Yang Xiang-Yun, Lai Xiao-Gang, Zhang Yong, Pei Jian-Ming, Yang An-Gang, Zhou Shi-Sheng
Department of Physiology, Xioan, Shaanxi, 710032, P. R. China.
Ai Zheng. 2006 Jul;25(7):805-10.
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 ClC-2 gene, on the proliferation of a human glioma cell line U-87.
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-ClC-21 and pSUPER. puro-shRNA-ClC-22, were transfected into U-87 cells using Lipofectamine2000 (Groups: PP0, PP1 and PP2, respectively). ClC-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.
The DNA fragments encoding siRNA targeting ClC-2-gene were successfully connected onto pSUPER. puro vector. ClC-2 mRNA expression and the cell growth rate in PP1 and PP2 groups were significantly inhibited compared to those in PP0 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 PP1 and 18.04% in PP2 vs. in control and PP0 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).
These results demonstrate that CIC-2 siRNA could inhibit the cell proliferation of a human glioma cell line U-87, thus ClC-2 gene may be used as a novel target for the suppression of the growth of human malignant glioma cells.
小分子干扰RNA（siRNA）已被用于抑制哺乳动物基因表达，是研究基因功能的有效工具。本研究旨在观察针对容积调控性氯通道ClC-2基因设计的siRNA对人胶质瘤细胞系U-87增殖的影响。
设计并构建了两种重组真核CIC-2 siRNA表达载体。通过限制性内切酶消化和DNA测序对序列进行鉴定和确认。使用Lipofectamine2000将空载体pSUPER.puro以及两种重组质粒pSUPER.puro-shRNA-ClC-21和pSUPER.puro-shRNA-ClC-22转染至U-87细胞中（分别为PP0、PP1和PP2组）。通过逆转录聚合酶链反应（RT-PCR）检测ClC-2 mRNA表达；采用MTT法测定细胞增殖率；通过流式细胞术（FCM）检测细胞周期；采用平板集落形成试验测定细胞集落形成率。
编码靶向ClC-2基因的siRNA的DNA片段成功连接到pSUPER.puro载体上。与PP0组和对照组相比，PP1组和PP2组中ClC-2 mRNA表达和细胞生长速率均显著受到抑制。同时，细胞周期阻滞于G1期，与对照组和PP0组相比，PP1组G1期细胞百分比增加约30.24%，PP2组增加18.04%，P<0.05。此外，siRNA处理组的细胞集落形成率在统计学上降低，PP1组为（11.0±1.0）%，PP2组为（20±3.1）%，而对照组为（46.5±1.6）%，PP0组为（47.5±2.8）%（P<0.01）。
这些结果表明CIC-2 siRNA可抑制人胶质瘤细胞系U-87的细胞增殖，因此ClC-2基因可作为抑制人恶性胶质瘤细胞生长的新靶点。
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