摘要
目的:应用特异性小干扰RNA(siRNA)下调97-H细胞中脑源性神经生长因子(BDNF)表达,观察对细胞凋亡和侵袭的影响并探讨相关分子机制。方法:在人肝细胞癌(HCC)细胞系97-H中,采用蛋白质印迹法检测BDNF的表达,采用ELISA方法检测培养上清上BDNF的分泌水平。特异性BDNF-siRNA转染细胞,采用FITC-phalloidin染色方法检测actin细胞骨架的变化,采用western blot方法检测细胞内RhoA、Rac1、Cdc42的活化情况。同时,流式细胞术检测细胞凋亡,Transwell小室测定细胞侵袭能力的变化。结果:97-H细胞培养上清中BDNF含量为(119.08±6.21)ρg/mL。在97-H细胞中,特异性BDNF-siRNA显著抑制BDNF的表达,干扰细胞内actin细胞骨架聚合,RhoA或Rac1活性受到抑制,同时与对照组相比,凋亡细胞百分比增加至(27.00±1.71)%,P=0.000,侵袭细胞数减少至(26.9±1.6)%,P=0.000。结论:干扰BDNF的表达能显著降低HCC细胞侵袭能力,其机制可能与阻断actin细胞骨架聚合、以及RhoA或Rac1活化相关。BDNF信号通路可能作为阻断HCC发展演进的新靶点,有待于进一步深入研究。
OBJECTIVE： To investigate the effects of siRNA specific for BDNF on apoptosis and invasion of 97-H cells and its potential molecular mechanism. METHODS： The expression of BDNF in cells was examined by western blot and BDNF secretion was evaluated by ELISA in human HCC cell lines of 97-H. BDNF knockdown was performed by spe- cific BDNF-siRNA transfection in 97-H cells and actin cytoskeleton was shown by FITC-phalloidin staining and the activa- tions of RhoA. Racl or Cdc42 were determined by western blot. Cell apoptosis and invasion were examined by flow cy- tometry and transwell assay respectively. RESULTS： The expression of BDNF was found in 97-H cells. BDNF concentra- tion in the supernatant of 97-H cells was （119. 08± 6. 21） ρg/mL. Inhibited expression of BDNF by Specific siRNA showed impaired aetin polymerization and decreased activations of RhoA or Racl in 97-H cells. BDNF knockdown also in- duced apoptosis and suppressed invasion of 97-H cells. Compared with the control group the apoptosis rate imcreased to （27. 00±1.71）% （P=0. 000） and the invasion rate decreased to （26.9±1.6）% （P=0. 000）. CONCLUSIONS： Our re- sults indicated that BDNF knockdown inhibited cell invasion probably through the blocked actin polymerization and the correlated inactivation of RhoA or Racl. Aiming at BDNF/TrkB signaling interruption may be an effective strategy to prevent HCC progression.
出处
《中华肿瘤防治杂志》
CAS
北大核心
2012年第17期1294-1297,1307,共5页
Chinese Journal of Cancer Prevention and Treatment
基金
辽宁省教育厅2009年度高等学校科研项目(2009A729)
关键词
癌
肝细胞
神经生长因子类
细胞骨架
细胞培养
carcinoma, hepatocellular
nerve growth factors
cytoskeleton
cell culture
