shRNA干扰技术在脑胶质瘤细胞TGFβ2／Smads通路中的作用  

shRNA interference on TGF - β2/Smads signal pathway in human glioma cell

作　　者：董程远[1] 米蕊芳[1] 金贵善[1] 周益强[1] 聂秀涛[1] 张国滨[1] 张晋[1] 刘福生[1] 

机构地区：[1]首都医科大学北京市神经外科研究所,首都医科大学附属北京天坛医院神经外科,100050

出　　处：《中华神经外科杂志》2014年第4期404-408,共5页Chinese Journal of Neurosurgery

基　　金：国家自然科学基金项目资助（81071776,81372354,81302186）;北京市自然科学基金项目资助（7102027,7132034）;北京市卫生系统高层次卫生技术人才培养项目资助（2013-2-018）

摘　　要：目的利用shRNA干扰技术抑制恶性脑胶质瘤细胞TGFβ2／Smads通路中Smad2／3基因的表达，探讨其在恶性胶质瘤细胞增殖中的作用。方法将表达Smad2／3shRNA干扰序列的特异性质粒及阴性对照质粒用脂质体转染法，分别转入到恶性脑胶质瘤细胞U251中。应用Real—TimePCR和Westernblot法分别检测细胞中Smad2／3基因mRNA和蛋白的表达；转染后的细胞经TGFl32（＋／-）处理，采用CCK-8法评价两组的增殖率。结果（1）两种特异性质粒能够分别下调细胞中Smad2／3基因的表达（P=0．01）；（2）Smad2／3的表达量在Smad3／2表达下调组中明显高于对照组（P=0．018，P=0．008）；（3）Smad2／3表达下调组的细胞增值率明显高于对照组（P=0．001）；（4）Smad3表达下调的细胞，在TGFl32（＋／-）处理的两组中增殖率差异无统计学意义（P=0．258）。结论在恶性胶质瘤细胞U251中，Smad2／3基因表达的下调能促进细胞的增殖，而TGFl32介导的细胞增殖依赖于TGFl32／Smad3通路；同时Smad2／3基因的表达下调增强了Smad3／2基因的表达。Objective Using shot hairpin RNA （shRNA） interference to specifically deplete cells expression of Smad2/3 gene of TGF - β2/Smads signal pathway and to investigate their effects on the proliferation of hunman glioblastoma mutiforme （GBM） cells. Methods GBM cell line U251 was transfected with plasmids taking psh - Smad2 or psh - Smad3 that expressed shRNA targeting Smad2 or Smad3 gene, and the negative control plasmid. The expression levels of Smad2/3 mRNA and protein were measured by Real - time fluorescence Quantitative PCR （ Real - time PCR） and Western blot. Ceils were treated by TGF - [32 （ ＋ / - ） after being transfected with plasmids. The proliferation rate was evaluated by CCK- 8 assay. Results （1）Real -time PCR and Western blot illustrated that the Smad2/3 expression levels were dramatically down - regulated in U251 cells by shRNA interference （ P = 0. 01 ）. （ 2 ） The proliferation of cells transfected with psh - Smad2 and psh - Smad3 group was obviously higher than that of negative control group（ P =0. 018, P =0. 008）. （3）The rate of cell proliferation was similar between the ceils treated with and without TGF - β2 when Smad3 was knocked down （ P = 0.001 ）. （4） Additionally, the expression level of Smad3 in psh - Smad2 group was significantly higher than that of the psh - NC group and the mRNA expression of Smad2 in psh - Smad3 group also was significantly higher than that of the psh - NC group（ P = O. 258）. Conclusions Knockdown of Smad2/3 both enhanced the cellular proliferation. U251 cell proliferation inducted by TGFβ2 was dependent on Smad3 but not Smad2. And depletion of Smad2 enhanced Smad3 expression, meanwhile depletion of Smad3 also enhanced Smad2 expression.

关 键 词：RNA干扰 神经胶质瘤 转化生长因子Β2 基因 

分 类 号：R739.41[医药卫生—肿瘤]