title: Sequencing of captive target transcripts identifies the network of regulated genes and functions of primate-specific miR-522.
abstract: Identifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical "seed" base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Shen Mynn
authors.initials: SM
authors.lastname: Tan
authors.affiliation: Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA.
authors.forename: Rory
authors.initials: R
authors.lastname: Kirchner
authors.affiliation: New England Biolabs, Ipswich, MA 01938, USA.
authors.forename: Jingmin
authors.initials: J
authors.lastname: Jin
authors.affiliation: Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofmann
authors.affiliation: New England Biolabs, Ipswich, MA 01938, USA.
authors.forename: Larry
authors.initials: L
authors.lastname: McReynolds
authors.affiliation: Department of Biostatistics, Harvard School of Public Health, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA.
authors.forename: Winston
authors.initials: W
authors.lastname: Hide
authors.affiliation: Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: judy.lieberman@childrens.harvard.edu.
authors.forename: Judy
authors.initials: J
authors.lastname: Lieberman
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D053263:Gene Regulatory Networks; D006801:Humans; D035683:MicroRNAs; D011379:Prognosis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D064726:Triple Negative Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism*
chemical_list: C560599:MIRN522 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger