title: A new plasmid-based microRNA inhibitor system that inhibits microRNA families in transgenic mice and cells: a potential new therapeutic reagent.
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.
authors.affiliation: Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA.
authors.forename: H
authors.initials: H
authors.lastname: Cao
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Yu
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.affiliation: Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.affiliation: Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA.
authors.forename: S
authors.initials: S
authors.lastname: Gao
authors.affiliation: Dows Institute of Dental Research, College of Dentistry, The University of Iowa, Iowa City, IA, USA.
authors.forename: N E
authors.initials: NE
authors.lastname: Holton
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.|Craniofacial Anomalies Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors.forename: S
authors.initials: S
authors.lastname: Eliason
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Sharp
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.|Dows Institute of Dental Research, College of Dentistry, The University of Iowa, Iowa City, IA, USA.|Craniofacial Anomalies Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
authors.forename: B A
authors.initials: BA
authors.lastname: Amendt
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000008:administration & dosage / Q000037:antagonists & inhibitors* / Q000235:genetics*; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering