Effect of VEGF antisense RNA on inducing apoptosis of myeloma cells and inhibition of angiogenesis in endothelial cells in vitro / 中国实验血液学杂志

ABSTRACT

This study was aimed to investigate the effect of VEGF antisense RNA on proliferation and apoptosis in myeloma cell line U266 as well as on angiogenesis in endothelial cell ECV304, and to explore the feasibility of gene therapy for multiple myeloma using VEGF antisense RNA. The VEGF121 cDNA was inserted into multiple clone site of eukaryotic expression vector pIRES2-EGFP to construct the recombinant plasmid AS-VEGF. Restriction endonuclease analysis and DNA sequencing were used to confirm the reverse orientation of the VEGF cDNA. The recombinant plasmid was transfected into human myeloma cell line U266 and the positive clone was screened by G418. The VEGF mRNA and protein expressions of the positive clone were detected by RT-PCR and Western blot respectively. The viability and apoptosis of U266 cells were observed by MTT assay, flow cytometry. Angiogenesis was tested by network formation of endothelial cells on matrigel. The results indicated that the recombinant plasmid AS-VEGF expressing VEGF antisense RNA were constructed successfully. VEGF expression in U266 cells was blocked partially by VEGF antisense RNA. Expression of VEGF mRNA and protein decreased more significantly in U266 cells transfected by AS-VEGF than that in control group. Then increasing of apoptosis and inhibition of proliferation in U266 cells transfected by AS-VEGF were observed. Vasoformation on matrigel in the supernatants of U266 culture group transfected by AS-VEGF decreased more significantly than that in control group. It is concluded that VEGF antisense RNA can inhibit the expression of VEGF gene in U266 cells, thereby inhibits the proliferation of U266 cells; increases the apoptosis of U266 cells; and inhibits angiogenesis in vitro.