric cancer cells were influenced by the expression of genes in the epithelial-mesenchymal transformation signaling pathway.The apoptosis of gastric cancer cells was influenced by BAX/BCL2 and Capase3/PARP.Part ? Differential gene and signal pathway analysis in gastric cancer cells after VEGFR3 knockdown expressionObjective:The methylation status of VEGFR3 gene promoter in early gastric cancer tissues and cells was detected,and the clinicopathological relationship between the methylation status and the expression of VEGFR3 in patients with early gastric cancer was analyzed,so as to explore the epigenetic regulation mechanism affecting VEGFR3 expression.Methods:1.The mRNA genes in gastric cancer cells transfected with VEGFR3 were analyzed by second-generation sequencing to screen out the differentially expressed genes and signal pathways with significant influence.2.Rt-qpcr and Western blot were used to detect the expressions of related genes in the selected signaling pathways in gastric cancer cells with VEGFR3 knockdown.Results:1.The second-generation sequencing results showed that 729 up-regulated genes and 803 down-regulated genes were found in gastric cancer cells after VEGFR3 knockdown.KEGG enrichment analysis showed that the difference genes were concentrated in Notch signaling pathway.2.Rt-qpcr and Western blot results showed that CREBBP,DLL4,Hey 1,MAML1 and Nrarp genes were down-regulated in gastric cancer cells(MKN45 and BGC823)after VEGFR3 knockdown.This indicates that downregulation of VEGFR3 has an inhibitory effect on Notch signaling pathway.Conclusion:VEGFR3 knockdown has a significant effect on Notch signaling pathway in MKN45 gastric cancer cells,and VEGFR3 knockdown has a certain inhibitory effect on Notch signaling pathway.Part ? Relationship between methylation and expression of VEGFR3 promoter in gastric cancerObjective:By detecting the methylation status of VEGFR3 gene promoter in early gastric cancer tissues and cells,the clinicopathological relationship between the methylation status and the expression of VEGFR3 in gastric cancer patients was analyzed,so as to explore the epigenetic regulation mechanism affecting VEGFR3 expression.Methods:MSP technique was used to detect the methylation status of VEGFR3 gene promoter in early gastric cancer tissues and their paired normal gastric mucosal tissues,and the clinicopathological relationship between VEGFR3 gene promoter methylation and early gastric cancer was analyzed.MSP,RT-QPCR and Westernblot were used to detect the promoter methylation status of VEGFR3 and its mRNA and protein expression levels before and after treatment with 5-Aza-CDR at different concentrations in gastric cancer cell line(MGC823).Results:The methylation frequency of VEGFR3 promoter in early gastric cancer tissues(48%)was significantly lower than that in normal gastric mucosa(85%),and the high expression of VEGFR3 protein was closely related to the hypomethylation of VEGFR3 gene promoter.The methylation status of VEGFR3 promoter was associated with lymph node metastasis(P<0.05),but not with gender,age,tumor size,degree of differentiation,or depth of tumor invasion(P>0.05)After treatment with 5-Aza-CDR at 10 mol/L for gastric cancer cell line(MGC823),abnormal methylation of VEGFR3 promoter was reversed,and mRNA and protein expression of VEGFR3 were increased.Conclusion:Hypomethylated status of VEGFR3 promoter is the main mechanism of VEGFR3 overexpression in gastric cancer tissues,and is related to lymph node metastasis in patients with gastric cancer.Using 5-Aza-CDR can reverse the methylation status of gastric cancer cells and increase the mRNA and protein expression of VEGFR3 gene.