Skp2 siRNA表达载体构建及其对喉鳞状细胞癌细胞Skp2基因表达的抑制作用

Construction siRNA expressing vector of Skp2 and the inhibitory effects on the expression of Skp2 in laryngeal carcinoma cell line Hep2 cell

目的:应用RNA干扰技术,构建针对Skp2的siRNA表达载体,抑制喉鳞状细胞癌细胞Skp2基因的表达。方法:根据设计siRNA的原则,针对人Skp2的mRNA序列,设计并合成编码siRNA的2条寡核苷酸序列,经退火成互补双链,再克隆到真核表达载体pGPU6/Neo中构建重组体pGPU6Skp2,转化DH5α菌株,提取质粒进行酶切鉴定后,进行序列测定。然后转染重组质粒至Hep2细胞中,应用流式细胞术检测Skp2基因的表达。结果:将合成的DNA序列退火后克隆到载体上,经酶切和测序鉴定确实为所需序列。pGPU6Skp2转染细胞后,Skp2基因在蛋白水平的表达量受到明显抑制。结论:成功构建了针对人Skp2的siRNA表达载体,通过转染Hep2细胞,可有效抑制细胞Skp2的表达,为后续基因治疗研究奠定了实验基础。

Objective： To construct the siRNA expression vector of Skp2 and inhibit the expression of Skp2 through RNA interference in laryngeal carcinoma cell line Hep2 cell. Method：According to the encoding sequence of mRNA of Skp2, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonu cleotide fragments were subcloned into pGPU6/Neo siRNA expression vector, and the recombinant plasmid was transformed into strain DHSa. The plasmid identified by restriction enzyme was used for sequencing. After being identified by sequencing , the recombinant plasmids pGPU6Skp2 were transfected into Hep2 cells. Skp2 expression in the transfected cells was assayed by flow cytometry. Result： DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGPU6 vector, and Skp2 expression in the transfected cells was downregulated significantly by pGPU6Skp2 at the protein level. Conclusion：The siRNA expression vector of Skp2 was successfully constructed and could inhibit Skp2 expression in Hep2 cells. This result will facilitate further studies of Skp2 in gene therapy for tumors.