Construction of Skp2 shRNA and its effect on lung cancer cell growth / 第三军医大学学报

ABSTRACT

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 MTT 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