Construction and screening of plasmid expression vectors containing short hairpin RNA targeting at vascular endothelial growth factor gene of GBC-SD cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Previous reseamh has proved that RNA interference can inhibit vascular endothelial growth factor (VEGF) gene expression of colon carcinoma, carcinoma of prostate, and retinoblastoma. However, RNA interference inhibiting VEGF of carcinoma of gallbladder was not reported. OBJECTIVE: To construct a plasmid expression vector coding for the short hairpin RNA (shRNA) targeting hVEGF165 mRNA. DESIGN, TIME AND SETTING: A gene engineering study was performed at National Hepatobiliary & Enteric Surgery Research Center, Xiangya Hospital, Central South University from 2008 to 2009.MATERIALS Human GBC-SD was provided by Tumor Research Institute of Tongji University. METHODS: Four pairs of shRNAs that targeted at VEGF gene were designed. The eukaryotic expression plasmids (named shRNA1-4) were constructed and identified using restriction enzyme analysis. The plasmids were then transfected into GBC-SD cells via liposome2000. The transfection rate of recombinant plasmids was measured at 48 hours after transfection. MAIN OUTCOME MEASURES: Enzyme analysis of recombinant plasmid; transfection rate; VEGF mRNA expression determined using fluorescent polymerase chain reaction. RESULTS: shRNA plasmid vector targeting at VEGF gene was successfully constructed, in particular, pDC316-EGFP-U6-shRNA2 was the most effective. The expression plasmids were confirmed by restriction enzyme analysis. The transfection rate of recombinant plasmids in GBC-SD cells was approximately 58.6%. shRNA could inhibit VEGF mRNA expression, in particular, the inhibitory rate of RNA2 was the highest by 86%.CONCLUSION: The shRNA eukaryotic expression plasmid targeting at VEGF gene is constructed and selected successfully, and it can remarkably inhibit VEGF expression of GBC-SD cells. Additionally, the inhibitory effect of RNA2 is the greatest.