Lentivirus-Mediated RNA Interference Targeting Vascular Endothelial Growth Factor Gene Enhances the Sensitivity of K562 Cells to Imatinib

Abstract

Background: Vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis, plays an important role in tumor cell proliferation and migration. Elevated VEGF levels are found in both the plasma and the bone marrow of patients with chronic myelogeneous leukemia(CML). Moreover, the CML-associated oncogene Bcr-Abl induces endogenous secretion of VEGF. The aim of this study was to construct a lentiviral vector-mediated RNA interference (RNAi) of vascular endothelial growth factor (VEGF) gene and to investigate the effects of RNAi inhibiting VEGF gene alone or in combination with imatinib on the proliferation and apoptosis of K562 leukemic cell line.Methods: A lentiviral expression vector containing short hairpin RNA (shRNA ) targeting VEGF was constructed and cotransfected with the pPACKH1 packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blot and ELISA. Cellular proliferation was determined by trypan blue exclusion and MTT assay. imatinib -induced apoptosis was analyzed by flow cytometry.Results: The lentiviral shRNA vector targeting VEGF has been constructed and transfected into K562 cells successfully. The expression of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562–con cells (mock transfection). The proliferation rate of K562-shVEGF cells slowed down. After imatinib treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562–con cells.Conclusion: Suppression of VEGF by lentivirus-mediated RNAi could effectively inhibit proliferation and increase the sensitivity of K562 cells to imatinib. These results indicate that Bcr/Abl-targeting compound in combination with anti-angiogenic therapies may provide a potent therapeutic strategy to CML.