Skp2靶向RNA干扰质粒的构建及其对人舌鳞癌Tca8113细胞的影响

目的运用RNA干扰技术阻断人舌鳞状细胞癌Tea8113细胞中Skp2基因的表达，观察Skp2基因沉默后对Tea8113细胞的影响。方法采用真核转录质粒pRNAT-U6．1／Neo构建针对Skp2基因的重组转染质粒。经聚乙烯亚胺法将其转染Tea8113细胞，通过反转录聚合酶链反应（reverse transcrip-tase polymerase chain reaction，RT-PCR）、Western blot检测Skp2、p27表达的变化；流式细胞仪、甲基噻唑基四唑（methyl thiazolyl terrazolium，MTT）法检测转染后Tea8113细胞的细胞周期、生长速度的变化。结果转染重组质粒Skp2shRNA-2、Skp2shRNA-3后，Tea8113细胞内Skp2基因在mRNA和蛋白水平上均下调表达（P〈0．01），p27基因蛋白水平上调表达（P〈0．01）；而各重组质粒转染后p27基因的mRNA水平无明显变化。重组质粒Skp2shRNA-2、Skp2shRNA-3转染Tea8113细胞后，与对照组相比，G1～G0期细胞增加了约22％（P〈0．01），G2～M期和S期细胞减少了约10％和12％（P〈0．01），细胞的生长速度明显变慢（P〈0．01）。结论初步证明了Skp2、p27基因在口腔鳞状细胞癌细胞分化增殖中所扮演的重要角色；筛选出了高效RNA干扰重组质粒Skp2shRNA-2、Skp2shRNA-3，为进一步研究以Skp2基因为靶点的人舌鳞状细胞癌基因治疗奠定了基础。

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.