| Objective:Research show that, Rho GDI plays a very important role in the regulation of Rho GTPases’ activity. Rho GDI2 is one of the three members of the Rho GDI2 family, which plays different roles in different tumors, such as inhibiting cancer or promoting cancer. Our previous experiments show that, DADS(diallyl disulfide)could cause down-regulation of Rho GDI2, and confirm that DADS may affect the Rac1/LIMK1 pathway, which leads to the inhibition of cell migration and invasion of gastric cancer. Rho GDI2 is located upstream of Rac1/LIMK1 pathway, the purpose of this experiment is to detect the effect of DADS on migration and invasion in Rho GDI2 silence MGC803 cells to confirm that Rho GDI2 may be used as a potential prognostic biomarker and a therapeutic target for gastric cancer.Methods:The eukaryotic expression vector encoding human RhoGDI2 gene sequence was constructed, and we transfected it into MGC803 cells to establish a stable cell line, which stably silenced Rho GDI2 protein. RT-PCR and Western blotting assay were used to test the expressions of Rho GDI2, Rac1, PAK1 and LIMK1 m RNA and protein level. The effect of Rho GDI2 silenced on migration and invasion of MGC803 cells with or without DADS treatment were tested by wound healing,transwell invasion chamber assays and nude mice xenograft experiments.Results 1.pc DNA6.2/Rho GDI2-mi RNA/MGC803 cells with stable low expression of Rho GDI2 pc DNA ? 6.2-GW/Em GFPmi R-Rho GDI2 interfering plasmid and pc DNA ? 6.2-GW/Em GFPmi RNA-negative interfering plasmid were established, and they were extracted from glycerol bacteria successfully. This work was implemented by the Invitrogen company in Shanghai. 4 groups of interference vector and empty vector were transfected into MGC803 cells. after 2 weeks screening with G418, cells giving out green fluorescent can be found using the inverted fluorescence microscope, which showed that the positive clone was successfully created. compared with the negative transfected group, the expression of Rho GDI2 protein and Rho GDI2 m RNA decreased in different degrees in each transfected group with Western-blotting and RT-PCR. The most obvious inhibitory effect of each group was Rho GDI2-mi RNA1/MGC803 cells(P<0.05), so Rho GDI2-mi RNA1 plasmid was chosen as subjects in the next experiments. 2. Effect of DADS on migration and invasion in Rho GDI2 silenced MGC803 cells The Scratch test showed that, when compared with untransfected group and negative transfected group, the scratch distance of interference group were significantly decreased(P<0.05); Treated with DADS, the scratch distance of each group all decreased than before(P<0.05); Treated with DADS,the scratch distance of interference group were decreased comparing with untransfected group;There was nodifference between untransfected group and negative transfected group(P>0.05); the results showed that, Rho GDI2 silenced can inhibit the ability of migration of MGC803 cells, and enhance the ability of DADS inhibiting the migration of MGC803 cells. The Transwell assays showed that, when compared with untransfected group and negative transfected group, the number of cells which went through the membrane in interference group were significantly decreased(P<0.05); Treated with DADS, the number of cells which went through the membrane of each group all decreased than before(P<0.05); Treated with DADS,the number of cells in interference group were decreased comparing with untransfected group; There was no difference between untransfected group and negative transfected group(P>0.05); the results showed that, Rho GDI2 silenced can inhibit the ability of invasion of MGC803 cells, and enhance the ability of DADS inhibiting the invasion of MGC803 cells. 3. Effect of Rho GDI2 silenced and DADS on the downstream gene, such as RAC1, PAK1 and LIMK1. Western Blotting and RT-PCR experiments showed that, when compared with untransfected group and negative transfected group, the expression of Rho GDI2, Rac1, Pak1 and Limk1 in Rho GDI2 silenced group were reduced significantly(P<0.05); Treated with DADS for 24 hours, the expression of Rho GDI2, Rac1, Pak1 and Limk1 were reduced than before(P<0.05); There was no difference between untransfected group and negative transfected group(P>0.05). 4.Effects of Rho GDI2 silenced and DADS on MGC803 cells in nude mice xenograftexperiments. The results of nude mice xenograft experiments showed that, compared with untransfected group, the volume and weight of in Rho GDI2 silenced group were much more smaller(P<0.05); Treated with DADS for 24 hours, the volume and weight of each group was smaller than before(P<0.05); The results showed that, Rho GDI2 silenced can inhibit the growth of nude mice xenograft tumor, and enhance the ability of DADS inhibiting the growth of nude mice xenograft tumor. 5.Effects of Rho GDI2 silenced and DADS on nude mice transplanted tumor cell morphology and the expression of Ki-67,CD34,Vimentin and E-cadherin. The HE stain experiment showed that, when compared with untransfected group,the cell atypia, karyoplasmic ratio, nucleus stain and nuclear fissionin in Rho GDI2 silenced group all decreased. Treated with DADS for 24 hours, the cell morphology showed the same change. Immunohistochemical experiments showed that, when compared with untransfected group, the expression of Ki-67, CD34 and Vimentin decreased in Rho GDI2 silenced group, while the expression of E-cadherin increased. Treated with DADS for 24 hours, the expression of Ki-67, CD34 and Vimentin decreased, while the expression of E-cadherin increased.It showed that, Rho GDI2 silenced and DADS could up-regulate the expression of E-cadherin, and down-regulate the expression of Ki-67, CD34 and Vimentin.Conclusion: 1. Rho GDI2 silenced can enhance the ability of DADS inhibiting migration and invasion in MGC803 cells.2. Rho GDI2 silenced and DADS can inhibit migration and invasion through Rac1/PAK1/LIMK1 pathway in MGC803 cells. 3. DADS and Rho GDI2 silenced can inhabit tumorigenicity in MGC803 cells in vivo. |
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