| Objective(1) To detect the expression of RhoC and IQGAP1 (IQ motif-containing GTPase activating protein 1) in gastric cancer tissue and gastric cancer cell lines, and analyze the correlation between RhoC expression and IQGAP1 expression, and the correlation between the expression of two proteins and the parameters of clinical pathology.(2) To identify the effect of RhoC and IQGAP1 on the proliferation and migration of gastric cancer cells, and to investigate the possible interaction between RhoC and IQGAPl.Methods(1) Recombinant adenovirus encoding IQGAP1, IQGAP1-C and constitively active RhoC gene were amplified in 293A cells. Western blotting was applied to detect the expression of target proteins. The cell lines with high sensitivity to the adenovirus were chosen for proliferation and migration assay.(2) RT-PCR, Western blotting and immunofluorescence assay were used to detect the expression of RhoC and IQGAP1 in gastric cancer tissue and cell lines. The correlation between RhoC and IQGAP1 was analyzed by Spearman's rank correlation analysis. Fisher's Exact Test was applied to analyze the correlation between expression of these proteins and parameters of clinical pathology.(3) The gastric cancer cell lines were infected with adenovirus encoding constitively active RhoC to increase the activity of RhoC. The proliferation effect of RhoC was analyzed by MTT method, and the migration effect was measured by transwell migration assay. (4) The plasmid encoding IQGAP 1, IQGAP 1-C or IQGAP 1-N was co-transfected with Green Fluorescent Protein (GFP) plasmid into BGC-823 cells. BrdU incorporation assay was used for detecting the effect of different structures of IQGAP1 on proliferation.(5) Adenoviral constructs encoding the full length IQGAP 1 and the C-terminal fragment of IQGAP1 gene were used to infect gastric cancer cells to increase the expression of these proteins. MTT method was applied to analyze the proliferation effect of IQGAP 1 and IQGAP 1-C, and the migratory activity was measured by a transwell migration assay.(6) The siRNA targeting RhoC or IQGAP 1 were transfected into gastric cancer cells to suppress the expression of these proteins. MTT method and transwell migration assay were applied to observe the influence of RhoC siRNA and IQGAP 1 siRNA on proliferation and migration of gastric cancer cells.(7) Western blotting was used to investigate the influence of RhoC and IQGAP1 on the expression of ICAM-1, vimentin, and MMP-2 in gastric cancer cells.(8) On one hand, gastric cancer cells were transfected with siRNA to interfere the expression of RhoC, and then infected with adenovirus encoding IQGAP 1, IQGAP 1-C to observe the influence on cell proliferation and migration. On the other hand, gastric cancer cells were transfected with siRNA suppressing the expression of IQGAP1, and then infected with adenovirus encoding RhoC-V14 to observe the effect on cell proliferation and migration.(9) COS-7 and BGC-823 cells were co-infected with pAd-IQGAP1 or pAd-IQGAP1-C and pAd-RhoC-V14. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. Results(1) In 22 patients (75.9%), the expression of RhoC and IQGAP1 in gastric cancer tissue was higher than that in adjacent non-neoplastic mucosa tissues. Spearman's rank correlation analysis indicated that the increases of IQGAP1 and RhoC were closely correlated. The expression of two proteins were reversely correlated with the differentiation of the gastric cancer tissues (P<0.05, P<0.05), and there were also some relevance with proliferation and migration, but the differences were not statistically significant.(2) Adenovirus encoding IQGAP1, IQGAP1-C and constitively active RhoC gene were successfully constructed and amplified. Western blotting showed that the RhoC, IQGAP1 and IQGAP1-C proteins were highly expressed in gastric cancer cells infected with above adenovirus. On account of the strong proliferation capacity and migration activity in BGC-823 and AGS and their sensitivity to the adenovirus vector, the two cell lines were chosen for the following study.(3) Both IQGAP1 and RhoC protein expression levels were higher in the gastric cancer cell lines BGC-823, AGS and SGC-7901 as compared with the human gastric epithelial cell line GES-1, and IQGAP1 was mainly located in cell membrane and cell-cell junctions.(4) RhoC significantly stimulated the migration and proliferation activity of gastric cancer cell lines, and depletion of RhoC by siRNA inhibited the traits.(5) The increased expression of full length IQGAP1 (by infecting with pAd-IQGAP1) or the C-terminal fragment of IQGAP1 (by infecting with pAd-IQGAP1-C) accelerated cell migration and proliferation. By contrast, reduction of endogenous IQGAP1 by siRNA attenuated cell migration and proliferation. (6) Both constitutively active RhoC and IQGAP1 could increase the expression of vimentin and MMP-2, but reduce the expression of ICAM-1.(7) The depletion of IQGAP1 expression by siRNA significantly blocked the migration and proliferation enhanced by constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced migration and proliferation in gastric cancer cells.(8) Co-IP and Immunofluorescence assays showed that RhoC and IQGAP 1 bound to each other.ConclusionsThe high expressions of IQGAP 1 and RhoC are closely related, and the expressions of two proteins are reversely correlated with the differentiation of the gastric cancer tissues. In gastric cancer cells, the overexpression of IQGAP 1, IQGAP 1-C and constitutively active RhoC could promote cell migration and proliferation. IQGAP 1 and RhoC bind to each other, and IQGAP 1 is a down-stream effector of RhoC in regulating the migration and proliferation activity of gastric cancer cells.
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