Effect of shRNA-mediated silencing of BDNF gene on VEGF expression of RPMI8226 cells.
- Author:
Jing HUANG
1
;
Zhangbo CHU
1
;
Lei CHEN
1
;
Yadan WANG
1
;
Lu ZHANG
1
;
Yu HU
1
;
Chunyan SUN
1
Author Information
- Publication Type:Journal Article
- MeSH: Apoptosis; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Gene Silencing; Genetic Vectors; Humans; Multiple Myeloma; Neovascularization, Pathologic; RNA, Messenger; RNA, Small Interfering; Transfection; Vascular Endothelial Growth Factor A
- From: Chinese Journal of Hematology 2015;36(5):403-407
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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).
CONCLUSIONBDNF 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.
