摘要
目的:探讨干扰肺腺癌SPC-A-1细胞中Skp2(S-phase kinase associated protein,Skp 2)基因的发夹结构RNA(smallhairpin RNA,shRNA)对肺癌细胞Skp2和p27基因表达的影响及对细胞生长的影响。方法:设计、合成特异性的shRNA序列并与pGenesil-1质粒载体连接转染SPC-A-1细胞,经过稳定筛选后的肺癌细胞,通过RT-PCR和Western印迹法分别检测细胞内Skp2、p27 mRNA和蛋白的表达情况,MTT法检测细胞生长情况并绘制生长曲线。结果:在转染干扰RNA后SPC-A-1细胞内的Skp2 mRNA及蛋白表达下降明显,而p27蛋白水平却明显上升,这与抑制了Skp2表达导致p27降解减少有关。生长曲线表明细胞生长受到了抑制。结论:成功构建了针对Skp2基因的shRNA真核表达载体,能有效抑制肺癌细胞生长,并使得Skp2基因表达下降,p27基因的表达增多,为肺癌细胞的基因治疗提供了实验依据。
Objective： To construct small hairpin （shRNA） vector targeting Skp2 and p27 and investigate its effect on the expressions of Skp2 and p27 as well as on the growth of lung cancer cells. Methods：The specific shRNA sequence was designed, synthesized and cloned into plasmid pGenesil-1. The lung cancer cells were selected by G418 screening. The mRNA and protein expressions of Skp2 and p27 were analyzed by RT-PCR and Western blotting. Cell proliferation was detected by MTT assay and cell growth curve was drawn to analyze the inhibitory effects on cell proliferation. Results：The Skp2 mRNA and protein expression was significantly decreased after transfection of interfering plasmid, p27 protein level was significantly increased, which resulted from decrease in p27 degradation caused by inhibition of Skp2 expression. The cell growth curves indicated that the proliferation of cells was inhibited significantly compared with the control group. Conclusion：The shRNA vector targeting Skp2 gene effectively inhibited the proliferation of lung cancer cells, down-regulated Skp2 gene expression, and up-regulated p27 gene expression. The method laid the an experimental basis for lung cancer gene therapy.
出处
《肿瘤》
CAS
CSCD
北大核心
2008年第6期485-488,493,共5页
Tumor
