应用RNA干扰技术抑制白血病细胞血管内皮生长因子基因表达及功能的研究

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

目的探讨用RNA干扰技术下调血管内皮生长因子(VEGF)基因表达对白血病细胞生长的影响.方法设计3段分别针对VEGF第3～5外显子的短发夹状RNA(shRNA)并构建其表达载体,分别转染人白血病细胞株NB4,用实时定量PCR及Western blot方法观察3段shRNA对细胞内VEGF基因表达的影响.抗性筛选稳定抑制VEGF表达的永久细胞克隆,应用MTT法、甲基纤维素半固体培养法及细胞周期动力学检测了解VEGF基因缄默对白血病细胞生长的影响.结果成功构建分别携带3段shRNA及对照随机片段的重组质粒pGenesil-VR1,2,3和pGenesil-con,3种shRNA重组质粒中pGenesil-VR3可明显降低细胞内VEGF mRNA的丰度及VEGF蛋白表达,筛选出的NB4-VR3细胞株增殖能力明显下降,细胞集落形成率为(13.3±3.8)%,与NB4-con的(21.3±6.4)%和NB4组的(24.5±5.2)%相比,差异有统计学意义(P＜0.05),细胞周期阻滞于G1期,3组细胞G1期比例分别为(53.2±4.6)%、(42.7±5.9)%和(40.5±5.3)%.结论应用RNA干扰技术能筛选出特异而高效阻断VEGF基因表达及功能的shRNA,VEGF基因表达下调能明显抑制白血病细胞生长.

Objective 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. Methods 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^TM 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. Results 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 cells transfected with pGenesil-VR3 （ NB4 -VR3） had a more significant decrease in proliferation ability than NB4 and that transfected with pGenesil-con（NB4-con）. The colony forming efficieneies of NB4 -VR3, NB4-con and NB4 cell were （ 13.3 ± 3.8 ） % , （21.3 ± 6.4） % and （24.5 ± 5.2） % , respectively （ P 〈 0.05 ）. Higher G1 and lower S proportion were found in cell cycle distribution in comparison with the control groups by FCM. Conclusions The shRNA can efficiently suppress VEGF expression in NB4 cells. Selective VEGF gene silence can inhibit the malignant proliferation of leukemia cells.