VEGF Directed siRNA Inhibits Hypoxia Induced VEGF Upregulation In Vitro

Abstract

Abstract: : Purpose: To determine if small interfering RNA (siRNA) directed towards human VEGF can inhibit hypoxia induced upregulation of VEGF in vitro. Methods: Human cell lines (293, Hela and ARPE19) and the mouse cell line (NIH3T3) were seeded into 24 well plates to 50% confluence. All cells were transfected with a dose range of 10 nM to 50nM of siRNA directed towards human or murine VEGF or alternatively siRNA targeting green fluorescent protein (GFP). Transfections were performed using a transit TKO transfection reagent (Mirus). As additional controls, some cells were treated with transfection reagent lacking siRNA or were left untreated. 24 hours after transfection, hypoxia was induced in the cells by the addition of desferoxamide mesylate to a final concentration of 130µm per well. Supernatant was removed 24 hours post hypoxia induction. VEGF protein was measured with a human or murine VEGF ELISA (R&D systems, Minneapolis, MN). Results: In the human cell lines, VEGF was significantly downregulated after transfection with siRNA directed towards human VEGF as compared to cells treated with siRNA directed to murine VEGF, GFP or with other solutions. In the murine cell lines siRNA directed towards murine VEGF was significantly downregulated as compared to cells treated with siRNA directed to human VEGF, GFP or with other solutions Conclusions: siRNA directed towards human or murine VEGF can downregulate only the species specific VEGF expression induced by hypoxia. This experiment demonstrates the specificity and the potency of siRNA's ability to downregulate its directed target rna.

Keywords: growth factors/growth factor receptors • gene/expression • gene transfer/gene therapy