Construction of shRNA Interfering Plasmid Targeting Pin1 Gene and Its Inhibitory Effect on Pin1 Gene of Colon Cancer SW620 Cells

Objective To construct the short hairpin RNA(shRNA)interfering plasmid targeting Pin1 gene and determine its inhibitory effect on the Pin1 gene of colon cancer SW620 cells. Methods The oligonucleotide sequence specific to human Pin1 gene was designed and synthesized then, after annealing, inserted into vector pGenesil-1. The constructed shRNA eukaryotic expression vector pGenesil-1-Pin1(+)was transfected to SW620 cells in mediation of liposome, using the SW620 cells transfected with plasmid pGenesil-1-Pin1(-)and the untransfected SW620 cells as controls. The green fluorescent protein(GFP)expressed in transfected cells were observed by fluorescent microscopy, based on which the transfection efficacy was calculated. The effects of plasmid pGene-sil-1-Pin1(+)on the transcription of Pin1 mRNA and expression of Pin1 protein in SW620 cells were determined by real-time fluores-cent quantitative PCR and Western blot. Results Both restriction analysis and sequencing proved that shRNA eukaryotic expression vector pGenesil-1-Pin1(+)was constructed correctly. The transfection efficacy of SW620 cells with pGenesil-1-Pin1(+)was 64%. The inhibiting rate on transcription of Pin1 mRNA in SW620 cells reached a peak value(70. 2%)72 h after transfection with plasmid pGenesil-1-Pin1(+), while that on expression of Pin1 protein was 60%. Conclusion The shRNA interfering plasmid targeting Pin1 gene was successfully constructed, which inhibited the transcription of Pin1 mRNA and expression of Pin1 protein in SW620 cells.