A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.

H Cao,N E Holton,T Sharp,B A Amendt,J Wang,S Gao,X Li,S Eliason,W Yu

doi:10.1038/gt.2016.22

Abstract

Current tools for the inhibition of microRNA (miR) function are limited to modified antisense oligonucleotides, sponges and decoy RNA molecules and none have been used to understand miR function during development. CRISPR/Cas-mediated deletion of miR sequences within the genome requires multiple chromosomal deletions to remove all functional miR family members because of duplications. Here, we report a novel plasmid-based miR inhibitor system (PMIS) that expresses a new RNA molecule, which inhibits miR family members in cells and mice. The PMIS engineered RNA optimal secondary structure, flanking sequences and specific antisense miR oligonucleotide sequence bind the miR in a stable complex to inhibit miR activity. In cells, one PMIS can effectively inhibit miR family members that share the same seed sequence. The PMIS shows no off-target effects or toxicity and is highly specific for miRs sharing identical seed sequences. Transgenic mice expressing both PMIS-miR-17-18 and PMIS-miR-19-92 show similar phenotypes of miR-17-92-knockout mice. Interestingly, mice only expressing PMIS-miR-17-18 have developmental defects distinct from mice only expressing PMIS-miR-19-92 demonstrating usefulness of the PMIS system to dissect different functions of miRs within clusters. Different PMIS miR inhibitors can be linked together to knock down multiple miRs expressed from different chromosomes. Inhibition of the miR-17-92, miR-106a-363 and miR-106b-25 clusters reveals new mechanisms and developmental defects for these miRs. We report a new tool to dissect the role of miRs in development without genome editing, inhibit miR function in cells and as a potential new therapeutic reagent.

Highlights

IntroductionMicroRNAs (miRs) are short noncoding RNA molecules, ~ 22 nucleotides (nts) long, that regulate messenger RNA (mRNA) transcripts post-transcriptionally through binding to complementary sequences on target mRNA.[1,2,3,4] The human genome may contain over 1500 miR species (miRBase, release 18) and it has been estimated that more than half of protein coding genes could be regulated by miRs.[5,6] Since the first discovery in 1993, miRs have been shown to be involved in the regulation of a broad range of biological processes and the malfunction of miRs are associated with many human diseases.[7,8,9,10,11,12,13,14,15,16]Given the importance of miRs during different biological processes, tools for repression of miR function will be useful for research and have therapeutic potential
The reduction of miR levels using a sponge miR inhibitor was previously reported to be an artifact of the detection method;[27] we performed Northern blots for miR expression after transduction of the plasmid-based miR inhibitor system (PMIS)-miR-17-18 construct in 293 cells (Figure 2b). These results show that miR-17 and miR-18 levels were reduced by PMIS-miR-17-18 and this is consistent with other studies demonstrating miR degradation by inhibitors.[36,37]
PTEN was identified in a screen for genes regulated by miR-17 and we show that inhibition of miR-17 by PMIS-miR-17-18 increased PTEN protein expression (Figure 2c)

Summary

Introduction

MicroRNAs (miRs) are short noncoding RNA molecules, ~ 22 nucleotides (nts) long, that regulate messenger RNA (mRNA) transcripts post-transcriptionally through binding to complementary sequences on target mRNA.[1,2,3,4] The human genome may contain over 1500 miR species (miRBase, release 18) and it has been estimated that more than half of protein coding genes could be regulated by miRs.[5,6] Since the first discovery in 1993, miRs have been shown to be involved in the regulation of a broad range of biological processes and the malfunction of miRs are associated with many human diseases.[7,8,9,10,11,12,13,14,15,16]Given the importance of miRs during different biological processes, tools for repression of miR function will be useful for research and have therapeutic potential. Other chemically modified antisense oligonucleotides with a 2′-fluoro/2′-methoxyethyl modified antisense oligonucleotide motif improved in vivo inhibition of miR activity.[24] A limitation of these miR inhibitors resides in their inability to be retained in the tissues after cell division and they must be reapplied to maintain their effectiveness To address these limitations and promote long-term repression of specific miRs, several plasmid and/or viral vectors expressing antagomirs, sponges, eraser and Tough Decoy (TuD) RNA molecules have been reported.[25,26,27,28] This system and others can inhibit miR activity without degradation of the miR.[24,29]

Methods

Results

Conclusion