RNAi沉默Glil基因对人胶质瘤细胞U251增殖与凋亡的影响  

Effect of RNA interference of Glil gene expression on proliferation and apoptosis of glioma cell line U251

作　　者：田海龙[1] 白靖平[1] 李惠武[1] 李卉[1] 柳琛[1] 段明军[1] 

机构地区：[1]新疆医科大学附属肿瘤医院神经外科,乌鲁木齐830011

出　　处：《中华神经医学杂志》2011年第8期768-773,共6页Chinese Journal of Neuromedicine

摘　　要：目的研究RNA干扰技术（RNAi）沉默Hedgehog（Hh）信号转导途径中核心转录因子Glil基因表达后对U251细胞增殖与凋亡以及下游因子Bcl-2、Bax、cycinD1表达的影响。方法合成、转染4对针对GlilmRNA不同位点序列的siRNA（s8、59、60、61）至人胶质瘤U251细胞．RT-PCR检测细胞Glil mRNA的表达，筛选最有效抑制Glil mRNA表达的siRNA干扰片段（siRNA-Gill）；RT-PCR、Western blotting分别检测转染SiRNA．Glil后不同时间U25l细胞Glil mRNA和蛋白的表达，确定转染干扰的时间规律；实验分为3组：siRNA-Gli1组（转染筛选后siRNA-Gli1片断）、siRNA-NC组（转染阴性对照siRNA片断1、siRNA-N组（空白对照）。RT-PCR、Western blotting分别检测各组转染48h后U251细胞cyclin D1、Bcl-2及BoxmRNA和蛋白的表达，MTT、流式细胞术分别检测细胞增殖、凋亡及细胞周期的变化。结果干扰片断58、59、60、61、NC的转染效率均达69．2％；RT—PCR检测显示转染后48h U251-60细胞无明显GlilmRNA表达，选择60作为最佳干扰片段siRNA-Glil转染U251细胞，48h时无明显Glil mRNA和蛋白表达，确定48h为转染干扰的最佳时间；转染48h后与siRNA-N和siRNA-NC组相比，siRNA-Glil组U25l细胞Bcl-2、cyclin D1 mRNA和蛋白表达下调、Boxm RNA和蛋白上调，差异均有统计学意义（P〈0．05）。MTT检测显示24、48和72h时siRNA—Glil组细胞增殖抑制率均明显高于siRNA-NC和siRNA-N组，差异均有统计学意义（P〈0．05）。流式细胞术结果显示siRNA-Glil组细胞凋亡率高于siRNA-NC和siRNA-N组，差异有统计学意义（P〈0．05），且siRNA．Glil组Gl／G0期细胞比例增加，S期细胞明显减少。结论针对Glil基因设计合成的siRNA-Glil能明显抑制人胶质瘤U251细胞生长并能诱导其凋亡，其机制可能与下调cyclin D1、Bcl-2的表达，上调Bax的表达有关。Objective To investigate the inhibitory effect of RNA interference （RNAi） on Glil, Bcl-2, Box and cycin DI gene expressions in U251 cell line and the proliferation of U251 cells. Methods Small interfering RNA （siRNA, at locus of 58, 59, 60 and 61） targeted for Glil gene was designed and transfected into U251 cells. RT-PCR was emplyed to detect the mRNA expression of Glil gene to select the siRNA interference fragment （siRNA-Glil） that could most efficiently inhibit the mRNA expression of Glil gene. The mRNA and protein expressions of Glil gene at different times after siRNA-Glil transfection were detected to determine the time law of this interference. U251 cells at logarithmic phase were divided into 3 groups： siRNA-Glil group （transfection of selected siRNA-Glil fragments）, siRNA-NC （transfection of siRNA fragments） and siRNA-N group （blank controls）. The mRNA and protein expressions of Bcl-2, Box and cycin D1 gene were assessed by RT-PCR and Western blotting. Proliferation of cells was measured by MTT assay, and cell apoptosis and cell cycles were detected by flow cytometry （FCM）. Results Transfection efficiency of interference fragments （at locus of 58, 59, 60 and 61, and NC） reached 69.2%; RT-PCR indicated that no obvious Glil mRNA expression was noted at U251-60 cells 48 h after the transfection, therefore, locus 60 was the best interference fi＇agment and 48 h was the best time. The mRNA and protein expressions of Bcl-2 and cycin D1 genes were obviously suppressed by siRNA, and the rnRNA and protein expressions of Bax gene were significantly up-regulated in the siRNA-Glil group as compared with those in the siRNA-N and siRNA-NC groups 48 h after transfection （P〈0.05）. Silencing Glil by RNAi significantly inhibited the proliferation and induced the apoptosis of U251 cells as compared with siRNA-N and siRNA-NC groups 24, 48 and 72 h after transfection （P〈0.05）. Cells at GO and G1 phases were obviously increased and those at S phase were significantly decreased i

关 键 词：HEDGEHOG通路 信号转导 RNA干扰 

分 类 号：R739.4[医药卫生—肿瘤]