| Objective:1.To explore the expression of vascular endothelial growth factor(VEGF) in mRNA and protein level between human leukemia cell lines K562 cell and K562/A02 cell.2.To construct the short-hairpin RNA (shRNA) specific targeting to human VEGF and observe the expression of VEGF in mRNA and protein level after the shRNAs were transfected into the K562/A02 cell for 24h,48h,72h by Lipofecatmine 2000.3.when the expression of VEGF in mRNA and protein level were reduced,the growth, apoptosis and sensitivity to anticancer agent of K562/A02 cell could be studied,and its possible molecular mechanisms were then explored,for example,the effect of PI3K/AKT and MAPK/ERK signal-transduction pathway on this process were studied.Methods:1.The different expression of vascular endothelial growth factor(VEGF) genes between K562 cell and K562/A02 cell were examined by reverse transcription-polymerase chain reaction(RT-PCR), and the cytoplasm extracts were prepared for Western blotting to detect the expression of VEGF in protein level between K562 cell and K562/A02 cell. VEGF shRNA and the random-sequence HK were introduced into K562/A02 cell by lipofectamine mediation.At the 24-hour,48-hour and 72-hour time point after transfection,RT-PCR and western blotting were employed to detect the expression of VEGF mRNA and protein in K562/A02 cell.2.The proliferative ability of K562/A02 cell was determined by MTT assay and cell cycles analysis.Apoptosis of K562/A02 cell was assayed by AnnexinⅤ-FITC/PI double labeled flow cytometry.The drug sensitivity to anticancer agent(doxorubicin)was determined by MTT assay.The expression of multidrug resistance-relating genes(MDR1,MRP1 and topoⅡ)and proteins were detected by RT-PCR,flow cytometry and western blotting,respectively.Intracellular Rhodamine 123(Rho123)retention assay was applied to test the function of P-gp and MRP1.Western blotting was used to detect the expression of ERK,P-ERK,AKT and P-AKT.Results:1 The expression of VEGF mRNA in K562/A02 cell was significantly higher than that in K562 cell by RT-PCR;The density value of VEGF gene standarded byβ-actin were 0.518±0.055,0.814±0.073(t=6.845, p＜0.001),respectively;And the similar result was yielded by western blotting,the level of VEGF protein in K562/A02 cell was significantly higher than that in K562 cell.2 The random sequence HK and the fragments of VEGF shRNA have been transfected into the human leukemia cell line K562/A02 cell,then five cell lines were obtained as followed:K562/A02,K562/A02-HK,K562/A02-VEGF shRNA1,K562/ A02 -VEGF shRNA2 and K562/A02-VEGF shRNA3.At the 24-hour, 48-hour and 72-hour time point after transfection,The expression of VEGF in mRNA and protein level decreased dramatically,specially in K562/A02 cell transfected with VEGF shRNA3,and the inhibitory effects of VEGF shRNA3 sequence targeting VEGF in mRNA level at different time point were(29.6±1.1)%,(45.9±1.6)%and(43.2±1.5)%,repectively.The introduction of exogenous VEGF shRNA gene into K562/A02 cell resulted in decreasing levels of the expression of VEGF in mRNA and protein level.2.The introduction of exogenous VEGF shRNA gene into K562/A02 cell resulted in decreaseing levels of the proliferative ability in K562/A02 cells,higher G1,lower G2 and S ratio in cell cycle distribution in comparison with the control groups(p＜0.05).AnnexinⅤ/PI detection indicated higher AnnexinⅤratio in three positively transfected cells after treated with As2O3 0.5μM for 24 hours.The ratio of apoptosis of the five cell lines were(1.52±0.56)%,(9.14±0.98)%,(11.09±1.47)%(t=2.206, p=0.07),(19.89±1.63)%(t=11.29,p＜0.001) and(32.30±2.16)% (t=19.529,p＜0.001),repectively,indicating that when compared with group HK,the apoptosis of K562/A02 cell transfected with VEGF shRNA2 or VEGF shRNA3 has significantly statistical difference.The IC50 values of doxorubicin in three positively transfected cells were lower than that in control groups.The IC50 values in five groups were 19.23±1.633,18.66±1.632,17.67±1.425(t=0.982,p=0.364),16.11±1.184 (t=2.553,p=0.043) and 11.46±1.061(t=7.394,p＜0.001),repectively. When compared with control groups,all the mRNA level of MDR1,MRP1 and topoⅡgenes in three positively transfected cells were differently down-regulated by RT-PCR,respecially in K562/A02 cell transfected with VEGF shRNA3.The protein levels of MRP1,topoⅡand P-gp in three positively transfected cells were lower than that in control groups,respectively,by western blotting and flow cytometry.By flow cytometry,the mean fluorescence intensity of intracellular Rho 123 in five groups were 11.823±1.611,11.784±1.633,15.683±1.633(t=3.377, p=0.015),16.158±1.45(t=3.613,p=0.011),699.294±73.485(t=18.707, p＜0.001)(n=4),respectively,indicating significantly statistical difference when compared with group HK.Both the phosphorylation of ERK and AKT were decreased in K562/A02 cell after transfection with VEGF shRNA by western blotting,specially VEGF shRNA3.Conclusion:The mRNA and protein level of VEGF in persister K562/A02 cell were significantly higher than that in sensitive strain K562 cell.Transfection with exogenous VEGF shRNA gene can inhibit the proliferation of leukemia cells by delaying the progression G1 into S stage in cell cycles and induce cell apoptosis by down-regulating the VEGF gene expression.After VEGF shRNA were transfected into K562/A02 cell,the expression of MDR1,MRP1 and topoⅡin mRNA and protein level were decreased that may responsible for the higher sensitivity to anticancer agent of transfected leukemia cells,and the MAPK/ERK or PI3K/AKT signal-transduction pathway may be play an important role in this process. Our study demonstrated that VEGF involved the drug resistance,cell proliferation and apoptosis of leukemia cells.then it would be a new target to reverse the multidrug resistance in human malignancies.
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