The Effects And Mechanism Of Scaffolding IQGAP1 On The Angiogenesis Of Human Esophageal Carcinoma Cells

Objective:1.Establish the stable cell lines of IQGAP1 gene overexpression and the cell lines of negative control;Establish the stable cell lines of IQGAP1 gene interference and its control cell line.2.The effects of IQGAP1 gene expression on angiogenesis were detected through vivo and vitro experiments.3.To further explore the mechanism of scaffold protein IQGAP1 on angiogenesis.Method:1.Transfercting the vector containing IQGAP1 gene labeled with green fluorescent protein into human esophageal cancer cell line EC9706 by Lipofectamin 2000,and G418 was used to obtain drug-resistant clones,then the stable transfected cell line would achieved after amplification.Cell localization was observed under fluorescence microscope,and the expression of GFP-IQGAP1 fusion protein was detected by western blot to determine whether IQGAP1 overexpression stable cell line was constructed successfully.2.KSYE150 cells were also transfected the plasmids which containing si RNA targeting IQGAP1 gene by Lipofectamine 2000,then we would obtained the drug-resistant clones by G418,later we would achieved the stable-transfected cell line after amplification.The expression of GFP in the cells was observed under fluorescence microscope.In interference group and its control group we used western blot to detect the expression of IQGAP1 to determine whether the IQGAP1 interference stable cell line was constructed successfully.3.By in vitro HUVEC tube formation assay and in vivo chick chorioallantoic membrane assay to analysis the effects of IQGAP1 on tumor angiogenesis.4.To detect the expression of angiogenesis related factors VEGFA、VEGFR2 and p-VEGFR2 in esophageal carcinoma cells by Western blot.5.To study the effect of IQGAP1 on the activation of Akt and ERK by western blot.6.Detecting the effects of AKT and ERK inhibitors on the expression of VEGFA and p-VEGFR2 in the over expression of IQGAP1 cells and its influence on angiogenesis.Results:1.Transfected the recombinant plasmid GFP-IQGAP1 could be observed in cytoplasm in EC9706 cells by fluorescence microscope;The results of Western blot showed that the expression of GFP-IQGAP1 fusion protein in IQGAP1 overexpressing cells,while in the control group cells do not.It can be proved that the overexpression cell lines of IQGAP1 have been successfully constructed.2.Fluorescence microscopy showed that GFP was found to be whole cell localization in KYSE150 cells which transfected with IQGAP1 interference plasmid and control plasmid.The expression of IQGAP1 protein was obviously reduced in interference group by western blot than its control group,and the difference was statistically significant by statistical analysis(P<0.0001).3.The results of in vitro HUVECs tubule formation showed that,compared with the control group,the number of tubules in IQGAP1 overexpression group was more,and the lumen of the tubules was more complete,and the difference was statistically significant(P<0.0001);above results indicated that overexpression of IQGAP1 could promote angiogenesis in vitro.Chorioallantoic membrane assay showed that the number of blood vessels in the IQGAP1 overexpression group was increased compared with the control group,and the vascular was more morphology stretch,the difference was statistically significant(P<0.001);these results suggested that overexpression of IQGAP1 could promote angiogenesis in vivo.The experiments through vitro and vivo both showed that overexpression of IQGAP1 could promote tumor angiogenesis.4.Using the same experimental method,the result of HUVEC tubular formation assay showed that,compared with the control group,the number of tubules in the interference of IQGAP1 group was less,and the lumen of the tubules was incomplete,and difference was statistically significant(P<0.0001);above result indicated that IQGAP1 interference can inhibit angiogenesis in vitro.In the chick chorioallantoic membrane assay showed the number of blood vessels in the IQGAP1 interference group was less than that in the control group,and the main blood vessel was less,and difference was statistically significant(P<0.001).Above results suggested that IQGAP1 interference can inhibit angiogenesis in vivo.The results are consistent with the in vivo and in vitro experiment,which indicate that interference of IQGAP1 gene expression can inhibit the formation of tumor angiogenesis.5.The expression of VEGFA and p-VEGFR2 proteins were increased in IQGAP1 overexpressing cells by Western blot assay,we can find the difference was statistically significant(P<0.0001);At the same time,the expression of VEGFR2 was not significantly different between the IQGAP1 overexpression group and the control group.The results indicated that IQGAP1 could promote the phosphorylation of VEGFR2 and further promote the production of blood vessels by the interact between VEGFA and VEGFR2.6.In the IQGAP1 interference group,the expression of VEGFA and p-VEGFR2 proteins were lower than that in the control group by Western blot assay,we can find that difference was statistically significant(P<0.0001);At the same time,we found the expression of VEGFR2 was not significantly different between the IQGAP1 interference group and the control group.These results suggest that IQGAP1 inhibit angiogenesis may through the way of inhibiting the interaction between VEGFA and VEGFR2.7.Western blot showed that,compared with the control group,the expression of p-Akt was significantly increased when IQGAP1 over expression,also the p-ERK,and the differences were statistically significant(P<0.0001);However,the expression of p-Akt and p-ERK in the IQGAP1 interference group were decreased significantly than those of its control group,we found the differences were statistically significant(P<0.0001).These results suggest that the mechanism of IQGAP1 on angiogenesis of tumor cells may play a role in the activation of Akt and ERK signaling pathway.8.The expression of VEGFA and p-VEGFR2 proteins were significantly decreased after Akt and ERK inhibitors were added into the IQGAP1 overexpressing cells;Moreover,the numbers of angiogenesis were decreased after intervention of inhibitor;This phenomenon indicates that IQGAP1 may influence tumor angiogenesis through Akt or ERK/VEGFA-VEGFR2 signaling pathway.Conclusion:1.The stable cell line of IQGAP1 overexpresson and the stable cell line of IQGAP1 interference were successfully constructed.2.Overexpression of IQGAP1 can promote angiogenesis in vitro and in vivo;Interfering the expression of IQGAP1 can inhibit the angiogenesis of tumor.3.The mechanism of IQGAP1 on angiogenesis in esophageal carcinoma may be related to the activation of VEGFR2 by the interaction between VEGFA and VEGFR2,as well as the activation of Akt and ERK signaling pathway.4.After the intervention of Akt and ERK inhibitors in IQGAP1 overexpression cells may block the Akt or MEK/VEGFA-VEGFR2 signaling pathway,which leads to inhibition of angiogenesis.