Given their capacity to self-renew and differentiate efficiently into the different cell types, Embryonic Stem Cells (ESCs) provide a valid model to understand the complex network of signaling interactions in the mammalian embryo and open up new possibilities for cell therapy. So a deeper understanding of the molecular mechanisms that regulate generation, self-renewal and differentiation of ESCs is become crucial not only to fulfill their clinical promise but also to get insight into the molecular mechanisms controlling early events of mammalian development. The emergence of microRNAs (microRNAs) as potent regulators of gene expression at the post-transcriptional level has broad implications in all facets of biology, including ESCs and early development.
In recent years, the role of microRNAs in ESCs and mammalian embryogenesis has begun to be explored but specific roles of the microRNAs in the regulation of ESC specific fate are still largely unknown. In this context, our interest is to identify microRNAs regulating ESC functions. We performed a systematic comparison of microRNA expression in undifferentiated versus differentiating mouse ESCs. We report that different microRNAs are increased upon the induction of differentiation. We compared the entire list of candidate mRNA targets of upregulated microRNAs with that of mRNA dowregulated in ESCs upon the induction of differentiation. Among the candidate targets emerged from this analysis, we found three genes Smarca5, Jarid1b and Sirt1, previously demonstrated to be necessary to sustain the undifferentiated phenotype in ESCs. On this basis, we first demonstrated that Smarca5 is a direct target of miR-100, Jarid1b of miR-137 and confirmed previously published data demonstrating that Sirt1 is a direct target of miR-34a in a different context. The suppression of these three microRNAs by anti-miRs caused block of ESC differentiation induced by LIF withdrawal. On the other hand, the overexpression of the three microRNAs resulted in an altered expression of differentiation markers. These results demonstrated that miR-100, miR-137 and miR-34a are required for proper differentiation of ESCs, and that they function by targeting, among the others, the mRNAs of Smarca5, Jarid1b and Sirt1.
In conclusion, we have characterized a subset of microRNAs that are necessary for proper differentiation of mouse ESCs. The identification of microRNAs that are up-regulated upon the induction of ESC differentiation suggests that they suppress, directly or indirectly, the expression of genes necessary to maintain ESCs in the undifferentiated state. The identification of targets of the microRNAs that we have studied may provide tools to drive ESC differentiation towards specific lineages
