Shen Hui-Ling, Xu Wenlin, Wu Zhao-Yang, Zhou Lei-Lei, Qin Ru-Juan, Tang Hua-Rong
Department of Oncology, The Affiliated People's Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, Jiangsu 212002, PR China.
Leuk Res. 2007 Apr;31(4):515-21. doi: 10.1016/j.leukres.2006.09.011. Epub 2006 Oct 10.
Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. K562 human leukemia cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. In the present study, three 19 bp reverse repeated motifs targeting exons 5 and 7 boundary of VEGF165 gene sequence with 9 bp spacer 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 K562 cells, respectively, through lipofectamine reagent. A vector-based small interfering RNA(SiRNA) inhibited VEGF165 mRNA expression by 72% and protein production by 67% in K562 cells. Human microvascular endothelial cell migration induced by conditioned medium from VEGFsi-transfected K562 cells was significantly less than that induced by conditioned medium from K562 cells and control vector-transfected K562 cells. Furthermore, the VEGF shRNA dramatically suppressed tumor angiogenesis and tumor growth in a K562 s.c. xenograft model. Vessel density as assessed by vWF immunohistochemical analysis was also decreased. This strategy provides a novel tool to study the function of various VEGF isoforms and may contribute to VEGF-specific treatment in cancer.
血管内皮生长因子（VEGF）在正常胚胎血管生成过程中以及在包括癌症在内的多种疾病中发生的病理性血管生成中都起着关键作用。K562人白血病细胞过度表达VEGF，其异构体产生从膜结合的VEGF189转变为更具可溶性的VEGF165。在本研究中，合成了三个靶向VEGF165基因序列外显子5和7边界的19 bp反向重复基序，间隔9 bp，并将其克隆到含有U6 shRNA启动子和RNA聚合酶终止信号的真核表达质粒pGenesil-1中。通过脂质体转染试剂将重组质粒pGenesil-VR1、pGenesil-VR2、pGenesil-VR3和pGenesil-con（含有随机DNA片段的质粒）分别转染到K562细胞中。基于载体的小干扰RNA（SiRNA）在K562细胞中抑制VEGF165 mRNA表达72%，抑制蛋白质产生67%。VEGF siRNA转染的K562细胞条件培养基诱导的人微血管内皮细胞迁移明显少于K562细胞和对照载体转染的K562细胞条件培养基诱导的迁移。此外，VEGF shRNA在K562皮下异种移植模型中显著抑制肿瘤血管生成和肿瘤生长。通过vWF免疫组织化学分析评估的血管密度也降低了。该策略为研究各种VEGF异构体的功能提供了一种新工具，并可能有助于癌症的VEGF特异性治疗。
