Silencing Of IQGAP1 On The Expression Of ERK Gene In Tongue Squamous Cell Carcinoma Cell,and The Effects Of Proliferation,Invasion,and Migration

Scaffold proteins are the key regulators of signaling pathways,playing important roles in the immune system.The IQ domain Ras GTPase-activating-like protein(IQGAP1)is a highly conserved cytoplasmic scaffold protein that regulates a variety of signaling pathways.The IQGAP1 scaffold protein is a multifunctional scaffold protein,binding to more than 100 other proteins.IQGAP1 has two closely related family proteins,IQGAP2 and IQGAP3,which have different functions.IQGAP1 is involved in skeletal reorganization and regulates cell adhesion,polarity and migration.IQGAP1 also regulates RAF and MEK via the IQ domain and the proline-rich WW domain to modulate RAF/MEK/ERK signaling.There is evidence that IQGAP1 is an important regulator of MAPK and Wnt/p-catenin signaling pathways.Recent studies have shown that IQGAP1 plays a key role in tumorigenesis.IQGAP1 is overexpressed in esophageal squamous cell carcinoma,and IQGAP1 knockdown can reduce cell proliferation and metastasis in vitro and in vivo.Furthermore,studies reported the AKT and ERK phosphorylation levels are up-regulated in cancer cells overexpressing IQGAP1.However,the role and mechanism of IQGAP1 in tongue squamous cell carcinoma is still unclear.This study investigated the correlation between IQGAP1 and ERK in tongue squamous cell carcinoma.The expression of IQGAP1 and ERK in tongue squamous cell carcinoma cell line SCC25 was detected by qPCR assay.The results showed that both IQGAPI and ERK were highly expressed in tongue squamous cell carcinoma.IQGAP1 and ERK were down-regulated in tongue squamous cell carcinoma when IQGAP1 was silenced.In addition,the biological behaviors of tongue squamous cell carcinoma including proliferation,invasion,migration and apoptosis were inhibited after IQGAP1 was silenced.Chapter Ⅰ Screening the Silencing S1-RNA for IQGAP1 GeneObjcet:To construct effective si-RNA vector for IQGAPlgene and to selected the optimized sequence and cells for the subsequent experiments.Methods:1.Culturing of tongue squamous cell carcinoma cell lines CAL27,SCC25,SCC9;2.Detecting the expression levels of IQGAP1 and ERK in tongue squamous cell carcinoma cell lines by qPCR assay;3.Constructing three different sequence si-RNA vectors to silence the IQGAP1 gene;and transfecting CAL27,SCC25 and SCC9.The expression of IQGAP1 in each group was detected by qPCR assay.4.Detecting the proliferation of each cell line by CCK8 assay.The optimal sequence and cell line were screened based on the CCK8 results for subsequent experiments.Results:1.IQGAP1 was highly expressed in tongue squamous cell carcinoma cell lines,and ERK also showed high expression in tongue squamous cell carcinoma cell lines;2.IQGAP1 showed low expression in CAL27,SCC25 and SCC9 cell lines after silencing IQGAP1 within three different sequences of si-RNA;3.Three different sequences of si-RNA in each cell group showed different degrees of difference in different time periods,which are statistically significant.The sequence coding si-RNA-3746 was found to be more stable in SCC25.Therefore,si-RNA-3746 and SCC25 were selected as the optimal sequence and cell line,respectively,for subsequent experiments.Chapter Ⅱ Effects of silencing of IQGAPI gene on expression of ERKgenein SCC25Object:To investigate whether there is a link between IQGAP1 and ERK,which both are highly expressed in the tongue squamous cell carcinoma.Methods:1.Silencing the IQGAP1 gene and verifying the expression levels of IQGAP1 and ERK genes by qPCR assay;2.Verifying the expression levels of IQGAP1 gene and ERK gene by qPCR assay after knockout of IQGAP1.Results:1.The results of qPCR assay showed that both the expressions of IQGAPI and ERK genes in SCC25 cancer cell line were decreased,after IQGAP1 was silenced.2.Results of Western Blot showed that both the expressions of IQGAP1 and ERX genes in SCC25 cancer cell line were decreased after IQGAP1 was silenced.Chapter Ⅲ Effect of Silencing IQGAP1 Gene on proliferation,invasion,and migration of SCC25Object:To investigate effects silencing IQGAPlon tongue squanous cell carcinoma cell lines.Methods:1.Testing cell proliferation on experimental group,negative control group and blank group;2.Investigating the invasiveness of experimental group,negative control group and the blank group by transwell experiment;3.Detecting cell migration in the experimental group,the negative control group and the blank group by wound healing experiment.Results:1.The CCK8 results showed that the proliferation of cancer cells in the experimental group was significantly decreased,the negative control group showed no significant change,and the blank group showed an upward trend.2.Results of Transwell assay showed that the cancer cells in the experimental group had no obvious invasiveness,while the cancer cells in both the negative control group and blank group were invasive.3.Woundhealing experimental test results showed that after 30h,the migration rate of cancer cells in the experimental group was significantly lower than that of the negative control group and the blank group.Conclusions1.Both IQGAP1 and ERK genes are highly expressed in tongue squamous cell carcinoma cell.IQGAP1 is associated with ERK,as ERX expression is decreased when the IQGAP1 gene is silenced;2.When the IQGAP1 gene was silenced in SCC25 cells,the expression of ERK gene was down-regulated,and the proliferation,invasiveness and migration ability of cancer cells decreased.3.The IQGAPI gene may be a potential new target for the treatment of tougue squamous cell carcinoma.