Fang Liang, Hu Qin-gang, Hua Zi-chun, Li Shu-feng
Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, The Affiliated Hospital of Medical School, Nanjing University, Nanjing 210008, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2007 Oct;42(10):624-8.
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.
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.
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).
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.
利用pRNAT-U6.1/Neo质粒载体构建表达Skp2短发夹RNA（shRNA）的重组质粒，并观察RNA干扰介导的Skp2基因沉默对Tca8113细胞的影响。
分别利用pRNAT-U6.1/Neo质粒载体成功构建5种重组真核表达载体。用聚乙烯亚胺将其转染至Tca8113细胞后，采用RT-PCR和蛋白质免疫印迹法检测Skp2和p27的干扰效果。采用流式细胞术检测Tca8113细胞的细胞周期。采用MTT法检测Tca8113细胞的增殖情况。
在Skp2shRNA-2和Skp2shRNA-3载体中，Tca8113细胞Skp2蛋白表达下调，p27蛋白表达上调（P<0.01）。G1/G0期细胞数量增加22%（P<0.01），G2/M期和S期细胞数量分别减少10%和12%（P<0.01）。Tca8113细胞增殖减缓，细胞数量减少（P<0.01）。
成功构建了针对Skp2的shRNA的Skp2shRNA-2和Skp2shRNA-3载体。它们可影响Skp2和p27基因的表达。Skp2可能是人类舌鳞状细胞癌基因治疗的一个有前景的靶点。
