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New microRNA Biotechnology to Inhibit Inflammation and Regenerate Bone
Author(s) -
Amendt Brad,
Cao Huojun,
Yu Wenjie,
Holton Nathan,
Eliason Steven,
Hong Liu
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.902.24
Subject(s) - dicer , microrna , biology , transgene , gene knockdown , argonaute , rna interference , oligonucleotide , function (biology) , computational biology , microbiology and biotechnology , gene , genetics , rna
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 families due to the duplication of miR seed sequences and family members. We report a novel plasmid‐based miR inhibitor system (PMIS) that inhibits miR family members in cells and mice. The PMIS engineered 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. A complete family of miRs can be inhibited with a single plasmid. Different PMIS miR inhibitors can be linked together to knockdown multiple miRs expressed from different chromosomes. The PMIS shows no off‐target effects or toxicity and is highly specific for miRs sharing identical seed sequences. PMIS constructs associate with Dicer and Argonaute to form a stable miR inhibitor complex. Transgenic mice expressing PMIS‐miRs reveal different developmental processes affected by miRs. Genome‐wide analyses of PMIS transgenic mice and cells identified new miR regulated gene networks. Complete inhibition of the miR‐17‐92 , miR‐106a‐363 , miR‐106b‐25 , and miR‐200 clusters reveals new mechanisms for bone regeneration and developmental defects for these miRs. These miRs also control inflammation through the direct targeting of pro‐inflammatory cytokines. We report a new tool to dissect the role of miRs in development without genome editing and as a potential new therapeutic reagent. Support or Funding Information This work was supported by grants from the State of Iowa Bio Venture group, College of Dentistry and Carver College of Medicine and NIH DE025328 to LH.