MicroRNA-363 negatively regulates the left ventricular determining transcription factor HAND1 in human embryonic stem cell-derived cardiomyocytes

Stem Cell Res Ther. 2014 Jun 6;5(3):75. doi: 10.1186/scrt464.

Abstract

Introduction: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / biosynthesis*
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Cell Differentiation / genetics
  • Cell Line
  • Embryonic Stem Cells / cytology*
  • Gene Expression Regulation, Developmental / genetics*
  • Heart Ventricles / cytology*
  • Humans
  • Immunoblotting
  • In Situ Hybridization, Fluorescence
  • Induced Pluripotent Stem Cells / cytology
  • MicroRNAs / genetics*
  • Myocytes, Cardiac / cytology*
  • Real-Time Polymerase Chain Reaction

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • MIRN363 microRNA, human
  • MicroRNAs
  • helix-loop-helix protein, eHAND