Paolella, Gaetana (2010) Investigating miRNA expression and function in Embryonic Stem Cells. [Tesi di dottorato] (Unpublished)

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MicroRNAs (miRNAs) are post-transcriptional modulators of gene expression and in recent years have been identified as important players in proper function and differentiation of mouse embryonic stem cells (ESCs). Given their capacity to self-renewal and differentiate into different cell types, ESCs provide a valid model to understand the network of signaling interaction in mammalian embryo and open new possibilities for cell therapy. In recent years, the role of miRNAs in ESC and mammalian embryogenesis has begun to be explored but the specific roles of the miRNAs in the regulation of ESC specific fate are still largely unknown. In this context, the aim of this thesis is to identify miRNAs regulating ESC functions. I performed a systematic analysis of miRNA expression in undifferentiated and neural differentiating ESCs by TaqMan assay and I performed a systematic comparison of them. I reported 319 miRNAs differentially expressed in undifferentiated and neural differentiated ESCs and selected for detailed studies some microRNAs that appeared to significantly change during ESC differentiation. In particular, I selected some miRNAs not present in ESCs and whose expression increases during differentiation (up-regulated miRNAs). By using bioinformatic’s tools we identified the candidate targets for these miRNAs, Smarca5, Jarid1b, and Sirt1, previously demonstrated to be involved in sustaining 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 we also confirmed previously published data demonstrating that Sirt1 is a direct target of miR-34a in a different context. Then, we demonstrated that the suppression of these three miRNAs by anti-miRNAs caused the block of ESC differentiation induced by LIF withdrawal. Then we focused on a subset of miRNAs (miR-467 cluster), already expressed in undifferentiated ESCs, up-regulated in the early stages of differentiation and down-regulated when neural differentiation occurs. This selection was based on the attempt to identify miRNAs that contribute to control ESC fate decisions by suppressing genes required for the late phase of differentiation. To study the ability of these miRNAs to modulate ESC differentiation we have developed a differentiation method that allow to identify molecules able to improve neuronal differentiation of ESCs. Here we show that, by suppressing of miR-467a and d in ESCs we strongly improved neuronal differentiation. These data suggest that these miRNAs regulate genes that enhance neuronal differentiation. Finally, we selected miRNAs already present in ESCs and whose expression increase upon differentiation and we performed a systematic analysis of the effect of miRNAs ectopic expression. Over-expression of miR-125a and 384 hindered normal decrease of stemness markers such as Oct4/3 and Nanog yielded a new population of cells characterized as epiblast cells.

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