Bcl-x mRNA前体剪接转换寡核苷酸诱导胶质瘤细胞凋亡的实验研究  

Apoptosis-promoting effect of splice-switching oligonucleotides targeting Bcl-x pre-mRNA of the human glioma cell line

作　　者：李朝晖[1] 田男[2] 付红[1] 李妍哲 韩亮[1] 田宇[1] 

机构地区：[1]吉林大学白求恩医学部中日联谊医院神经外二科,长春市130031 [2]浙江中医药大学生命科学学院

出　　处：《中国肿瘤临床》2014年第6期359-362,共4页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金(编号:30672159);教育部"新世纪优秀人才支持计划"(编号:NCET-06-0306);吉林省科技发展计划国际科技合作项目(编号:20130413028GH)资助~~

摘　　要：目的:探讨Bcl-x mRNA前体剪接转换寡核苷酸(Bcl-x splice-switching oligonucleotides,Bcl-x SSO)对胶质瘤细胞株U251增殖和凋亡的影响。方法:设计靶向Bcl-x基因下游选择性5'端剪接位点的Bcl-x SSO,β-globin SSO作为阴性对照SSO。SSO经2'-甲氧乙基(2'-O-methoxyethyl,MOE)、全硫代磷酸化(phosphorothioate,PS)修饰。采用阳离子脂质体将不同的SSO转染至U251细胞。采用四甲基偶氮唑蓝比色法(MTT法)检测Bcl-x SSO对U251细胞的增殖抑制率。流式细胞术定量检测U251细胞的凋亡率。逆转录聚合酶链反应(RT-PCR)方法检测Bcl-x SSO对Bcl-xL、Bcl-xS mRNA表达水平的影响,Western blot方法检测Bcl-x SSO对Bcl-xL、Bcl-xS蛋白表达的影响。结果:MTT结果显示Bcl-x SSO显著抑制U251细胞的增殖,且抑制作用呈剂量依赖性。流式细胞术检测Bcl-x SSO能够明显促进U251细胞凋亡。RT-PCR检测Bcl-x SSO处理组Bcl-xL mRNA表达水平下降,Bcl-xS mRNA表达水平升高。Western blot检测Bcl-x SSO处理组Bcl-xL蛋白表达水平下降,Bcl-xS蛋白表达水平升高。结论:在胶质瘤细胞U251中,Bcl-x SSO可特异性的作用于Bcl-x mRNA前体,调节其选择性剪接模式从Bcl-xL转换至Bcl-xS,进而促进U251细胞凋亡。Objective： To investigate the proliferation inhibition and apoptosis-promoting effects of splice-switching oligonucle-otides targeting the Bcl-x pre-mRNA （Bcl-x SSO） of the human glioma cell line U251. Methods： Bcl-x SSO was designed to bind to the 5＇-splice site of exon Ⅱ in Bcl-x pre-mRNA. An oligonucleotide targeted to aberrantly splice human β-globin intron was used as a control SSO. SSOs were modified using 2＇-O-methoxyethyl and phosphorothioate, and were delivered together with lipofectamine into the human glioma cell line U251 via cationic liposomes. The proliferation inhibition rate of the cell human cell line U251 was assessed via MTT assay. Flow cytometry was performed to detect the apoptosis rate. Modulation from Bcl-xL to Bcl-xS was analyzed via re-verse transcription polymerase chain reaction and Western blot. Results： The study showed that Bcl-x SSO caused proliferation inhibi-tion and induced apoptosis in a dose-dependent manner in the human glioma cell line U25 l, whereas the control SSO did not show any evident effect. The expression of Bcl-xL mRNA and protein increased, whereas the expression level of Bcl-xS mRNA decreased in the human glioma cell line U251 treated with Bcl-x SSO. Conclusion： The study demonstrated that Bcl-x SSO can induce apoptosis in hu-man glioma cell line U251. The mechanism involves the redirection of Bcl-x splicing from Bcl-xl to Bcl-xs. Bcl-x SSOs have the poten-tial to be used as anti-cancer drugs for glioma therapy.

关 键 词：选择性剪接 寡核苷酸 凋亡 流式细胞术 胶质瘤细胞 

分 类 号：R739.41[医药卫生—肿瘤]