Micro-RNA regulation of embryonic stem cell differentiation

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MicroRNAs (miRNAs) are short, single-stranded RNAs that represent a novel level of genetic regulation, whereby gene activity is attenuated post-transcriptionally by inhibiting translation and/or destabilising messenger-RNA. During development, these non-coding RNAs appear to play essential roles in regulating many distinct developmental processes, for example, neural development, myocyte differentiation and haematopoietic differentiation. We have recently initiated a study to investigate the role of miRNAs in embryonic stem cell differentiation. Our approach is aimed at identifying micro-RNA changes associated with the transition from an undifferentiated to a differentiated state. We have utilised the ZHBT4 cell line whereby cells can either be maintained in an undifferentiated state or can be rapidly induced to differentiate simultaneously, en masse. In this system, expression of the essential stem cell master regulator Oct4 can be rapidly switched off in mouse ES cells through the addition of the drug doxycycline. This initiates a differentiation cascade that directs ES cells along the trophoblast lineage. Our analysis identified a large number of different miRNAs, some of which had previously been identified in the mouse and many which were entirely novel miRNAs. Of these, the majority were expressed at similar levels in treated and untreated cells while a small number were either up-regulated or down-regulated as a result of switching off Oct4. The differentially expressed miRNAs were mapped to the mouse genome and this analysis revealed that around half of the miRNAs that are induced upon differentiation are encoded within a single cluster. This cluster is comprised of a mix of previously identified miRNAs and novel miRNAs. Further analysis revealed that this cluster is contained within an intron of a recently identified gene that is known to be expressed in the developing trophoblast and placenta of the mouse embryo.

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