[Construction of targeting-Skp2 shRNA plasmids and observation of their inhibitory effect on Tca8113 cells].
OBJECTIVE
To 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.


METHODS
Five 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.


RESULTS
In 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).


CONCLUSIONS
Skp2shRNA-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.
Fang
Liang
L
Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China.
Hu
Qin-gang
QG
Hua
Zi-chun
ZC
Li
Shu-feng
SF
D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D014062:Tongue Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D014162:Transfection
D044786:S-Phase Kinase-Associated Proteins