MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells
MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells
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DOI：
10.1038/nmeth1079
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发表时间：
2007-09-01
期刊：
影响因子：
48
通讯作者：
Sharp, Phillip A.
中科院分区：
文献类型：
--
作者：
Ebert, Margaret S.;Neilson, Joel R.;Sharp, Phillip A.
微小RNA（MicroRNAs）预计可调控数千个哺乳动物基因，但经实验验证的靶点相对较少，且微小RNA功能缺失的表型也鲜有确定。作为化学修饰的反义寡核苷酸的一种替代方法，我们开发了可在细胞中表达的微小RNA抑制剂，它们是由转基因产生的RNA。这些被称为“微小RNA海绵”的竞争性抑制剂是由强启动子表达的转录本，包含多个与感兴趣的微小RNA的串联结合位点。当将编码这些海绵的载体瞬时转染到培养细胞中时，海绵对微小RNA靶点的去抑制作用至少与化学修饰的反义寡核苷酸一样强。它们特异性地抑制具有互补七聚体种子序列的微小RNA，因此单个海绵可用于阻断整个微小RNA种子家族。RNA聚合酶II启动子（Pol II）驱动的海绵包含一个荧光报告基因，用于识别和分选经海绵处理的细胞。我们设想在疾病和发育的动物模型中使用稳定表达的海绵。
MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.