Abstract 408: Mir148a Family Regulates Cardiac Differentiation of Human Embryonic Stem Cells by Inhibiting The Dll1 -mediated Notch Signaling Pathway

Abstract

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can differentiate into spontaneously beating cardiomyocytes in vitro, and hold great promise for cardiovascular disease modeling, therapy and drug discovery. However, these applications were hampered by low yield and purity of the in vitro differentiated cardiomyocytes. Although miRNAs represent a type of potential candidates to promote cardiac differentiation, most of cardiomyocyte-enriched miRNAs have not been functionally investigated until now. This study investigated the roles of MIR148A family in cardiac differentiation from hESCs. The MIR148A family is composed of MIR148A , MIR148B and MIR152 , three highly conserved miRNAs sharing same seed sequences. The expression levels of all MIR148A family members were progressively increased during cardiac differentiation in vitro. By using CRISPR-Cas9-mediated knockout, we demonstrated that triple knockout of MIR148A family ( MIR148A -TKO), rather than individual knockout of each members, could grossly inhibited TNNT2 + cardiomyocyte generation. Ectopic expression of MIR152 significantly restored the cardiac differentiation efficiency of MIR148A -TKO hESCs. The transcriptome analysis identified a total of 1071 upregulated genes and 766 down-regulated genes in the MIR148A -TKO hESC-derived cells compared to wild-type hESC-derived cells at day 4 of cardiac differentiation. Gene Ontology analysis revealed that most genes down-regulated in MIR148A -TKO hESC-derived cells were involved in the events of lateral/cardiac mesoderm development, while the up-regulated genes were mainly involved in the events of paraxial/somitic development. Furthermore, the NOTCH ligand Delta-like1 ( DLL1 ) was validated as the target gene of MIR148A family, and the knockdown of DLL1 could inhibit target gene expression of Notch signaling pathway, and promote cardiac differentiation of MIR148A -TKO hESCs. Our findings demonstrate a new function of MIR148A family during cardiac differentiation. Synergistic inhibition of DLL1 -mediated Notch signaling represents a major mechanism for the MIR148A family to inhibit undesired lineage formation and promote hESC differentiation into cardiomyocytes.