VEGF shRNA enhances the sensitivity of multidrug-resistant leukemia cells to anticancer agent / 中国实验血液学杂志

ABSTRACT

This study was aimed to explore the effect of vascular endothelial growth factor (VEGF) on sensitivity of leukemia cell line K562/A02 to doxorubicin by using RNA interference, and to investigate its mechanism. The 3 shRNA targeting human vegf gene were synthesized, then transfected into K562/A02 cells by lipofectamine 2000 reagent. RT-PCR was used to detect the expression of vegf and mrp1 at the mRNA level;Western blot was used to analyze the expression of VEGF, MRP1, AKT, P-AKT at the protein level; MTT was used to determine the IC(50) value of transfected cells to doxorubicin (DOX); flow cytometry was used to detect cell apoptosis and intracellular Rho123 retention. The results showed that after vegf shRNA were transfected into K562/A02 cells, the expression of vegf at the mRNA level decreased, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was statistically significant (p < 0.05), the greatest decrease was observed in the cells transfected with vegf shRNA3; and the protein level of VEGF was also down-regulated. The IC(50) value of positively transfected group was lower than that of control groups, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was significant (p < 0.05). The retention of intracellular Rho123 was enhanced in three positively transfected groups (p < 0.05). Cell apoptosis increased in positively transfected groups, and there was statistically difference between vegf shRNA2 group or vegf shRNA3 group and HK group (p < 0.05). The expression of mrp1 at the mRNA level were decreased, and there were statistical difference between vegf shRNA3 group and HK group (p < 0.05), and the protein level of mrp1 was also down-regulated; the expression of P-AKT at protein level decreased in positively transfected groups, and the greatest decrease was seen in vegf shRNA3 group. It is concluded that the transfection with exogenous vegf shRNA can inhibit the expression of vegf at both mRNA and protein levels, and enhance the sensitivity of K562/A02 cell to doxorubicin, the mechanism of which may be the inhibition of apoptosis and down-regulation of MRP1 by inactivating PI3K/AKT signaling pathway.