Skp2-RNAi suppresses proliferation and migration of gallbladder carcinoma cells by enhancing p27 expression

Abstract

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 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 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.

Keywords: Cell cycle; Gallbladder carcinoma; Gene therapy; S-phase kinase-associated protein-2; p27.

Figure 1

Detection of downstream gene and protein expression after kinase-associated protein-2-RNAi transfection. A: Expression of p27 mRNA did not change following down-regulation of S-phase kinase-associated protein-2 (Skp2); B and C: Protein expression of p27 was upregulated. Lane 1: Control; Lane 2: Scr-RNAi; Lane 3: Skp2-RNAi-L; Lane 4: Skp2-RNAi-H.

Figure 2

Proliferation curves for the gallbladder carcinoma cell line cells after the inhibition of kinase-associated protein-2 expression. Cell proliferation was significantly inhibited after down-regulation of S-phase kinase-associated protein-2 (Skp2) by Skp2-RNAi. GBC-SD: The gallbladder carcinoma cell line.

Figure 3

Colony formation assays after inhibition of kinase-associated protein-2 expression. A: Colony formation assays; B: Colony formation of the gallbladder carcinoma cell line (GBC-SD) cells was significantly inhibited after transfection with S-phase kinase-associated protein-2 (Skp2)-RNAi.

Figure 4

Results of the wound healing assay after inhibition of kinase-associated protein-2 expression. A: The wound healing assay after inhibition of kinase-associated protein-2 (Skp2) expression; B: The migrated cells in the two S-phase Skp2-RNAi groups were markedly decreased as compared with the two control groups.

Figure 5

Proportion of cells in the cell cycle stages. A: Detection of the proportion of cells in the cell cycle stages after S-phase kinase-associated protein-2 (Skp2) expression was inhibited; B: The proportion of cells in the S phase of the cell cycle increased, and the proportion of cells in the G₂/M phase decreased in Skp2-RNAi-L and Skp2-RNAi-H groups.

Figure 6

Tumorigenicity experiments in nude mice. A: Tumorigenicity experiments; B: Transfection with S-phase kinase-associated protein-2 (Skp2)-RNAi inhibited the growth of tumor cells.