Abstract
Our previous research results showed that both Ras homolog family member C (RhoC) and IQ-domain GTPase-activating protein 1 (IQGAP1) were over-expressed in gastric cancer tissues and cells, but their role in tumorigenensis has not been addressed clearly. Herein we reported the proliferation stimulating effect of RhoC and IQGAP1 on gastric cancer cells and the interaction between two proteins in regulating the proliferation of gastric cancer cells. Plasmids and viral constructs encoding target siRNA and DNA were used to alter the expression of RhoC and IQGAP1. MTT method and BrdU incorporation assay were used for analyzing the effect of RhoC and different structures of IQGAP1 on proliferation. Protein levels of IQGAP1 and RhoC in cell lines were detected by Western blotting. Immunofluorescence and Co-Immunoprecipitation assays were applied to investigate the localization and binding between RhoC and IQGAP1. The results showed that RhoC, IQGAP1 and the C-terminal fragment of IQGAP1 significantly stimulated the proliferation of gastric cancer cells, and enhanced the expression of cyclin E and cyclin D1. By contrast, reduction of endogenous IQGAP1 or RhoC by siRNA attenuated cell proliferation. The depletion of IQGAP1 expression by siRNA significantly blocked the proliferative activity of constitutively active RhoC, while RhoC silencing by siRNA had no effect on IQGAP1-induced proliferation in gastric cancer cells. Co-immunoprecipitation and Immunofluorescence assays showed that RhoC and IQGAP1 bound each other. In conclusion, our results suggest that RhoC stimulates the proliferation of gastric cancer cells through recruiting IQGAP1 as an effector.
Highlights
Rho GTPases can induce certain intracellular signal transduction and impact various cellular activities, including reorganization of the actin cytoskeleton, gene transcription, survival, and proliferation [1,2]
Interference of IQ-domain GTPase-activating protein 1 (IQGAP1) expression with siRNA reduced cell proliferation by 43%, compared with negative control (*P,0.05, Fig. 2C and D). These results indicated that IQGAP1 was able to accelerate cell proliferation; the active domain was located in the C-terminal fragment of the protein
In order to elucidate the possible association between Ras homolog family member C (RhoC) and IQGAP1 in regulating cell proliferation, we firstly investigated if IQGAP1 and RhoC affect their expression each other
Summary
Rho GTPases can induce certain intracellular signal transduction and impact various cellular activities, including reorganization of the actin cytoskeleton, gene transcription, survival, and proliferation [1,2]. Three Rho isoforms, RhoA, RhoB, and RhoC, exhibit more than 85% amino acid identity, they confer differences in function in cells [3]. The results from Pile laboratory indicated that RhoA and RhoC siRNA represent powerful tools for inhibiting cancer cell proliferation, invasiveness, and angiogenesis both in vitro and in vivo [9]. Sun et al found that intratumoral injection of RhoA or RhoC siRNA to nude mice can inhibit tumor growth [13]. A study by Ikoma and colleagues showed that RhoC did not affect tumor growth but enhances the metastatic nature of lung cancer by stimulating cell motility [10]. Further study is needed to identify the role of RhoC in regulating biological activities of tumor cells
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