Construction of targeting-Skp2 shRNA plasmids and observation of their inhibitory effect on Tca8113 cells.
- Author:
Liang FANG
1
;
Qin-gang HU
;
Zi-chun HUA
;
Shu-feng LI
Author Information
- Publication Type:Journal Article
- MeSH: Carcinoma, Squamous Cell; genetics; metabolism; pathology; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Genetic Vectors; genetics; Humans; Plasmids; genetics; RNA Interference; S-Phase Kinase-Associated Proteins; genetics; metabolism; Tongue Neoplasms; genetics; metabolism; pathology; Transfection
- From: Chinese Journal of Stomatology 2007;42(10):624-628
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the recombinant plasmids expressing Skp2 short hairpin RNA (shRNA) by pRNAT-U6.1/Neo plasmid vector and observe the effects of RNAi-mediated Skp2 gene silencing on Tca8113 cells.
METHODSFive recombinant eukaryotic expression vectors were successfully constructed using pRNAT-U6.1/Neo plasmid vector separately. After they were transfected into Tca8113 cells with PEI, the interference effects no Skp2 and p27 were detected by RT-PCR and Western blot. The cell cycle of Tca8113 cells were tested by flow cytometry. The proliferation of Tca8113 cells were examined by MTT.
RESULTSIn Skp2shRNA-2 and Skp2shRNA-3 vectors, the expression of Skp2 protein of Tca8113 cells was down-regulated and p27 protein up-regulated (P < 0.01). The cell number during G1/G 0 phases increased 22% (P < 0.01) and during G(2)/M and S phases the number decreased 10% and 12% (P < 0.01). The proliferation of Tca8113 cells slowed down and the cells number decreased (P < 0.01).
CONCLUSIONSSkp2shRNA-2 and Skp2shRNA-3 vectors of shRNA for Skp2 were successfully constructed. They could influence expression of Skp2 and p27 gene. Skp2 may be a promising target of gene therapy on human tongue squamous cell carcinoma.
