MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells

Abstract

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 spongetreated cells. We envision the use of stably expressed sponges in animal models of disease and development.MicroRNAs are 20-24-nucleotide RNAs derived from hairpin precursors. Through pairing with partially complementary sites in 3′ untranslated regions (UTRs), they mediate posttranscriptional silencing of a predicted 30% of protein-coding genes in mammals 1 . MicroRNAs have been implicated in critical processes including differentiation, apoptosis, proliferation, and the maintenance of cell and tissue identity; furthermore, their misexpression has been linked to cancer and other diseases [2][3][4][5][6][7] . But relatively few micro-RNA-target interactions have been experimentally validated in cell culture or in mouse models, and the functions of most microRNAs remain to be discovered. Creating genetic knockouts to determine the function of microRNA families is difficult, as individual microRNAs expressed from multiple genomic loci may repress a common set of targets containing a complementary seed sequence. Thus, a method for inhibiting these functional classes of paralogous micro-RNAs in vivo is needed. Presently, loss-of-function phenotypes are induced by means of chemically modified antisense oligonucleotides-2′ O-methyl, locked nucleic acid (LNA) and others-which are presumed to pair with and block mature microRNAs through extensive sequence complementarity [8][9][10] . Typically, oligonucleotide

In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression

“…Conversely, vectors expressing 'microRNA sponges' could be used to inhibit miRNA sequences. 18 In a similar manner, vectors expressing short hairpin RNA could be administered to the fetal brain to knockdown a gene's expression and evaluate its function in development. This would have particular relevance if the subsequent phenotype provided a useful model for neurodegenerative disease, circumventing the need for producing complex transgenic models.…”

miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression

“…To further address whether endogenous miR-764-5p acts as an attenuator for the CHIP expression, we designed an antagomiR sequence against miR-764-5p (named as miR-sponge) (19) and stably expressed the sequence using pcDNA3.1 vector in MC3T3-E1 cells. The result showed that the protein level of CHIP was increased when miR-sponge was stably expressed (Fig.…”

MicroRNAs as potential cancer therapeutics

“…In an alternative strategy to single anti-miRs, Ebert et al (2007) recently developed miRNA inhibitors called 'miRNA sponges', which can be transiently expressed in cultured cells. These molecules are transcripts expressed from strong promoters, containing multiple tandem binding sites to specific miRNAs, and competitively inhibit them.…”