title: Inhibiting the growth of malignant melanoma by blocking the expression of vascular endothelial growth factor using an RNA interference approach.
abstract: BACKGROUND
  Vascular endothelial growth factor (VEGF) is overexpressed in malignant melanoma (MM).
  
  
  OBJECTIVE
  To develop an RNA interference approach that specifically targets VEGF by constructing a eukaryotic expression plasmid containing short interfering RNA (siRNA), and to evaluate the effects of this vector on the proliferation and apoptosis of MM in vitro and in vivo.
  
  
  METHODS
  pU-VEGF-siRNA plasmid was transfected into MM cell line A375 and colorectal carcinoma cell line Lovo by electroporation. Expression of VEGF mRNA and protein in A375 and Lovo cells after gene transfer was detected by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Proliferation of pU-VEGF-siRNA-transfected A375 and Lovo cells and control cells was observed by cell counting through the microscope. The proliferation of human umbilical vein endothelial cells (ECV-304) cultured in medium containing supernatants of transfected and control A375 cells was measured by the cell counting method. Flow cytometry (FCM) was used to analyse the apoptosis of transfected and control groups. In a mouse model, tumorigenicity and tumour growth of transfected cells were studied in vivo. VEGF expression and microvessel density (MVD) in tumour tissue were measured by immunohistochemistry. Apoptosis in tumours was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling.
  
  
  RESULTS
  Expression of VEGF mRNA and protein in pU-VEGF-siRNA-transfected A375 and Lovo cells was significantly decreased on days 3, 10, 17 and 24 post-transfection, compared with controls. The greatest suppression occurred on days 3 and 10 post-transfection. The proliferation of transfected A375 cells and ECV-304 cocultured with supernatants of transfected A375 cells was inhibited. FCM analysis showed that a hypodiploidy peak was found only in A375 cells transfected by pU-VEGF-siRNA. After subcutaneous inoculation with pU-VEGF-siRNA-transfected A375 cells, tumour growth in mice was inhibited, VEGF expression and MVD were decreased, and tumour apoptosis was increased significantly, in comparison with mice inoculated with untransfected A375 cells.
  
  
  CONCLUSIONS
  The delivery of siRNA directed against VEGF was shown not only to give efficient and specific downregulation of the expression of VEGF, inhibit proliferation of A375 and ECV-304 cells and induce apoptosis of A375 cells in vitro, but also to suppress growth of MM in vivo. These results suggest that a strategy based on siRNA targeting of VEGF may build the foundation to the clinical management of MM.
authors.affiliation: Department of Dermatology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: J
authors.initials: J
authors.lastname: Tao
authors.forename: Y-T
authors.initials: YT
authors.lastname: Tu
authors.forename: C-Z
authors.initials: CZ
authors.lastname: Huang
authors.forename: A-P
authors.initials: AP
authors.lastname: Feng
authors.forename: Q
authors.initials: Q
authors.lastname: Wu
authors.forename: Y-J
authors.initials: YJ
authors.lastname: Lian
authors.forename: L-X
authors.initials: LX
authors.lastname: Zhang
authors.forename: X-P
authors.initials: XP
authors.lastname: Zhang
authors.forename: G-X
authors.initials: GX
authors.lastname: Shen
mesh_terms: D000818:Animals; D017209:Apoptosis; D049109:Cell Proliferation; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D051379:Mice; D008833:Microcirculation; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A