The suppression of miR-23a, miR-24 and miR-27a induces apoptosis during ESC neural differentiation by modulating BMP4 signaling

Abstract

Bone morphogenetic protein 4 (BMP4) plays an important role in mouse embryonic stem cells (ESCs) by sustaining pluripotency and blocking differentiation towards neural fate. In this study, we have analyzed the miRNAs regulated by BMP4 in ESCs. We have found that BMP4 signaling directly regulates miR-23a, miR-24-2 and miR-27a through the binding of phospo-Smads on the promoter of the gene encoding all these miRNAs. The suppression of miR-23a, miR-24-2 and miR-27a, together with the suppression of miRNAs of the same families, miR-23b, miR-24-1 and miR-27b, does not impair ESC stemness maintenance and epiblast stem cells (EpiSCs) derivation from ESCs. However, this suppression affects ESC differentiation, thus resulting in the increase of the number of cells undergoing apoptosis soon after the transition from ESCs to EpiSCs. We have demonstrated that the block of BMP4 signaling completely rescues the apoptosis induced by the suppression of miRNAs. Considering that it was already known that BMP4 induces apoptosis during ESC differentiation, our observation suggests that the apoptotic phenotype provoked by miRNA suppression is due to an enhancement of BMP4 signaling. We also demonstrated that miR-23a and miR-23b clusters target Smad5, a downstream effector of BMP4 pathway; this phenomenon explains how the suppression of miRNA clusters enhances BMP4 signaling. In conclusion, the results unveil the existence of a feedback loop, involving Smad5 and miR-23a clusters, that regulates the apoptosis induced by BMP4 during ESC neural differentiation.