Project description:MicroRNAs (miRNAs), a class of small, non-coding RNA molecules, play an important role in the posttranscriptional regulation of gene expression, thereby influencing important cellular functions. In adipocytes, miRNAs show import regulatory features and are described to influence differentiation as well as metabolic, endocrine, and inflammatory functions. We previously identified miR-27a being upregulated under inflammatory conditions in human adipocytes and aimed to elucidate its function in adipocyte biology. Both strands of miR-27a, miR-27a-3p and -5p, were downregulated during the adipogenic differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells, human multipotent adipose-derived stem cells (hMADS), and human primary adipose-derived stromal cells (hASCs). Using miRNA-mimic transfection, we observed that miR-27a-3p is a crucial regulator of adipogenesis, while miR-27a-5p did not alter the differentiation capacity in SGBS cells. In silico screening predicted lipoprotein lipase (LPL) and peroxisome proliferator activated receptor γ (PPARγ) as potential targets of miR-27a-3p. The downregulation of both genes was verified in vitro, and the interaction of miR-27-3p with target sites in the 3' UTRs of both genes was confirmed via a miRNA-reporter-gene assay. Here, the knockdown of LPL did not interfere with adipogenic differentiation, while PPARγ knockdown decreased adipogenesis significantly, suggesting that miR-27-3p exerts its inhibitory effect on adipogenesis by repressing PPARγ. Taken together, we identified and validated a crucial role for miR-27a-3p in human adipogenesis played by targeting the essential adipogenic transcription factor PPARγ. Though we confirmed LPL as an additional target of miR-27a-3p, it does not appear to be involved in regulating human adipogenesis. Thereby, our findings call the conclusions drawn from previous studies, which identified LPL as a crucial regulator for murine and human adipogenesis, into question.

Project description:Post-transcriptional regulation of gene expression accomplished by microRNAs (miRNAs) importantly affects the complex gene regulatory network. In particular, miRNAs are known to be involved in recurrent motifs named miRNA-mediated feed forward loops (FFLs) where a transcription factor (TF) regulates a miRNA and they both regulate the expression level of a target RNA. Here, we focus on the identification of active FFLs during adipogenic differentiation. A list of putative feed-forward loops was generated based on sequence analysis of conserved and overrepresented motifs in the regulatory regions. Since this approach is not specific for adipogenesis and is known to generate false positive feed-forward loops, an experiment was designed to select active ones based on their dynamics using a model-based approach. Microarray time series of gene and miRNA expression data were collected at seven time points on human multipotent adipose-derived stem (hMADS) cells upon adipogenic differentiation. Three different dynamic models, sharing the same FFL topology but incorporating descriptions of increasing complexity of miRNA and mRNA dynamics, are identified on miRNA and mRNA expression data and compared based on identification criteria, namely: goodness of fit, precision of the estimates and comparison with submodels. 24 FFLs, able to properly reproduce data, are selected as active out of the 329 putative ones. This method considerably reduces the search space for new interactions between TFs, miRNAs and mRNAs and provides interesting biological results identifying genes known from the literature to be regulators in adipogenesis and adipocyte-related functions that can be interpreted as positive control of the validity of the apporoach. Therefore, the genes in the selected FFLs that are not yet known to be involved in this context are potential novel players in this regulatory network of adipogenesis and adipocyte function. Two independent cell culture experiments were performed as biological replicates during adipogenic differentiation of human mesenchymal stem cell. Cells where harvested at the pre-confluent stage as reference (day -3) and at seven subsequent time points during human adipogenic differentiation: day -2 and 0 before, and 1, 2, 5, 10, 15 days after induction of differentiation. All hybridizations were repeated with reversed dye assignment (dye-swap).

Dataset Information

MicroRNA-27a/b-3p and PPARG regulate SCAMP3 through a feed-forward loop during adipogenesis.

Publications

MicroRNA-27a/b-3p and PPARG regulate SCAMP3 through a feed-forward loop during adipogenesis.

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