The Effect Of Scaffold Protein IQGAP1 On Migration,invasion And Expression Of MMP-2 In Esophageal Squamous Carcinoma Cell And Its Mechanism

Objective:1.To investigate the effects of IQGAP1(IQ motif containing GTPase activating protein 1)on the migration and invasion of esophageal squamous cell carcinoma cells.2.To investigate the effect of IQGAP1 on the expression and activity of MMP-2 gene.3.To explore the molecular mechanism by which IQGAP1 affects the migration and invasion of esophageal squamous cell carcinoma cells and the expression and activity of MMP-2 gene.Methods:1.The GFP-IQGAP1 plasmid and control plasmid were transfected into EC9706 cells,and the IQGAP1 short hair pin RNA plasmid and the negative control plasmid were transfected into KYSE150 cells.The establishment of the high expression cell line of IQGAP1 and the knockdown cell line of IQGAP1 gene was verified by Western blot experiment.2.MTT assay and plate cloning assay were used to detect the effect of IQGAP1 overexpression or gene knockdown on the proliferation ability of esophageal squamous cell carcinoma cells.The effects of high expression of IQGAP1 or gene knockdown on migration,migration and invasion of esophageal squamous cell carcinoma cells were detected by wound healing assay and Transwell assay.3.The expression levels of MMP-2 protein and m RNA in cells with high expression of IQGAP1 or knockdown genes were detected by Western blot assay and Real-Time PCR assay.The activity of MMP-2 in cells with high expression of IQGAP1 or knockdown gene was detected by gelatin enzyme assay.4.The influence of IQGAP1 on NF-κB signaling pathway and the influence of PDTC on NF-κB signaling pathway were detected by Western blot assay and cellular immunofluorescence assay.NF-κB inhibitor PDTC was used to treat IQGAP1 cell lines with high expression.The effects of PDTC on migration and invasion of esophageal squamous cell carcinoma cells were detected by wound healing assay and Transwell assay.The effects of PDTC on the expression and activity of MMP-2 in esophageal squamous cell carcinoma cells were detected by Western blot assay,Real-Time PCR assay and gelatin enzyme assay.Results:1.Western blot results showed that the cell lines with high IQGAP1 expression could express the GFP-IQGAP1 fusion protein,while the control cells did not.The expression level of IQGAP1 in knockdown cells was significantly lower than that in control cells.2.The results of MTT assay and plate cloning assay showed that the proliferation abilityof IQGAP1-overexpressing cells was significantly higher than that of control cells(P < 0.05).The proliferation ability of IQGAP1 knockdown cells was significantly lower than that of control cells(P < 0.05).The results of wound healing assay and Transwell assay showed that compared with the control group,cells in the high expression group of IQGAP1 had stronger migration and invasion ability(P < 0.05),while the migration and invasion ability of cells in the knockdown group of IQGAP1 was weakened(P < 0.05).3.Western blot assay,Real-Time PCR assay and gelatin enzyme assay showed that compared with the control group,the cell lines with high IQGAP1 expression had higher MMP-2 expression level and functional activity(P < 0.05).However,the low expression of IQGAP1 cells showed lower MMP-2 expression level and functional activity(P < 0.05).4.Western blot results showed that the expression of NF-κB(p65)in the cell lines with high IQGAP1 expression was increased(P < 0.05).The expression of NF-κB(p65)in IQGAP1 knockdown cell lines was decreased(P < 0.05).Immunofluorescence showed that the nuclear localization level of NF-κB(p65)was increased in the cell lines with high IQGAP1 expression,and decreased in the knockdown cell lines with IQGAP1 gene.Immunofluorescence showed that the nuclear localization level of NF-κB(p65)decreased after PDTC treatment of cells with high IQGAP1 expression.Western blot assay,wound healing assay and Transwell assay showed that the expression of NF-κB(p65)was decreased and the invasion and migration of IQGAP1-overexpressing cells were inhibited after PDTC treatment.Western blot assay,Real-Time PCR assay and gelatin enzyme assay showed that the expression level and functional activity of MMP-2 were inhibited after PDTC treatment in IQGAP1-overexpressing cells.Conclusions:1.IQGAP1 promotes proliferation,migration and invasion of esophageal squamous cell carcinoma cells.2.IQGAP1 promotes the expression and functional activity of MMP-2 in esophageal squamous cell carcinoma cells,suggesting that IQGAP1 may affect the migration and invasion of tumor cells by regulating MMP-2.3.IQGAP1 regulates the migration,invasion ability and the expression and activity of MMP-2 of esophageal squamous cell carcinoma cells through the NF-κB signaling pathway.