The Function And Mechanism Of ARHGEF7 Targeting Wnt/?-catenin Signaling Pathway To Promote Invasion And Migration Of Hepatoma Cells

Background and purpose:Hepatocellular carcinoma?HCC?is one of the most common malignant tumor with the biological characteristics of high invasion and metastasis in the world.Recent studies indicate that ARHGEF7 may play a critical role in HCC.Our early study have already confirmed that the liver cancerous tissues have the highest mRNA and protein expression level of ARHGEF7 compared with normal liver tissues and the tissues adjacent to carcinoma.Furthermore,we found that the mRNA and protein expression level of ARHGEF7 in HCCLM3 cell lines are observably higher than that in any other non-metastatic hepatoma cell?Huh7,Hep3B,HepG2,etc?.Subsequently,we constructed sh-RNA mediated inhibition of ARHGEF7 by lentivirus.In this study,the biological functions of ARHGEF7 in hepatoma cell was observed by us,which will provid a foundation for the subsequent experiment in vivo and the mechanism of ARHGEF7 promoting the invasion and metastasis of HCC.Methods:1.The optimal multiplicity of infection?MOI?and infection time were determined:different MOI of LV-GFP-RNAi were used to infect HCCLM3 cells,then observe the number of fluorescent cells per 24h by fluorescence microscope.2.HCCLM3 cells were divided into three groups:control groups,LV-GFP-RNAi groups and LV-ARHGEF7-RNAi groups,then the ARHGEF7 mRNA and protein level were examined by qRT-PCR and Western blot.3.Cell migration were observed by scratch test.Cell invasion were observed by Transwell.Cell proliferation were observed by MTT and clone formation assay.Results:1.We observed the maximum fluorescent cells when the HCCLM3 cells were infected by LV-GFP-RNAi(MOI=5×10⁵ v.g./cell)for 72h.Consistently,the ARHGEF7 mRNA and protein level were also showed prominent decrease compared to control and LV-GFP-RNAi groups?P<0.01?.2.Scratch test showed that the LV-ARHGEF7-RNAi group perform a marked decrease in cell migration compared to the controlandvectorgroups.3.Transwellindicatedthatthe LV-ARHGEF7-RNAi group perform a remarkable decrease in cell invasion compared to the control and vector groups.4.MTT and clone formation assay revealed that there is no significant difference in cell proliferation among LV-ARHGEF7-RNAi group,the control and vector groups.Conclusion:Slience of ARHGEF7 could dramatically inhibit cell migration and invasion,but not cell proliferation in HCC.Background and purpose: Rho family acts as a molecular in some of he basic features of the cells,involving cell motility,ttumor invasion and malignant transformation,regulation of transcription factors and tumor angiogenesis,etc.Rac-the subfamily of Rho including many family members,such as Rac1,Rac2,Rac3 and shear mutant of Rac1-Rac1 b.Rac1 is a important signal transduction molecule in the cell.Studies have shown that Rac1 can promote ?-catenin into the nuclear by phosphorylatng ser191 and ser605 locus of ?-catenin,which subsequently activate the downstream target genes of Wnt/?-catenin signaling pathway relate with cell migration and invasion.Interestingly,ARHGEF7 also plays an important role in HCC,which may regulate multiple signaling transduction pathways,while,the mechanism of ARHGEF7 between signaling pathways has not been addressed thus far.Given that we investigate the possible relationship between ARHGEF7 and Rac1,as well as Wnt/?-catenin.Here,we preliminarily discuss the mechanism of ARHGEF7 promoting the invasion and metastasis.Methods: 1.The m RNA and protein level of GTP-Rac1 and total-Rac1 were checked by q RT-PCR and Western blot in LV-ARHGEF7-RNAi,LV-GFP-RNAi and control group.2.The protein level of ?-catenin as well as the downstream target genes?C-myc?Cyclin D1 and MMP-7?of Wnt/?-catenin signaling pathways were determined by Western blot in LV-ARHGEF7-RNAi,LV-GFP-RNAi and control group.3.After be treated with the Rac1 agonist FMLP,the protein level of ?-catenin as well as the downstream target genes?C-myc?Cyclin D1 and MMP-7?of Wnt/?-catenin signaling pathways were determined by Western blot in LV-ARHGEF7-RNAi,LV-GFP-RNAi and control group.Results : 1.The m RNA and protein level of GTP-Rac1 in LV-ARHGEF7-RNAi group were observably down-regulated compared to the control and vector groups,although the expression of total-Rac1 had no significant difference among the three groups?P<0.01?.2.The protein level of ?-catenin as well as the downstream target genes?C-myc?Cyclin D1 and MMP-7?of Wnt/?-catenin signaling pathway were signally down-regulated compared to the control and vector groups.3.The Rac1 agonist FMLP could ablate the inhibition of ?-catenin as well as the downstream target genes?C-myc?Cyclin D1 and MMP-7?of Wnt/?-catenin signaling pathways in LV-ARHGEF7-RNAi HCCLM3.Conclusion: ARHGEF7 promote the invasion and metastasis of HCC via activating the Rac1/Wnt/?-catenin signaling pathway.