Skp2 shRNA表达质粒的构建及其对肺癌细胞生长的影响

Construction of Skp2 shRNA and its effect on lung cancer cell growth

目的构建Skp2基因的RNAi真核表达质粒,观察其对SPC-A-1肺癌细胞内Skp2基因表达及细胞生长的影响。方法根据GenBank中Skp2cDNA序列设计针对Skp2基因的shRNA序列,合成序列并克隆入pGenSil-1质粒中,构建Skp2 pshRNA重组质粒。脂质体介导重组质粒转染SPC-A-1肺癌细胞。RT-PCR检测肺癌细胞Skp2 mRNA的表达,Western blot检测Skp2蛋白表达。MTT检测各组细胞生长情况,流式细胞仪检测细胞周期改变。结果重组质粒经酶切鉴定和测序,证实表达干扰质粒构建成功。转染重组质粒的肺癌细胞Skp2 mRNA和蛋白的表达明显下降(P<0.05),细胞生长增殖受到抑制,阻滞在G0/G1期的细胞比例增加,而进入S期的细胞比例明显下降(P<0.05)。结论构建的Skp2 shRNA表达质粒能有效下调Skp2基因的mRNA及蛋白的表达、抑制细胞的生长增殖。

Objective To construct and identify the RNAi eukaryotic vector of Skp2 gene and to observe its interfering effect on the growth of SPC-A-1 lung cancer cells. Methods The specific shRNA sequence was designed and synthesized according to the Skp2 cDNA sequence in GenBank. The sequence was cloned into plasmid pGenesil-1. Then recombinant vector was transfected into SPC-A-1 lung cancer cells by Lipofectamine 2000. The expressions of Skp2 mRNA were analyzed by RT-PCR and the levels of Skp2 protein were detected by Western blot. The cell growth suppression was analyzed by MTF assay. Distribution of cell cycle was assessed by flow cytometry. Results The sequence of template and specific siRNA was correct by sequence analysis. Obvious decrease was observed in the levels of Skp2 mRNA and Skp2 protein after Skp2 shRNA transfection （P 〈 0.05 ）. The subcloned recombinant plasmid expressing Skp2 shRNA effectively inhibited lung cancer cell growth, while empty plasmid had no such specific effect（P 〈0.05）, induced cell cycle arrest in G0/G~ phase with a significant decrease of cells in S phase （P 〈 0.05）. Conclusion The successful construction of Skp2 shRNA vector, which could effectively abate the expression of Skp2 and inhibit the growth of SPC-A-1 lung cancer cells, laid the foundation for further study on the function of Skp2 gene and gene therapy of lung cancer.