Effect And Mechanism Of VEGFR2 On Promoting Growth And Metastasis Of Gastric Cancer By Regulating VTN

Part I Expression of VEGFR2 and VTN in gastric cancer and its relationship with clinical and prognosisObjective:To investigated the expression of Vascular Endothelial Growth Factor Receptor 2(VEGFR2)and Vitronectin(VTN)in gastric cancer,and to investigated the relationship between the expression of VEGFR2 and VTN and its clinical features and prognosis.Methods:1、During December January 2010 to 2012 in the First Affiliated Hospital of Soochow University and Suzhou Xiang cheng people’s hospital accepted surgical treatment of 156 cases of gastric cancer patients,used immunohistochemistry to detect the expression of VEGFR2 and VTN in gastric cancer tissue.2、The patients were divided into two groups with high and low expression of VEGFR2 and VTN.3 、 Analysis of the relationship between VEGFR2 and VTN in patients with clinical pathological data and the correlation with prognosis.The prognostic significance in relation to VEGFR2 and VTN together with various clinicopathological factors was evaluated by multivariate analysis.Results:1、There was no significant relationship between VEGFR2 expression and gender(P=0.331),age(P=0.427),Tumor size(P=0.153),Lauren type(P=0.739)and tumor differentiation(P=0.912),but it was related with T staging(P<0.001),N staging(P<0.002)and disease stage(P<0.001).There was no significant relationship between VTN expression and gender(P=0.305),age(P=0.494),Tumor size(P=0.120),Lauren type(P=0.826)and tumor differentiation(P=0.687),but it was related with T staging(P<0.003),N staging(P<0.003)and disease stage(P<0.001).2、The median value of VTN in the high expression group of VEGFR2 was 9,which was significantly higher than that of VEGFR2 low expression group(P<0.001).All patients were followed up for 36 months at least.The median OS was 42 months and median DFS was about 36 months in 156 patients with胃癌.The medium OS for the higher and lower expression of VEGFR2 groups were 35 and 48 months respectively(χ2=4.158,P=0.041).The medium DFS for the higher and lower expression groups were 28 and 40 months respectively(χ2=4.346,P=0.037).The group of VEGFR2low expression had longer OS and DFS.The medium OS for the higher and lower expression of VTN groups were 34 and 52 months respectively(χ2=4.223,P=0.040).The medium DFS for the higher and lower expression groups were 26 and 44 months respectively(χ2=4.114,P=0.043).The group of VTN low expression had longer OS and DFS.3、Multivariate Cox regression analysis revealed that tumor stage and the expression of VEGFR2 and VTN are prognostic factors affecting DFS and OS.Conclusion:The expression of VEGFR2 and VTN can provide important diagnostic and prognostic results in patients with gastric cancer.Part II The mechanism of VEGFR2 involved in malignant biological behavior of gastric cancerObjective: To investigate the expression of vascular endothelial growth factor receptor 2(VEGFR2)in different gastric cancer cell lines and the effect of VEGFR2 gene expression on the proliferation and invasion of gastric cancer cells and the growth of tumor in vivo.Our present study may provide a basis for further development of targeting VEGFR2 as a promising therapeutic agent for the treatment of gastric cancer.Methods: 1、The expression of VEGFR2 in different gastric cancer cell lines was detected by real-time quantitative PCR and Western blotting.2、Construction of VEGFR2-sh RNA targeting VEGFR2 and VEGFR2 overexpression of eukaryotic expression plasmid VEGFR2-pc DNA3.1.3、The effect of regulation of VEGFR2 expression on proliferation of gastric cancer cells was investigated using CCK-8 colorimetry.4、The effect of regulation of VEGFR2 on invasion of gastric cancer cells was investigated using Transwell invasion assay.5、The effect of VEGFR2 expression on the adhesion of gastric cancer cells was investigated using adhesion test.6、The effect of VEGFR2 expression on the cell cycle of gastric cancer was investigated using flow cytometry.7、The effect of regulation of VEGFR2 expression on the growth of implanted tumor in vivo as investigated using imaging technique in vivo.Results: 1、VEGFR2 m RNA and protein expression were significantly up-regulated in gastric cancer cells.2、The expression of VEGFR2 m RNA and protein in VEGFR2-sh RNA transfection group was significantly decreased in MKN-45 cells.3、The down-regulation of VEGFR2 could significantly inhibit the proliferation,invasion,adhesion and arrest the cell cycle in G2 / M phase in MKN-45 cells.4、The overexpression of VEGFR2 m RNA and protein in VEGFR2-pc DNA3.1 transfection group was significantly up-regulated in SCH cells.5 、 The overexpression of VEGFR2 significantly promoted the proliferation,invasion,adhesion and increased the number of cells in G0 / G1 phase and S phase in SCH cells.6、Down-regulation of VEGFR2 expression can significantly inhibit the growth of tumor in vivo.7、Overexpression of VEGFR2 expression can significantly promote the growth of tumor in vivo.Conclusion: The expression of vascular endothelial growth factor receptor 2(VEGFR2)in gastric cancer cells was significantly up-regulated.Over-expression of VEGFR2 gene plays a key role in the process of invasion and metastasis of gastric cancer,which is of great significance to promote invasion and metastasis of gastric cancer.Part III Bioinformatics Study on downstream signaling pathways effects by interfering with VEGFR2Objective: To investigate the effect of downstream signaling pathways by interfering with VEGFR2.Methods: The downstream signaling pathways were observed by interfering with VEGFR2 by human expression profiling,and the valuable downstream molecules were screened by bioinformatics studies.Results: 1、Gene chip data is reliable and reproducible,and there is a great difference between KD group and NC group.2、Gene chip data showed that 37 genes were up-regulated and 19 genes were down-regulated.3、Classic pathway analysis showed that NGF Signaling was significantly enriched in the first signal pathway.Upstream regulatory factor analysis showed that MAPK1 was predicted to be strongly activated and IFNA2 was predicted to be strongly inhibited.The function of obvious activation was as follows: cell death of connective tissue cells,-1.583, cell death of fibroblasts,-1.486,etc.The analysis of the interaction network shows that the first network diagram mainly affects Endocrine System Development and Function,Cardiovascular System Development and Function,Tissue Development.And predicting that VTN is a valuable downstream signaling molecule.The above is the result of bioinformatics analysis.Conclusion: It is suggested that the downstream of the target gene regulates the NGF Sign signaling pathway on MKN-45 human gastric cancer cells and regulates the downstream signal molecule VTN and suggests that the gene is involved in the pathogenesis and function of binding of endothelial tissue,glucose tolerance and cell death of connective tissue cells.PartⅣ VEGFR2 promotes the growth and metastasis of gastric cancer by regulating VTNObjective: To investigate the mechanism of VEGFR2 promoting the growth and metastasis of gastric cancer by regulating VTNMethods: 1、The expression of VEGFR2 and VTN in different gastric cancer cell lines was detected by real-time quantitative PCR and Western blotting.2、The expression of VEGFR2 and VTN in gastric cancer cell lines by regulation of VEGFR2 was detected by real-time quantitative PCR and Western blotting.3、The expression of VEGFR2 and VTN in gastric cancer cell lines by regulation of VEGFR2 and VTN was detected by real-time quantitative PCR and Western blotting.4、The effect of regulation of VEGFR2 and VTN expression on proliferation of gastric cancer cells was investigated using CCK-8 colorimetry.5、The effect of regulation of VEGFR2 and VTN on invasion of gastric cancer cells was investigated using Transwell invasion assay.Results: 1、The expression of VEGFR2 and VTN m RNA and protein in gastric cancer cells were consistent.2、The expression of VTN m RNA and protein was significantly decreased in MKN-45 cells by interference of VEGFR2.The expression of VTN m RNA and protein was significantly upgrade in SCH cells by overexpression of VEGFR2.3、The expression of VTN m RNA and protein was significantly decreased in MKN-45 cells by interference of VEGFR2 and VTN.The expression of VTN m RNA and protein was significantly upgrade in SCH cells by overexpression of VEGFR2 and VTN.4 、 The down-regulation of VEGFR2 and VTN could significantly inhibit the proliferation in MKN-45 cells.The overexpression of VEGFR2 and VTN could significantly promote the proliferation in SCH cells.5、The down-regulation of VEGFR2 and VTN could significantly inhibit the invasion in MKN-45 cells.The overexpression of VEGFR2 and VTN could significantly promote the invasion in SCH cells.Conclusion: VEGFR2 regulates the expression of VTN in gastric cancer cells,and VEGFR2 promotes the growth and metastasis of gastric cancer cells by positively regulating VTN.