Shen Hui-ling, Xu Wen-lin, Wu Zhao-yang, Qi Yan-yan, Zhong Xi-ming, Huang Wei-bing, Xiao Ming
Department of Oncology, The Affiliated People's Hospital of Jiangsu University, Zhenjiang 212002, China.
Zhonghua Xue Ye Xue Za Zhi. 2005 Dec;26(12):710-4.
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.
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 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.
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 efficiencies 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 G(1) and lower S proportion were found in cell cycle distribution in comparison with the control groups by FCM.
The shRNA can efficiently suppress VEGF expression in NB4 cells. Selective VEGF gene silence can inhibit the malignant proliferation of leukemia cells.
探讨应用血管内皮生长因子（VEGF）短发夹RNA（shRNA）干扰技术选择性抑制VEGF表达的可行性，并观察VEGF基因沉默对NB4细胞生长的影响。
合成分别靶向VEGF基因外显子3、4、5的3个19bp反向重复序列，克隆至含U6 shRNA启动子和RNA聚合酶终止信号的真核表达质粒pGenesil-1中。重组质粒pGenesil-VR1、pGenesil-VR2、pGenesil-VR3和pGenesil-con（含随机DNA片段的质粒）分别经脂质体转染试剂转染入NB4细胞。采用荧光实时RT-PCR和Western blot检测VEGF表达的变化。通过台盼蓝拒染法、MTT法、集落形成试验和细胞周期分析检测白血病细胞的增殖能力。
成功构建了含3种shRNAs和随机片段的重组质粒，并通过脂质体介导的基因转移方法分别转染入NB4细胞。与pGenesil-VR1、Genesil-VR2和pGenesil-con相比，pGenesil-VR3细胞中的shRNA以序列特异性方式显著降低了VEGF mRNA和蛋白的表达。转染pGenesil-VR3（NB4-VR3）的NB4细胞增殖能力比NB4及转染pGenesil-con（NB4-con）的细胞明显降低。NB4-VR3、NB4-con和NB4细胞的集落形成效率分别为（13.3±3.8）%、（21.3±6.4）%和（24.5±5.2）%（P<0.05）。流式细胞术检测显示，与对照组相比，细胞周期分布中G1期比例升高，S期比例降低。
shRNA能有效抑制NB4细胞中VEGF的表达。选择性VEGF基因沉默可抑制白血病细胞的恶性增殖。
