详细记录  
题名: Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells
作者:Hui-Ling Shen, Wenlin Xu, Zhao-Yang Wu, Lei-Lei Zhou, Ru-Juan Qin and Hua-Rong Tang
来源:Leukemia Research[IF=2.372], Volume 31, Issue 4, April 2007, Pages 515-521
URL :http://dx.doi.org/10.1016/j.leukres.2006.09.011
日期:070415
摘要: Hui-Ling Shena, Wenlin Xu, a, , Zhao-Yang Wua, Lei-Lei Zhoua, Ru-Juan Qina and Hua-Rong Tanga

aDepartment of Oncology, The Affiliated People s Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, Jiangsu 212002, PR China

Received 6 July 2006; revised 3 September 2006; accepted 5 September 2006. Available online 10 October 2006.

Summary

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. 12.

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