siRNA沉默USP22基因对胶质瘤细胞增殖的抑制作用

si RNA-mediated silencing of the USP22 gene inhibits cell proliferation in human glioma cells

目的探讨小干扰RNA(si RNA)沉默泛素特异性肽酶22(ubiquitin specific protease 22,USP22)对胶质瘤细胞增殖的影响和作用机制。方法合成USP22 si RNA及阴性对照si RNA(control si RNA),用脂质体Lipofectamine 2000转染至人胶质瘤U87和U251细胞,分别设为实验组和阴性对照组。通过逆转录聚合酶链反应(RT-PCR)和Western blot检测两组胶质瘤细胞USP22 mRNA和蛋白表达水平的变化。采用四甲基偶氮唑蓝比色法(MTT法)检测USP22 si RNA对两组胶质瘤细胞的增殖抑制率。流式细胞术定量检测USP22 si RNA对胶质瘤细胞凋亡及细胞周期分布的影响。Western blot检测胶质瘤细胞凋亡蛋白和周期调控蛋白的表达。结果与阴性对照组比较,实验组胶质瘤细胞中USP22 mRNA和蛋白表达显著下降(P<0.05),细胞增殖明显受到抑制(P<0.05),凋亡率明显上升(P<0.05),细胞周期中G2/M期的细胞比例明显增高(P<0.05)。Western blot结果显示:细胞凋亡蛋白Procaspase-3、Procaspase-8、Procaspase-9表达明显下降(P<0.05);细胞周期蛋白CDK1、CDK2、Cyclin B1表达水平明显下降(P<0.05),Cyclin D1表达水平无明显变化(P>0.05)。结论 USP22基因在胶质瘤细胞的增殖中发挥重要作用,其机制可能为调节细胞凋亡和细胞周期。

Objective To explore the influence and action mechanism of ubiquitin-specific protease 22（USP22） gene silencing by small interference RNA（ si RNA） on proliferation of human glioma cells. Methods USP22 si RNA and a negative control si RNA were designed and transfected into glioma cell lines U87 and U251 by using Lipofectamine 2000. The cells which were transfected with USP22 si RNA and control si RNA were set as experimental and negative control groups respectively. RT-PCR and Western blot were used to detect the expressions of USP22 mRNA and protein in glioma cells in two groups. Cell proliferation was measured by MTT. The apoptosis rate and the distribution of cell cycle of U87 and U251 were determined by flow cytometry. The apoptosis proteins and cell cycle proteins were detected by Western blot. Results Compare with negative control group, the expressions of USP mRNA and protein were decreased significantly（P〈0.05）, cell proliferation was obviously inhibited（P〈0.05）, apoptosis rate rose markedly（P〈0.05）, the proportion of G2/M in cell cycle was higher（P〈0.05）. Western blot results showed that the expressions of apoptosis proteins such as Procaspase-3, Procaspase-8 and Procaspase-9 were decreased（P〈0.05）, cell cycle proteins such as CDK1, CDK2 and Cyclin B1decreased（P〈0.05）, but the expression of Cyclin D1 remained stable（P〉0.05）. Conclusion USP22 gene plays a key role in the proliferation of human glioma cells by regulating the apoptosis and cell cycle.