title: MicroRNA-363 negatively regulates the left ventricular determining transcription factor HAND1 in human embryonic stem cell-derived cardiomyocytes.
abstract: BACKGROUND
  Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes.
  
  
  METHODS
  We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification.
  
  
  RESULTS
  From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3'-untranslated regions (3'UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3'UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1.
  
  
  CONCLUSIONS
  These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.
authors.forename: Vilas
authors.initials: V
authors.lastname: Wagh
authors.forename: Alexander
authors.initials: A
authors.lastname: Pomorski
authors.forename: Karlijn J
authors.initials: KJ
authors.lastname: Wilschut
authors.forename: Sebastian
authors.initials: S
authors.lastname: Piombo
authors.forename: Harold S
authors.initials: HS
authors.lastname: Bernstein
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis* / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D006352:Heart Ventricles / Q000166:cytology*; D006801:Humans; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D035683:MicroRNAs / Q000235:genetics*; D032383:Myocytes, Cardiac / Q000166:cytology*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D035683:MicroRNAs; C574861:MIRN363 microRNA, human; C097341:helix-loop-helix protein, eHAND