RasGRF2 Promotes Migration And Invasion Of Colorectal Cancer Cells By Modulating Expression Of MMP9 Through Src/Akt/NF-?B Pathway

Background:Ras family GTPases regulate a wide variety of cellular processes including cell proliferation,apoptosis,differentiation and an array of differentiated functions unique to specific cell types.Ras GTPases exist in two conformations,GTP bound(active)and the GDP bound(inactive)states.Activation of Ras into its GTP-bound conformation is regulated by proteins known as Ras-specific guanine-nucleotide releasing factors(RasGRFs).One of the members of RasGRFs,RasGRF2 was found to be abnormally expressed in various cancers.However,the role of RasGRF2 in cancer is rarely reported,especially in colorectal cancer(CRC).Therefore,exploring the function of RasGRF2 in CRC and its related molecular mechanisms is helpful for further understand the process of tumor development.Objective:To investigate the expression of RasGRF2 in CRC clinical samples and CRC cells.To study the effects of RasGRF2 on CRC cell functions,including proliferation,apoptosis,migration and invasion.And to explore the molecular mechanisms of RasGRF2 on CRC cell functions.Methods:1.RasGRF2 expression in CRC tissues and CRC cellsReal-time quantitative PCR and Western Blot were performed to detect the expression of RasGRF2 in 26 paired tumor and non-tumor colorectal tissues after CRC surgical resection.Western Blot was used to measure the protein level in one normal colorectal cell line FHC and four CRC cell lines(SW480,HCT116,LS174 T and LoVo).Tissue microarrays were used to determine RasGRF2 protein level in 97 paired paraffin-embedded CRC tissues collected from 2005 to 2010.2.The effect of RasGRF2 knockdown on CRC cell functionsThe human rasgrf2 gene shRNA interference lentiviral vector was constructed,and the rasgrf2 gene was silenced in CRC cell lines(SW480,HCT116 and LS174T).The knockdown efficiency was evaluated by RT-qPCR and Western Blot.Then we analyzed the effect of RasGRF2 knockdown on CRC cell functions,including cell proliferation ability by MTT assay,apoptosis analysis by Annexin V-APC/7-AAD flow cytometry,cell migration and wound healing assay by Transwell,as well as invasion ability by Matrigel-Transwell.3.Molecular mechanism of RasGRF2-mediated CRC migration and invasionWestern Blot was used to detect EMT molecular markers(?-catenin,Vimentin),MMP9 and FAK/Src,Akt,NF-?B,Erk signaling pathway-related proteins.Results:We found that rasgrf2 mRNA and protein were increased in CRC tissues,compared to adjacent non-tumor tissues.The expression level of rasgrf2 mRNA and protein were also raised with comparison of FHC cell line.Silencing of rasgrf2 in CRC cells reduced their migration and invasion without affecting proliferation and apoptosis.RasGRF2 knockdown did not alter ?-catenin and Vimentin expression,but inhibited the activation of FAK/Src,Akt,NF-?B and Erk signaling pathway,and also impaired the expression of MMP9.Conclusion:RasGRF2 plays a role in controlling migration and invasion of CRC cells by modulation of the expression of MMP9 through Src/Akt/NF-?B pathways.This effect may not be associated with EMT.