人肿瘤坏死因子α特异性结合miR-146b的新生物学特性研究  

Novel Biological Characteristics of hTNF-α as miR-146b Binding Protein

作　　者：沈雪莲[1] 李洁[2] 黄皑雪[2] 王超男[2] 孙乐桥 李少华[2] 丁红梅[2] 李慧[2] 郭晓华[2] 郑伟[2] 董洁[2] 邵宁生[1,2] 

机构地区：[1]广西医科大学,广西南宁530021 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《生物技术通讯》2016年第2期183-187,共5页Letters in Biotechnology

基　　金：国家自然科学基金(31570817;31370794)

摘　　要：目的:探讨人肿瘤坏死因子α(TNF-α)作为micro RNA(mi RNA)结合蛋白的生物学特性。方法:Western印迹检测LPS激活的U937细胞中TNF-α蛋白表达水平,RT-PCR检测几种炎症相关的mi RNA在激活前后表达水平的变化;用RNA结合蛋白免疫共沉淀(RIP)技术钓取U937细胞中TNF-α结合的mi RNA,RT-PCR检测RIP产物中上述几种mi RNA的含量;用凝胶阻滞实验检测TNF-α与mi RNA是否直接结合。结果:LPS激活的U937细胞中能检测到膜结合型和可溶性2种形式的TNF-α,细胞激活前后mi R-16和mi R-21的表达水平在检测的几种mi RNA中最高,mi R-146b和mi R155次之;而RIP产物中mi R-146b的水平在实验组和对照组中有显著差异,其他mi RNA差异不明显;凝胶阻滞实验结果显示mi R-146b能与人TNF-α直接结合。结论:首次证实人TNF-α能够直接特异地结合mi R-146b,提示TNF-α可能作为mi RNA结合蛋白发挥生物学功能。Objective： To explore the novel biological characteristics of human TNF-α as mi RNA-binding pro-tein. Methods： The expression of TNF-α in non-activated or LPS-activated U937 was examined by Western blot,and the inflammation-associated mi RNAs were detected by RT-PCR simultaneously. RNA-binding protein immuno-precipitation（RIP） was used to map mi RNAs binding to TNF-α, and then RT-PCR was performed to quantify themi RNAs in the RIP products. To confirm the direct interaction of mi RNAs with TNF- α, rlectrophoretic mobilityshift assay（EMSA） was performed. Results： The results of Western blot suggested that the membrane-bound andsoluble forms of TNF- α were expressed in U937 cells activated by LPS. Meanwhile, the results of RT- PCRshowed that in activated or non-activated U937 cells, mi R-16 and mi R-21 were expressed highly, while mi R-146 and mi R155 expressed moderately. In RIP products, much more mi R-146 b was immunoprecipitated by the anti-TNF- α antibody than the isotype control. Furthermore, the results of EMSA confirmed TNF- α directly bindingwith mi R-146 b. Conclusion： The results validated for the first time that human TNF-α could bind to mi R-146 bdirectly, which prompt us to address the potential biological roles of TNF-α acting as mi RNA-binding protein.

关 键 词：人肿瘤坏死因子Α MICRORNA RNA结合蛋白 RNA结合蛋白免疫共沉淀 

分 类 号：Q502[生物学—生物化学] Q25