Effect Of DADS On Proliferation, Migration And Invasion In XIAP Silenced SGC-7901 And HCG-27 Cells

Objective: We investigated the effects of DADS on proliferatio n,migration and invasion in XIAP silenced SGC-7901 and HCG-27 cells by constructing XIAP silenced SGC-7901 and HCG-27 cells.Methods:The eukaryotic expression vector silence XIAP gene was constructed, and transfected it into SGC-7901 and HCG-27 cell s to establish a stable cell line, which stably silence XIAP protein expression. q RT-PCR and Western blotting assay were performed to detect the expressions of XIAP m RNA and protein level. The effe ct of XIAP silenced SGC-7901 and HCG-27 cells on proliferation,apoptosis,migration and invasion of SGC-7901 and HCG-27 cells wi th or without DADS treatment were detected by MTT, flow cytome try, wound healing, transwell invasion chamber assays and nude mic e xenograft experiments,western blot and q RT-PCR assay were perf ormed to detect the expression of EGFR、PAK1、XIAP and E-cadh erin,immunohistochemistry to detect the expression of E-cadherin, v imentien, Ki67, CD34 in Nude mice Xenografts.Results:1. The successful construction of XIAP silenced SGC-7901 and H CG-27 cells with stably silence XIAP.DNA sequencing showed thatthe pc DNA6.2-GW/Em GFP-mi RNA vec tor of human XIAP were successfully constructed. The four pc DNA6.2-GW/Em GFP-mi XIAP vector was transfected into SGC-7901 and HCG-27 cells. q RT-PCR and Western blotting assays showed that t he Sequence of three expressions of XIAP m RNA and protein was the lowest, and blasticidin S HCL was added into the transfected c ells of SGC-7901 and HCG-27 cell to filter out Stably silencing XI AP cells.2. Effect of DADS on the proliferation, migration and invasion in SGC-7901 and HCG-27 cells after XIAP silence MTT and flow cytometry assays showed that the proliferation capab ility of XIAP silensing cell were decreased comparing with SGC-7901 and HCG-27 cells(P<0.05). DADS inhibited cellular proliferatio n of XIAP silensing cells and SGC-7901 cells, and induced G2/M ar rest(P<0.05) and apoptosis,XIAP silenced enhance the ability of D ADS inhibit the proliferation capability of SGC-7901 cells(P<0.05), HCG-27 cells can get the same conclusion.Wound healing, Transwell migration and invasion chamber assays sh owed that the migration and invasion of XIAP silencing cell were significantly decreased comparing with no silence cell. DADS inhibit ed the migration and invasion in SGC-7901 cells and XIAP silen sing cells, Silencing XIAP can enhance the ability of DADS inhibi t the migration and invasion of human gastric cancer cells.3. Effect of DADS and XIAP silencesing regulated expression of EGFR、PAK1 and E-cadherin.Cellular immunofluorescence showde that the protein expression of XIAP were decreased in XIAP silencing and DADS treated cell co mparing with control group;Western blot and RT-PCR showed that the protein and expressions of EGFR、PAK1、XIAP were decrease d in DADS treated cells,Whereas the E-cadherin protein level were increased(P<0.05).the protein expression of EGFR、PAK1 were not affected in XIAP silensing cells compare with control group(P>0.05),wheras the E-cadherin protein level were increased(P>0.05).4. Effect of DADS and XIAP silencing on SGC-7901 cells in nud e mice xenograft experiments.Nude mice xenograft tumor model was successfully established,Nude mice xenograft experiments showed that the volume and wei ght of xenograft tumor of DADS treated cells groups were significa ntly smaller than untreated groups(P<0.05). XIAP silencing signific antly inhibited the growth of nude mice xenograft tumor(P<0.05).Conclusion1. XIAP silencing and DADS inhibit the proliferation, migration and invasion in human gastric cancer SGC-7901 and HCG-27 cells.2. The inhibition effects of DADS on the proliferation, migration and invasion in SGC-7901 and HCG-27 cells were enhanced by the XIAP silencing.3. DADS downregulate the expression of XIAP through EGFR/PAK1 pathway inhibiting the proliferation, migration,invasion and tumor igenicity in SGC-7901 cells in vivo.