miRNA-376c-3p mediates TWIST-1 inhibition of BMSC osteogenesis and can reduce aberrant bone formation of TWIST-1 haploinsufficient calvarial cells

Abstract

Key transcription factors, which activate or repress master gene regulators and signalling pathways, tightly regulate self-renewal and cell lineage differentiation of bone marrow stromal cells (BMSC). Among these factors is the basic helix-loop-helix (bHLH) transcription factor Twist-related protein 1 (TWIST-1), which is important in BMSC self-renewal, life span and differentiation. Another layer of gene regulation comes from MicroRNAs (miRNAs). MiRNAs are short non-coding RNAs that interfere with translation of specific target mRNAs and thereby regulate diverse biological processes including BMSC lineage commitment. However, little is known of how TWIST-1 regulated miRNAs control osteogenic commitment, and influence the fate of bone precursor cells. In this study, we have discovered a novel TWIST-1 regulated miRNA, miR-376c-3p. Reduced miR-376c-3p expression by a miR-376c-3p inhibitor or due to TWIST-1 haploinsufficiency promotes alkaline phosphatase activity, mineral deposition and expression of osteoblast-associated genes in BMSC and calvarial cells. Conversely, over-expression of miR-376c-3p using a miR-376c-3p mimic inhibited BMSC proliferation and the osteogenic potential of BMSC and TWIST-1 haploinsufficient calvarial cells. This was demonstrated by a decrease in Insulin Growth factor 1 receptor (IGF1R) levels, Akt signalling, alkaline phosphatase activity, mineral deposition and expression of osteoblast-associated genes. Thus, miR-376c-3p reduces IGF1R/Akt signalling in BMSC and is one mechanism by which osteogenesis may be inhibited. Overall, we have identified miR-376c-3p as a TWIST-1 regulated miRNA, which plays an important role in the osteogenesis of bone precursor cells and can mediate TWIST-1 inhibition of osteogenesis. Furthermore, over-expression of miRNA-376c-3p in TWIST-1 haploinsufficient calvarial cells can decrease the aberrant osteogenesis of these cells, which contributes to increased calvaria bone volume and premature fusion of the coronal sutures.