Knock-down of VEGF by adenovirus-delivered siRNA in human gastric adenocarcinoma cells

Objective: To construct recombinant adenovirus containing small interfering RNA (siRNA) targeting human VEGF mRNA (Ad-siRNA-VEGF), and to knock down VEGF in human gastric adenocarcinoma cells (MGC-803). Methods: ① The specific siRNA sequence targeting human VEGF mRNA was selected, and the homologous double-strand DNA was designed and synthesized. After such DNA was inserted into plasmid pSUPER, psiRNA-VEGF was obtained. The siRNA-VEGF fragment was derived from psiRNA-VEGF digested by NotⅠand XhoⅠ and cloned into the shuttle plasmid pAdTrack. pAdTrack-siRNA-VEGF was co-transfected into the E.coli strain BJ5183 with the bone plasmid pAdEasy-1, then Ad-siRNA-VEGF was generated by homologous recombination. Ad-siRNA-VEGF was packaged and amplified in cells 293; ② Ad-siRNA-VEGF was transfected into MGC-803 cells. The level of VEGF mRNA in cells was tested by Real Time PCR, and the concentration of VEGF protein in the cell culture supertant was determined by ELISA 96 hours later. Results: The recombinant adenovirus Ad-siRNA-VEGF was successfully constructed. One day after the linearized Ad-siRNA-VEGF in 293 cells being packed, green fluorescent protein(GFP) was observed. Ad-siRNA-VEGF with the titer of 3.6×108efu/ml was got by CsCl gradient purification. Ninty-six hours after Ad-siRNA-VEGF being transfected MGC-803 cells, the VEGF mRNA in cells reduced by 67.1% and the VEGF protein concentration in the cell culture supernatant decreased by 72.5%. Conclusion: The adenovirus based-RNAi is capable of knocking down remarkable VEGF in human gastric adenocarcinoma cells, which will benefit for further anti-angiogenesis therapy for cancer.