Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells.
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.
Shen
Hui-Ling
HL
Department of Oncology, The Affiliated People's Hospital, Jiangsu University, 8 Dianli Road, Zhenjiang, Jiangsu 212002, PR China.
Xu
Wenlin
W
Wu
Zhao-Yang
ZY
Zhou
Lei-Lei
LL
Qin
Ru-Juan
RJ
Tang
Hua-Rong
HR
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002465:Cell Movement; D049109:Cell Proliferation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D020014:K562 Cells; D007942:Leukemia, Experimental / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D015996:Survival Rate; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays / Q000379:methods*
D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A