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AIM:To explore the role of S-phase kinase-associated protein-2(Skp2)in gallbladder carcinoma and to identify whether depletion of Skp2 by Skp2-RNAi could attenuate proliferation and migration of gallbladder carcinoma.METHODS:Skp2-RNAi was transduced into cells of the gallbladder carcinoma cell line GBC-SD,using a lentiviral vector.The effect of Skp2-RNAi on the proliferation,migration,invasion and cell cycle of GBC-SD cells was studied using in vitro assays for cell proliferation,colony formation,wound healing and cell cycle.The expression of Skp2 and p27 was detected by real-time polymerase chain reaction and Western immunoblotting.The effect of Skp2-RNAi on the proliferation of GBC-SD cells in vivo was investigated by tumorigenicity experiments in nude mice.RESULTS:Lentivirus-mediated RNAi reduced the expression of Skp2 in cultured cells.The expression of the p27 protein increased along with the down-regulation of Skp2,although no significant difference was found in p27 mRNA expression.Flow cytometry revealed that Skp2-RNAi transfection significantly increased the proportion of cells in the S phase and significantly decreased the proportion of cells in the G 2 /M phase.No significant difference in the frequency of cells in the G0/G1 phase was observed.The results from the cell proliferation,colony formation and wound healing assays revealed that Skp2-RNAi transfection markedly inhibited the proliferation and migration of GBC-SD cells in vitro.Additionally,tumorigenicity experiments showed that suppression of Skp2 significantly decreased the weights of the tumors(0.56 ± 0.11 and 0.55 ± 0.07 g in the control and Scr-RNAi groups vs 0.37 ± 0.09 and 0.35 ± 0.08 g in the Skp2-RNAi-L and Skp2-RNAi-H groups).CONCLUSION:The expression of Skp2 in GBC-SD cells was inhibited following Skp2-RNAi transfection.Silencing of the Skp2 gene inhibited proliferation,migration and invasiveness of GBC-SD cells by mechanisms dependent on enhanced expression of the p27 protein.
AIM: To explore the role of S-phase kinase-associated protein-2 (Skp2) in gallbladder carcinoma and to identify whether depletion of Skp2 by Skp2-RNAi could attenuate proliferation and migration of gallbladder carcinoma. METHODS: Skp2-RNAi was transduced into cells of the gallbladder carcinoma cell line GBC-SD, using a lentiviral vector. The effect of Skp2-RNAi on the proliferation, migration, invasion and cell cycle of GBC-SD cells was studied using in vitro assays for cell proliferation, colony formation, wound healing and cell cycle. The expression of Skp2 and p27 was detected by real-time polymerase chain reaction and Western immunoblotting. The effect of Skp2-RNAi on the proliferation of GBC-SD cells in vivo was investigated by tumorigenicity experiments in nude mice. RESULTS: Lentivirus-mediated RNAi reduced the expression of Skp2 in cultured cells. The expression of the p27 protein increased along with the down-regulation of Skp2, although no significant difference was found in p27 mRNA expressi on. Flow cytometry revealed that Skp2-RNAi transfection significantly increased the proportion of cells in the S phase and significantly decreased the proportion of cells in the G2 / M phase. No significant difference in the frequency of cells in the G0 / G1 phase was observed.The results from the cell proliferation, colony formation and wound healing assays revealed that Skp2-RNAi transfection markedly inhibited the proliferation and migration of GBC-SD cells in vitro. Additionally, tumorigenicity examples showed that suppression of Skp2 significantly decreased the weights of the tumors (0.56 ± 0.11 and 0.55 ± 0.07 g in the control and Scr-RNAi groups vs 0.37 ± 0.09 and 0.35 ± 0.08 g in the Skp2-RNAi-L and Skp2-RNAi-H groups) .CONCLUSION: The expression of Skp2 in GBC-SD cells was inhibited following the Skp2-RNAi transfection. Silencing of the Skp2 gene inhibited proliferation, migration and invasiveness of GBC-SD cells by mechanisms dependent on enhanced expression of the p27 protein.