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目的运用 RNA 干扰技术阻断人舌鳞状细胞癌 Tca8113细胞中 Skp2基因的表达,观察Skp2基因沉默后对 Tca8113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 Tca8113细胞,通过反转录聚合酶链反应(reversetranscrip-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,Tca8113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 Tca8113细胞后,与对照组相比,G_1～G_0期细胞增加了约22%(P<0.01),G_2～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。
Objective To detect the expression of Skp2 gene in human tongue squamous cell carcinoma Tca8113 cells by RNA interference and to observe the effect of Skp2 gene silencing on Tca8113 cells. Methods Recombinant plasmids for Skp2 gene were constructed by eukaryotic transcription plasmid pRNAT-U6.1 / Neo. The cells were transfected into Tca8113 cells by polyethyleneimine method, and the changes of Skp2 and p27 expression were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Flow cytometry The changes of cell cycle and growth rate of Tea8113 cells were detected by methyl thiazolyl terrazolium (MTT) assay. Results After transfection of Skp2shRNA-2 and Skp2shRNA-3, the expression of Skp2 gene in Tca8113 cells was down-regulated (P <0.01) and the level of p27 protein was up-regulated (P <0.01) After transfection p27 gene mRNA levels did not change significantly. Compared with the control group, the number of cells in G_1 ~ G_0 phase increased by about 22% (P <0.01) after transfection with recombinant plasmids Skp2shRNA-2 and Skp2shRNA-3, while the cells in G_2 ~ M phase and S phase decreased about 10% And 12% (P <0.01), the cell growth rate was significantly slower (P <0.01). Conclusion Skp2 and p27 genes were initially demonstrated to play an important role in the differentiation and proliferation of oral squamous cell carcinoma cells. Skp2shRNA-2 and Skp2shRNA-3 were screened by high efficient RNA interference plasmids. To further study the effect of Skp2 gene Human tongue squamous cell carcinoma gene therapy laid the foundation.