PMID: 16620571Apr 20, 2006Paper

Inhibition of vascular endothelial growth factor gene expression and proliferation of leukemia cells by RNA interference

Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi
Huiling ShenMing Xiao

Abstract

To explore the feasibility of selective inhibiting VEGF expression using VEGF short hairpin RNA (shRNA) interference, and observe the effects of VEGF gene silencing on NB4 cells growth. Three 19 bp reverse repeated motifs targeting exons 3, 4, 5 respectively of VEGF gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into NB4 cells respectively through lipofectamine reagent. The alteration of VEGF expression was examined by fluorescent real time RT-PCR and Western blot. The proliferation capacity of leukemia cells was measured by trypan blue exclusion, MTT assay, colony formation assay and cell cycles analysis. Recombinant plasmids containing three shRNAs and random fragment were successfully constructed and transfected into NB4 cells respectively by liposome-mediated gene transfer method. shRNA in pGenesil-VR3 cells knocked down the expression of VEGF mRNA and protein dramatically in a sequence-specific manner when compared with that of pGenesil-VR1, Genesil-VR2 and pGenesil-con. The NB4 cell...Continue Reading

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