Inhibition effect of short hairpin RNA on VEGF receptor flt-1 gene expression in leukemia cell line K562 / 中国实验血液学杂志

ABSTRACT

This study was purposed to investigate the inhibitory effect of short hairpin RNA (shRNA) on expression of vascular endothelial growth factor (VEGF) receptor flt-1 gene in leukemia cells line K562, and to explore the influence of shRNA invasive ability on leukemia cells and its mechanism. The recombinant eukaryotic expression plasmid containing flt-1 shRNA gene was transfected into K562 cells by lipofectamine mediation and positive clones were screened by G418. shRNA gene in K562 cells was confirmed by PCR. RT-PCR and Western blot were employed to detect the expression of flt-1 mRNA and protein in leukemia cells. The invasive ability of K562 cells was studied by Boyden chamber invasion assay before and after flt-1 shRNA transfection. MMP-2 and MMP-9 mRNA expressions were detected by RT-PCR after transfection of the recombinant plasmid C1/U6/FltS2 into K562 cells through liposome. The results showed that the recombinant eukaryotic expression plasmid had been transfected into the human leukemia cell line K562 and positive clones had been screened by G418 for 2 weeks. PCR detection revealed the stable expression of the shRNA gene in K562 cells. Flt-1 gene and protein expressions were inhibited by plasmid-expressed shRNA after transfection of recombinant vetors C1/U6/FltS into K562 cells. The inhibitory efficiency of two different shRNA sequences targeting Flt-1 gene were 46.1% and 65.4% respectively. The expression of MMP-2 and MMP-9 mRNA decreased, and the mean invasion rate in C1/U6/fltS2-transfected K562 cells was lower than that in nontransfected cells. It is concluded that shRNA eukaryotic expression vector specific to VEGF receptor flt-1 gene can high efficiently be transfected into leukemia cell line K562, effectively inhibits the expression of flt-1 gene, weakens the in vitro invasive ability of leukemia cells and the expression levels of MMP-2 and MMP-9 mRNA, which suggests that the VEGF involves in the migration of leukemia cells by regulating the MMP-2 and MMP-9 through joints with the receptor.