shRNA干扰VEGF表达对白血病细胞多药耐药的影响

Effect of knockdown of VEGF expression by shRNA on multidrug resistance of leukemia cell line K562/A02

目的:探讨血管内皮细胞生长因子(VEGF)与人白血病耐药细胞株K562/A02细胞多药耐药的关系及其机制。方法:针对VEGF基因设计合成3条干扰片段shRNA1,2,3,采用脂质体2000分别将其转染入K562/A02细胞,RT-PCR法检测VEGF和多药耐药相关基因MDR1,MRP1,topoⅡ,GST的mRNA水平;蛋白质印迹法检测VEGF蛋白水平;MTT法检测各组细胞对多柔比星的半数抑制浓度(IC50值)。结果:K562/A02细胞VEGFmRNA表达水平显著高于敏感株K562细胞,差异有统计学意义(P<0.01),且VEGF蛋白水平也显著高于K562细胞;转染shRNA后,K562/A02细胞中VEGFmRNA水平出现不同程度下调,其中shRNA2组和shRNA3组与转染随机片段组比较差异有统计学意义(P<0.05),以shRNA3组下调最显著,并且VEGF蛋白水平也出现相应的下调;转染48 h后K562/A02细胞中MDR1,MRP1及topoⅡ的mRNA水平均出现不同程度下调,以shRNA3组下调最显著,分别下调(39.7±1.21)%,(29.1±0.97)%,(28.2±1.04)%,GST基因表达则无明显变化;阳性转染组细胞对多柔比星的敏感性明显增加,其中以shRNA3组最显著。结论:VEGFshRNA可以抑制K562/A02细胞VEGF基因与蛋白的表达,不同干扰片段的沉默效果不同,并增强K562/A02细胞对多柔比星的敏感性,其机制可能与MDR1,MRP1及topoⅡ的表达下调有一定的关系。

Objective： To study the relationship between vascular endothelial growth factor（VEGF） and multidrug resistance of human leukemia cell line K562/A02 by RNA interference, and then the mechanism was explored. Methods： The VEGF shRNA chainl ,2 and 3 were designed, synthesized, then transfected into K562/A02 cells line by lipofectamine 2000 reagent. RT-PCR was used to detect the expression of VEGF and MDR-relating genes MDR1, MRP1, topo 11 , GST in mRNA level; Western blotting was used to analyzed the expression of VEGF in protein level ; MTT was used to determine the IC50 of transfectional cells to doxornbicin. Results： Compared to K562 cell, the expression of VEGF in K562/A02 cell was even more （P 〈 0.01 ） , and after VEGF shRNAs were transfected into K562/A02 cells, the expression of VEGF at the mRNA level were decreased, and there were statistical difference between VEGF shRNA2 group or VEGF shRNA3 group and HK group （ P 〈 0.05 ）, the greatest decrease was seen in cells transfected with VEGF shRNA3 ; and the protein level of VEGF were also down-regulated. The MDR1 ,MRP1, topo Ⅱ mRNA of K562/A02 cell were significantly decreased; the greatest decrease was seen in VEGF shRNA3 group. The ICs0 value of positively transfected group were lower than that of control groups. Conclusion： After silence of VEGF by shRNA, the mRNA expression of MDR-associated genes such as MDR1, MRP1 and topo Ⅱ in K562/A02 cells were down-regulated, and the sensitivity of K562/A02 cell to doxorubicin were increased.