阻断Eag-1通道活性抑制胶质瘤细胞增殖的研究

Eag-1 channel blocking inhibits the proliferation of glioma cells

目的 探讨阻断Eag—1通道活性对胶质瘤细胞生物活性的影响。方法设计3对Eag-1通道蛋白的特异性小分子干扰RNA（siRNA）并转染人U87细胞，RT—PCR及Westernblotting检测其对U87细胞Eag-1 mRNA和蛋白表达的影响；将50nmol／LsiRNA1、siRNA2转染U87细胞，同时设5、10、20、30和40mmol／L奎尼丁（Eag-1通道特异性阻断剂）组和空白对照组，MTT法观察作用24、48、72h后U87细胞增殖情况，流式细胞仪检测作用48h后细胞周期、细胞凋亡率、胞内活性氧簇（ROS）浓度的变化。结果RT—PCR及Westernblotting结果显示siRNAl和siRNA2转染U87细胞12h后细胞Eag-1mRNA、蛋白的表达产物带明显弱于空白对照组，而siRNA3转染组产物条带虽也弱于空白对照组，但条带仍清晰；MTT检测结果显示与空白对照组比较，50nm01／LsiRNA1、siRNA2转染组和10、20、30、40mmol／L奎尼丁组细胞培养24、48和72h后吸光度值均降低，差异有统计学意义（P＜0．05），奎尼丁IC。为33．7mmol／L。流式细胞仪分析显示与空白对照组比较，50nmol／LsiRNA1、siRNA2转染组和33．7mmol／L奎尼丁组G1期细胞百分比、细胞凋亡率和胞内ROS水平均增加，差异有统计学意义（P＜0．05）。结论阻断Eag-1通道活性能明显抑制胶质瘤细胞增殖，使G1期细胞比例、胞内ROS水平明显升高并诱导其凋亡。

Objective To evaluate the influence of Eag-1 channel blocking on bioaetivity of glioma cells in vitro. Methods Different small interfering RNAs （siRNAs） targeting for Eag-1 channel were designed and transfected to the U87 cells, and the blocking effects of those siRNAs were further confirmed on mRNA and protein levels by RT-PCR and Western blotting. The 50 nmol/1 siRNAs （siRNA1 and siRNA2） and quinidine （5, 10, 20, 30 and 40 mmol/1） were used to block the activity of Eag-1 channel, respectively; and blank control group was also established. The proliferation of U87 cells 24, 48 and 72 h after the treatments was detected by MTT method; the changes of generation cycle, apoptosis ratio and intracellular reactive oxygen species （ROS） concentration were detected by flow cytometry. Results High mRNA and protein levels of Eag-1 channel on glioma cell line U87 were confirmed in the blank control group, however, siRNA1 and siRNA2 transfection groups showed significantly lower mRNA and protein levels of Eag-1 channel on glioma cell line U87. MTT method indicated that, 24, 48 and 72 h after the treatments, the proliferation of U87 cells in the siRNA1 and siRNA2 transfection groups, and quinidine treatment groups （10, 20, 30 and 40 mmol/1） was significantly inhibited as compared with that in the blank control group （P〈0.05）. The IC50 value of quinidine is 33.7mmol/1. As compared with the blank control group, 50 nmol/L siRNA1 and siRNA2 transfection groups, and 33.7 mmol/1 quinidine treatment group enjoyed a significantly increased cell percentage at GI stage, cell apoptosis ratio and intracellular ROS level （P〈0.05）. Conclusion Eag-1 channel blocking can obviously inhibit the proliferation of glioma cells, increase the cell percentage at G1 stage and intracellular ROS level, and induce apoptosis ofglioma cells.