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


CONCLUSIONS
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.
Department of General Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China.
Bin
B
Zhang
Lin-Hua
LH
Ji
Wei
W
Liu
Gang
G
Zhao
Zhi-Yong
ZY
Wu
D000818:Animals; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism / Q000556:secondary / Q000628:therapy*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D005706:Gallbladder Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D034622:RNA Interference; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D047368:Tumor Burden; D015854:Up-Regulation; D014945:Wound Healing; D023041:Xenograft Model Antitumor Assays
C436291:CDKN1B protein, human; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27
Cell cycle; Gallbladder carcinoma; Gene therapy; S-phase kinase-associated protein-2; p27