p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling

Biochim Biophys Acta. 2015 Jul;1852(7):1520-30. doi: 10.1016/j.bbadis.2015.04.006. Epub 2015 Apr 15.

Abstract

Transforming growth factor-β (TGF-β) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-β signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-β1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-β1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-β1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFβ-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.

Keywords: DICER; Myocardial fibrosis; Pre-miR-21; Pressure overload; TGF-β; p-SMAD2/3.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Animals
  • Aortic Valve Stenosis / metabolism*
  • Cells, Cultured
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism
  • Protein Binding
  • RNA Processing, Post-Transcriptional*
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Smad2 Protein / genetics
  • Smad2 Protein / metabolism*
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism*
  • Transforming Growth Factor beta / pharmacology
  • Ventricular Remodeling*

Substances

  • MIRN21 microRNA, mouse
  • MicroRNAs
  • Smad2 Protein
  • Smad2 protein, mouse
  • Smad3 Protein
  • Smad3 protein, mouse
  • Transforming Growth Factor beta
  • Dicer1 protein, mouse
  • Ribonuclease III
  • DEAD-box RNA Helicases