Construction and Identification of pU-VEGF-siRNA Expression Vector

Objective To constructed short interfering RNA(siRNA) eukaryotic expression vector for VEGF and to transfect into malignant melanoma cells.Methods A VEGF-siRNA targeting human VEGF mRNA common sequence was synthesized and was inserted into BamH I-Hind III linearized pSilencer~(TM)neoU_6 2.1 vector.The sequence of pU-VEGF-siRNA plasmid was analyzed by DNA sequencer.The recombinant plasmid was transfected into A375(human malignant melanoma cell line) and LOVO cells(human colorectal carcinoma cell line) by electroporation,and cells with stable expression of VEGF-siRNA were obtained by G418 selection.Results It was verified that the sequence of constructed recombinant plasmid was correct by DNA sequencing.We obtained cells with stable expressions of VEGF-siRNA.The expressions of VEGF mRNA and protein in A375 and LOVO cells of experimental groups were significantly decreased,compared to that of controls(P0.01).Conclusion It indicated that hairpin siRNA eukaryotic expression vector for VEGF would be successfully established,and it might play a specific inhibitory role in two kinds of tumor cell lines.At the same time we obtained cells with the stable expression of VEGF-siRNA.This study laid experimental foundation for further research of the therapy of pU-VEGF-siRNA vector in malignant melanoma.