脑源性神经营养因子基因沉默对RPMI8226细胞表达VEGF的影响

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 P(0) and P(1), 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: ①The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. BDNF mRNA expression and protein level in P(1) group were significantly inhibited compared to those in non-transfected group (P(n)) and P(0) groups (P < 0.05); ②MTT tests demonstrated that the cellular proliferation activities were obviously decreased in P(n) (0.42±0.06) vs P(0) (0.56±0.06) and P(1) (0.50±0.04) groups (P <0.05); ③The early cell apoptosis rates were statistically increased in P(1) [(53.84±9.95)%] vs P(n) [(5.23±2.46)%] and P(0) [(9.10±3.46)% ] groups (P<0.01); ④The silence of endogenous BDNF significantly decreased the expression of VEGF in RPMI8226 cells: the relative expression level of VEGF(121), VEGF145 and VEGF165 in P(1) group were (0.62± 0.07), (0.47±0.09) and (0.57±0.02) folds compared to P(n) group (P <0.05); ⑤ELISA demonstrated that secretion of VEGF in P(1) group were (0.36±0.05) and (0.44±0.06) folds compared to P(n) and P(0) group, respectively (P <0.05). CONCLUSION: 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.