title: [Effect of shRNA-mediated silencing of BDNF gene on VEGF expression of RPMI8226 cells].
abstract: OBJECTIVE
  To investigate the expression of vascular endothelial growth factor (VEGF) of human multiple myeloma (MM) cell line RPMI8226 regulated by brain-derived neurotrophic factor (BDNF), and preliminarily approach the close relationship between BDNF and angiogenesis of MM.
  
  
  METHODS
  The recombinant eukaryotic BDNF siRNA expression vector was designed and constructed. The empty vector pGenesil-1, and the recombinant plasmid, pGenesil-shRNA-BDNF were transfected into RPMI8226 cells using Lipofectamine™ 2000 (groups P0 and P1, respectively). BDNF mRNA and protein level in RPMI8226 cells were detected by RT-PCR and Western blotting, respectively; the cellular proliferation activity was determined by MTT assay, while the cell apoptosis was measured by flow cytometry; the variation of VEGF mRNA level in RPMI8226 cells via transfection was determined by RT-PCR, the secretion of VEGF was detected by ELISA.
  
  
  RESULTS
  (1)The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. BDNF mRNA expression and protein level in P1 group were significantly inhibited compared to those in non-transfected group (Pn) and P0 groups (P<0.05); (2)MTT tests demonstrated that the cellular proliferation activities were obviously decreased in Pn (0.42 ± 0.06) vs P0 (0.56 ± 0.06) and P1 (0.50 ± 0.04) groups (P<0.05); (3)The early cell apoptosis rates were statistically increased in P1 [(53.84 ± 9.95)%] vs Pn [(5.23 ± 2.46)%] and P0 [(9.10 ± 3.46)%] groups (P<0.01); (4)The silence of endogenous BDNF significantly decreased the expression of VEGF in RPMI8226 cells:the relative expression level of VEGF121, VEGF145 and VEGF165 in P1 group were (0.62 ± 0.07), (0.47 ± 0.09) and (0.57 ± 0.02) folds compared to Pn group (P<0.05); (5)ELISA demonstrated that secretion of VEGF in P1 group were (0.36 ± 0.05) and (0.44 ± 0.06) folds compared to Pn and P0 group, respectively (P<0.05).
  
  
  CONCLUSIONS
  BDNF gene silence can obviously increase apoptosis of RPMI8226 cells, inhibit their proliferation and decrease the expression of VEGF. BDNF might mediate the expression of VEGF in MM cells, which may be involved in MM angiogenesis.
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Zhangbo
authors.initials: Z
authors.lastname: Chu
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Yadan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Sun
mesh_terms: D017209:Apoptosis; D019208:Brain-Derived Neurotrophic Factor; D045744:Cell Line, Tumor; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D009101:Multiple Myeloma; D009389:Neovascularization, Pathologic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C579455:BDNF protein, human