The Role and Molecular Regulation of microRNAs and their Targets in Adipogenesis

Adipose tissue, rather than being just a passive storage for excess energy, is a highly dynamic organ actively balancing energy storage and release, and besides liver and muscle is a core contributor to the systemic metabolic state. With the surge in obesity and related metabolic comorbidities in recent years, adipogenesis comes into focus. Overfeeding can lead to overloading of adipocytes, the detrimental consequences of which can in turn impair one of the potential alleviators to this situation: recruitment of preadipocytes to adipocyte differentiation. Adipocyte differentiation is controlled not only by protein-coding genes, but also by non-coding RNAs, and particularly microRNAs (miRNAs): ~22nt RNAs that regulate gene expression by targeting the microRNA-induced silencing complex (miRISC) to the miRNA recognition element (MRE) in target mRNAs and basepairing to their seed match. miR-29a had been identified in preliminary studies to be downregulated during adipocyte differentiation of our human adipogenesis model, human Multipotent Adipose-Derived Stem (hMADS) cells. Previous studies had implicated it in insulin resistance and diabetes, however, none of these addressed its role in adipocyte differentiation. Thus, the goal of this thesis was to elucidate the role of miR-29 in human adipocyte differentiation, and identifying a direct target that mediates this effect. We found that miR-29a was downregulated during adipogenesis, and that increasing its abundance by transfection into preadipocytes inhibited subsequent adipocyte differentiation. From a gene expression screen, we identified eight potential target genes, five of which were directly bound by the miR-29a in vitro. One candidate, mesoderm-specific transcript (MEST), unexpectedly had the opposite of the expected effect on adipocyte differentiation and was pursued as a stand-alone project, since silencing MEST, contrary to published data in murine cell lines, enhanced adipogenesis in hMADS cells. Out of the remaining four candidates, only silencing of NR3C1, the glucocorticoid receptor (GR) gene, phenocopied the miR-29 effect on differentiation. In search of an upstream regulatory mechanism of miR-29 expression, we found that miR-29a is induced by glucocorticoids in vitro and in vivo, and that GR binds upstream of the miR-29a gene locus, thus creating a negative feedback loop between the target and the targeting miRNA. The identification of this novel and unexpected feedback loop opens up interesting research perspectives on the phenomenon of GR downregulation upon long-term treatment with glucocorticoids. In summary, this dissertation examines the role of the miR-29 and its targets in human adipocyte differentiation in vitro, associated correlations in murine and human tissue and identifies a novel feedback mechanism between miR-29 and GR that could serve as a starting point for exciting further research.