Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Aug 14, 2013; 19(30): 4917-4924
Published online Aug 14, 2013. doi: 10.3748/wjg.v19.i30.4917
Skp2-RNAi suppresses proliferation and migration of gallbladder carcinoma cells by enhancing p27 expression
Bin Zhang, Lin-Hua Ji, Wei Liu, Gang Zhao, Zhi-Yong Wu
Bin Zhang, Lin-Hua Ji, Wei Liu, Gang Zhao, Zhi-Yong Wu, Department of General Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China
Author contributions: Zhang B, Zhao G and Wu ZY designed research; Zhang B, Ji LH and Liu W performed research; Zhang B and Ji LH analyzed data; Zhang B and Zhao G co-wrote the paper.
Correspondence to: Gang Zhao, MD, PhD, Department of General Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, No. 1630 Dongfang Road, Shanghai 200127, China. zhaogang74313@163.com
Telephone: +86-21-68383732 Fax: +86-21-68383090
Received: March 19, 2013
Revised: May 13, 2013
Accepted: June 8, 2013
Published online: August 14, 2013
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: Gallbladder carcinoma, S-phase kinase-associated protein-2, p27, Gene therapy, Cell cycle

Core tip: The association between S-phase kinase-associated protein-2 (Skp2)/p27 and gallbladder carcinoma has rarely been reported. This study investigated the effects of Skp2-RNAi on in vitro and in vivo growth and the invasive potencies of gallbladder carcinoma cells. The authors proposed that the effects were due to the accumulation of the p27 protein following Skp2-depletion.