靶向沉默Gli1基因表达对K562细胞增殖的影响及其机制探讨  

Effect of Glil gene silencing on proliferation of K562 cells and its mechanisms

作　　者：苏文霞[1] 陈永红[1] 曾雯[1] 刘文励[1] 孙汉英[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院血液科,武汉430030

出　　处：《中华血液学杂志》2012年第7期570-573,共4页Chinese Journal of Hematology

基　　金：教育部博士点基金（200804870008）

摘　　要：目的探讨靶向沉默Sonichedgehog（Shh）信号通路的转录因子Gli1基因表达对K562细胞增殖的影响及其机制。方法将针对Gli1的小干扰RNA（smallinterferingRNA，siRNA）通过脂质体（Lipofectamine’”2000）导入K562细胞作为实验组，并以非特异性siRNA为对照组。实时定量PCR和Westernblot法检测Gli1mRNA和蛋白表达，MTY检测细胞增殖，碘化丙锭（PI）检测细胞周期，实时定量PCR检测c—myc、p21基因的表达变化。结果转染后24h实验组Gli1mRNA表达水平为对照组的（52．60±3．57）％（P〈0．01），转染后48h实验组Gli1的蛋白表达水平为对照组的（79．31±5．58）％（P〈0．01）。转染后24h实验组细胞增殖率为对照组的（94．41±3．58）％（P〈0．05），48h为对照组的（90．22±3．34）％（P〈0．01）。转染后24、48h实验组细胞出现G2／M期阻滞且c—myc的表达水平下降，而p21的表达水平升高（P值均〈0．05）。结论靶向沉默Gli1基因表达抑制了K562细胞的增殖，该抑制作用是通过下调c—myc基因、上调p21基因的表达实现的。Objective To investigate the effect of Glil gene silencing by RNA interference （RNAi） on proliferation of K562 ceils and its mechanisms. Methods The small interference RNA （siRNA） was syn- thesized in vitro. K562 cells were transfected with Glil siRNA by the way of lipofection （lipofectamine 2000）. Non-specific siRNA transfected cells were used as control. Transfection efficiencies of different siR- NA concentrations were detected by flow cytometry and the best siRNA concentration was selected. The silen- cing effect of siRNA was demonstrated by real time PCR and Westem blot analysis. Cell proliferation was measured by MTI＂ method, cell cycle by PI assay, c-myc and p21 mRNA level was detected by real time PCR analysis. Results Transfection efficiency of siRNA was increased in a dose-dependent manner when siRNA concentration was below 200 pmol, and the highest transfection efficiency reached （ 80.11 ± 5.63 ） %. Both the mRNA and protein level of Glil was down-regulated in Glil specific siRNA group, the mRNA level was （52.60 ±3.57 ） % of that of control group after 24 h （ t = 20. 33, P 〈 0.01 ） and the protein level was （79.31 ± 5.58 ） % of that of control group after 48 h （ t = 6. 54, P 〈 0.01 ）. The cell proliferation rate in Glil siRNA group was （94.41_±3.58）% （t=2.40, P=0.05） and （90.22±3.34）% （t=4.37, P〈 0.01 ） of that of control group after 24 h and 48 h, respectively. Gz/M cell cycle arrest was observed, the mRNA level of c-myc was down-regulated while p21 was up-regulated in Glil siRNA group after 24 h and 48 h （P 〈 0.05 ）. Conclusions Targeted silencing of Glil gene by RNAi inhibits the proliferation of K562 cells, which acts through the down-regulation of c-myc and up-regulation of p21 expression.

关 键 词：RNA干扰 SHH信号通路 基因 Gli1 细胞增殖 

分 类 号：R733.72[医药卫生—肿瘤]