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).
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.
目的: 研究脑源性神经营养因子（BDNF）对多发性骨髓瘤（MM）细胞表达血管内皮生长因子（VEGF）的调控作用，初步探讨BDNF与MM血管新生的关系。
方法: 设计和构建针对人BDNF基因的siRNA表达载体，用脂质体Lipofectamine™2000介导转染，将空载体质粒pGenesil-1和重组质粒pGenesil-shRNA-BDNF分别转染MM细胞株RPMI8226细胞（分别命名为P0组、P1组）。采用RT-PCR法和Western-blot法鉴定干扰后BDNF的表达；MTT法和流式细胞术检测细胞的增殖、凋亡；RT-PCR法和ELISA法检测干扰BDNF对细胞表达VEGF的影响。
结果: ①成功构建了针对BDNF的siRNA真核表达载体。与未转染组（Pn组）及P0组相比，P1组BDNF mRNA水平及蛋白水平显著下调（P值均<0.05）；②P1组细胞的增殖活性（0.42±0.06）显著低于Pn组（0.56±0.06）和P0组（0.50±0.04）（P值均<0.05）；③P1组细胞的早期凋亡率[（53.84±9.95）%]明显高于Pn组[（5.23±2.46）%]和P0组[（9.10±3.46）%]（P值均<0.01）；④P1组细胞VEGF121、VEGF145和VEGF165 mRNA表达水平分别为Pn组的（0.62±0.07）、（0.47±0.09）和（0.57±0.02）倍（P值均<0.05）。⑤P1组细胞VEGF的分泌水平分别为Pn组的（0.36±0.05）倍和P0组的（0.44±0.06）倍（P值均<0.05）。
结论: BDNF基因沉默可促进RPMI8226细胞凋亡并抑制其增殖，下调RPMI8226细胞VEGF的表达，BDNF可能通过调控MM细胞VEGF的表达而参与MM血管新生的病理过程。