microRNA Profiling during endoderm differentiation from mouse embryonic stem cells

The role of microRNAs (miRNAs) during mouse early development, especially in endoderm germ layer formation, is largely unknown. Here, using miRNA profiling, we discovered that miR-124a negatively regulates endoderm lineage commitment in mouse embryonic stem cells (mESCs). We showed that miR-124a inhibits endoderm differentiation in vitro through targeting the 3’ untranslated region (UTR) of Sox17 and Gata6, revealing the existence of an interplay between miR-124a and Sox17/Gata6 transcription factors in hepato-specific gene regulation. Besides, we proposed an in vivo system that utilizes teratoma to evaluate the functional role of miRNA in lineage specification. We demonstrated that ectopic expression of miR-124a in teratomas suppressed endoderm and mesoderm lineage differentiation while augmented the differentiation of ectoderm lineage. Collectively, our findings suggested that miR-124a plays a significant role in mESCs lineage commitment. To study the miRNA expression profiles during endoderm differentiation in mouse, we first induced definitive endoderm (DE) from mESC using Activin A, Noggin, and GSK-3 inhibitor (CHIR99021) (ActA-NG-CHIR). Next, RNA samples were harvested at 6 different time points along endoderm differentiation (Day 0, 2, 3, 4, 5 and 6). Experiment was performed in duplicate.