基因TOB1通过BCL-2和BCL-xl抑制胶质瘤细胞的生长

Inhibition of gene TOB1 on the cell growth in glioma through BCL-2 and BCL-xl

目的 :探讨基因TOB1在胶质瘤中的作用及其调控机制。方法 :通过免疫组织化学法对胶质瘤组织及正常组织中TOB1、BCL-2(BCL2 apoptosis regulator)、BCL-xl(BCL2 like 1)的表达进行检测。采用Western Blot和荧光素酶检测系统对靶向基因TOB1的microRNA进行筛选分析。用含TOB1过表达、干扰或对照的慢病毒感染胶质瘤细胞株U251。采用噻唑蓝比色法(methyl thiazolyl tetrazolium, MTT)或流式细胞术检测TOB1对U251细胞活力、细胞周期及凋亡的影响。实时聚合酶链式反应(real time polymerase chain reaction, Real-time PCR)和Western Blot分别检测TOB1对凋亡相关因子表达的影响。结果:胶质瘤组织中TOB1基因表达被抑制,miR-92a-3p可以通过结合TOB1基因的3′-非翻译区来调控基因TOB1的表达。并且,过表达TOB1可降低细胞活力,抑制细胞增殖,促进细胞凋亡,而抑制TOB1表达则具有相反作用。此外,TOB1过表达组的抗凋亡因子BCL-2和BCL-xl表达显著下调;相反,TOB1被干扰后,抗凋亡因子BCL-2和BCL-xl表达则上调。免疫组织化学法显示,胶质瘤组织中BCL-2和BCL-xl的表达增加。结论:TOB1在胶质瘤中表达受到抑制,并通过调控BCL-2和BCL-xl基因的表达影响胶质瘤的增殖和凋亡。

Objective: To evaluate the role of gene TOB1 in glioma and its regulation mechanism. Methods: The immunohistochemistry for the TOB1, BCL2 apoptosis regulator(BCL-2), and BCL2 like 1(BCL-xl) was performed in the glioma and normal tissues. The microRNAs targeting gene TOB1 were investigated by Western Blot and luciferase assay system. The glioma cell line U251 was infected with lentivirus containing gene TOB1 or lentivirus harboring RNAi sequence or control lentivirus.Then, the effects of TOB1 on the cell viability, cell cycle distribution and apoptosis of cell line U251 were determined with methyl thiazolyl tetrazolium(MTT) assay and flow cytometry analysis. Moreover, the effects of TOB1 on the expression of apoptosis related factors were also measured by real time polymerase chain reaction and Western Blot. Results: Our results indicated that the TOB1 was suppressed in the glioma tissues and miR-92a-3p could regulate the expression of gene TOB1 by binding its 3′-untraslated region. The overexpression of TOB1 could decrease cell viability, suppress cell proliferation and promote cell apoptosis, whereas knockdown of TOB1 took on an opposite effect. The anti-apoptotic factors BCL-2 and BCL-xl could be reduced by TOB1 and the knockdown of TOB1 had an opposite effect. The immunohist-ochemistry indicated that BCL-2 and BCL-xl were increased in the glioma tissue. Conclusion: TOB1 was inhibited in the glioma and closely correlated with the attenuation of cell viability, proliferation and promotion of apoptosis in glioma cells by regulating the expression of gene BCL-2or gene BCL-xl.