miR-322/503 Cluster Drives Cardiac Differentiation by Inhibiting CUG-binding Protein 1



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Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration after injury. MicroRNAs (miRNAs) involved in the process are particularly interesting due to their small profile and relatively shorter path to clinic. With Mesp1 as the marker, we used a Mesp1-Cre/Rosa-EYFP reporter system to track the earliest cardiac progenitors, and identified the miRNAs enriched in these cells. Among them, the miR-322/503 cluster is found to be a powerful regulator of the cardiac program: (1) in a screening of more than 20 cardiac progenitor cell (CPC) enriched miRNAs, miR-322/503 was the most powerful in driving calcium flux activity in mouse embryonic stem cells (mESCs) differentiation; (2) induced ectopic expression of miR-322/503 to mimic the natural course in mESCs led to α-actinin expression and significant increases of cardiac transcription factors (Tbx5, Mef2C, Nkx2-5 and α-MHC); and (3) inhibitors of miR-322 and miR-503 significantly reduced expression of α-actinin and the above cardiac TFs. Remarkably, miR-322/503 regulates the cardiac program by inhibiting an RNA-alternative splicing/decay factor, CUG-binding protein 1 (Celf1), which is also known for a role in myotonic dystrophy pathogenesis. The evidences include: (i) miR-322 and miR-503 had a shared target site at the 3’UTR of Celf1; (ii) expression patterns of miR-322/503 and Celf1 were mutually exclusive, with the highest Celf1 expression in the brain; (iii) miR-322/503 repressed Celf1 protein expression in a dose-dependent manner; (iv) Celf1-shRNA induced up-regulation of cardiac transcription factors and α-actinin, mimicking the function of miR-322/503; and (v) the ectopic expression of Celf1 repressed expression of cardiac transcription factors, while promoted expressions of early neural markers, including Sox1, Notch3, Nestin and Pax6. In summary, we have identified a miR-322/503-Celf1 pathway that promotes cardiac differentiation by preventing activation of other lineages. This new regulatory mechanism may be used to direct cardiac regeneration after heart injury, and treat myotonic dystrophy where Celf1 up-regulation is responsible for skeletal muscle wasting and other symptoms.



MicroRNAs (miRNA), Celf1, Cardiac differentiation, Neural differentiation