siRNA干扰ClC-2表达对人胶质瘤U-87细胞增殖的抑制作用

siRNA-mediated Silencing of ClC-2 Gene Inhibits Proliferation of Human U-87 Glioma Cells

背景与目的:利用小干扰RNA(smallinterferingRNA,siRNA)抑制哺乳动物基因表达已成为研究基因功能的一种有效方法。本研究采用siRNA抑制人神经胶质瘤细胞系U-87细胞上容积调控氯通道ClC-2基因的表达,观察其受抑制后对细胞增殖能力的影响。方法:设计和构建两个针对人ClC-2基因的siRNA真核表达载体,并给予酶切鉴定和DNA序列分析鉴定;用脂质体LipofectamineTM2000介导转染,将空载体质粒pSUPER.puro-shRNA和两个重组质粒pSUPER.puro-shRNA-ClC-21、pSUPER.puro-shRNA-ClC-22分别转染入U-87细胞(依次为PP0组、PP1组和PP2组);采用RT-PCR检测ClC-2mRNA表达变化;MTT分析检测细胞活性;流式细胞仪检测细胞周期;平板克隆形成实验检测克隆形成率。结果:目的片段成功地连接到真核细胞表达载体pSUPER.puro上。PP1组、PP2组与对照组、PP0组相比较,ClC-2基因的mRNA水平明显降低,细胞生长速度明显减慢,细胞周期进程被阻滞在G1期:细胞的G1期百分含量分别增加了约30.24%、18.04%(P<0.05)。平板克隆形成试验发现,克隆形成率PP1组[(11.0±1.0)%]、PP2组[(20±3.1)%]明显低于对照组[(46.5±1.6)%]和PP0组[(47.5±2.8)%](P<0.01)。结论:干扰人神经胶质瘤细胞系U-87细胞的ClC-2基因表达可以抑制细胞的增殖,提示ClC-2基因可能成为控制人恶性胶质瘤生长的新靶点。

BACKGROUND ＆ OBJECTIVE： Small interfering RNA （siRNA）, which has been used to inhibit mammalian gene expression, is demonstrated to be an effective tool for gene function investigation. The aim of the present study was to observe the effect of siRNA, which was designed to target volume-regulated chloride channel CIC-2 gene, on the proliferation of a human glioma cell line U-87. METHODS： Two recombinant eukaryotic CIC-2 siRNA expression vectors were designed and constructed. Sequences were identified and confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector, pSUPER, puro, and two recombinant plasmids, pSUPER, puro-shRNA-CIC-21 and pSUPER, puro-shRNA-CIC-22, were transfected into U-87 cells using LipofectamineTM2000 （Groups： PP0, PP1 and PP2, respectively）. CIC-2 mRNA expression was detected by reverse transcription polymerase chain reaction （RT-PCR）; the cellular proliferation rate was determined by MTT assay; the cell cycle was measured by flow cytometry （FCM）; and the cell colony formation rate was measured by plate colony formation assay. RESULTS： The DNA fragments encoding siRNA targeting CIC-2-gene were successfully connected onto pSUPER, puro vector. CIC-2 mRNA expression and the cell growth rate in PP1 and PP2 groups were significantly inhibited compared to those in PPo and control groups. Meanwhile, cell cycle was arrested in G1 phase and the percentage of G1 phase cells were increased by about 30.24% in PP~ and 18.04% in PP2 vs. in control and PPo groups, P 〈 0.05. Moreover, the cell colony formation rates were statistically decreased in siRNA treated groups, which were （11.0± 1.0）% in PP1 and （20±3.1）% in PP2 vs. （46.5±1.6）% in control and （47.5± 2.8）% in PP0 groups （P〈0.01）. CONCLUSION： These results demonstrate that CIC-2 siRNA could inhibit the cell proliferation of a human glioma cell line U-87, thus CIC-2 gene may be used as a novel target for the suppression of the growth of human malignant glioma cells.