砷对人胶质瘤细胞周期调控相关基因表达的影响

Effect of As_2O_3 on the expressions of genes relating to cell cycle regulation in human glioma cells

目的比较三氧化二砷(As_2O_3)对两种不同p53表型胶质瘤细胞U87MG、T98G细胞周期调控相关基因表达的影响。方法应用功能分类细胞周期基因芯片技术,筛选出As_2O_3作用于两种胶质瘤细胞U87MG、T98G后与细胞周期调控表达有差异的基因。并对3条有共性表达的基因进行Western印迹分析;四甲基偶氮唑蓝(MTT)法检测细胞生长抑制率;流式细胞仪分析细胞周期改变。结果MTT分析表明:As_2O_3对2个细胞系的生长有抑制作用,且抑制率具有浓度和时间的依赖性,砷作用72h后,U87MG和T98G细胞系的生长抑制率为50%(IC_(50))时,其浓度分别为1.78、3.55μmoL/L。As_2O_3可以将U87MG和T98G细胞周期阻滞于G_0/G_1和G_2/M期。U87MG细胞在加入As_2O_3后,差异表达2倍以上的基因共11条,表达上调的有5条,下调的有6条。T98G细胞在加入As_2O_3后。差异表达的基因共13条,表达上调的有9条,下调的有4条。Western分析显示,As_2O_3能上调2个胶质瘤细胞系的P53蛋白水平,同时降低细胞周期蛋白B1、D1表达。结论As_2O_3通过将细胞周期阻滞于G_0/G_1和G_2/M期来抑制U87MG和T98G细胞的增殖;p53、Cyclin B、Cylin D等基因表达与As_2O_3诱导胶质瘤细胞周期阻滞的发生有关。

Objective To investigate the effect of As203 on the changes of expression of genes relating to cell cycle regulation in two glioma cell lines differing in p53 status（U87MG,T98G）. Methods We selected the cell cycle related genes in U87MG and T98G whose expressed level were changed after exposure to As2O3 by functional classification cell cycle gene array. The protein expression level of three commonness genes were analyzed by Western Blotting, proliferation rate detected by methy thiazolyl tetrazolum（MTT）and cell cycle aborogation analyzed by flow cytometry. Results MTT results indicated that As2O3 inhibited the two cell lines in a time and concentration-dependent manner. The concentration of As2O3 in U87MG and T98G cell lines 72 hours later were 1.78 and 3.55 μmol/L when growth inhibition ratio was 50%（IC50）. Cell cycles of U87MG and T98G were blocked on Go/G1 and GJM stage by As2O3. Five genes were upregulated and six genes downregulated at least 2-fold by As2O3 in U87-MG cells; whereas, nine genes were upregulated and four genes downregulated at least 2-fold in T98G cells by As2O3. Western blotting indicated that As2O3 upregulated p53 protein level of the two glioma cell lines, whereas, downregulated the expression of cyclin B1 and D1. Conlnsions As2O3 inhibits the proliferations of U87MG and T98G cells via blocking the cell cycles on Go/G1 and G2/M stage, p53, Cyclin B and Cylin D genes are involved in the mechanisms of glioma cell cycle blockge induced by As2O3.