Abstract:
The current epidemic of obesity has resulted in a renewed interest in the developmental origins of adipose tissue. Adipose tissue is formed from mesenchymal stem cells (MSCs) in a process known as adipogenesis. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit translation by binding to the 3¿UTR of their target genes. The aim of this study was to determine whether miRNAs play a role in the adipogenesis of bone marrow derived MSCs. Microarray analysis identified 20 miRNAs which were significantly regulated in adipogenically differentiating MSCs compared to undifferentiating MSCs, of which 11 were significantly downregulated and 9 were significantly upregulated. MiR-221 and miR-222 were significantly downregulated during hMSC differentiation to adipocytes. MiR-221 and miR-222 were overexpressed using lentiviral vectors to determine whether their reduction is necessary for adipogenic differentiation. MSCs overexpressing miR-221 and miR-222 displayed a significantly reduced ability to differentiate to adipocytes compared to control MSCs, as measured by lipid accumulation and expression of adipogenesis-related transcripts. MiRZip short hairpin RNAs (shRNAs) against miR-221 and miR-222 were lentivirally delivered to determine the effects of reduced miR-221 and miR-222 activity on hMSC adipogenesis. The reduction in activity of either miRNA caused a significant reduction in cell number. P27 is an experimentally confirmed target of miR-221 and miR-222 in cancer models. The overexpression of miR-221 and miR-222 reduced the levels of p27 protein but not mRNA 72 hours after the induction of adipogenesis. Direct interactions between the miRNAs and the 3¿UTR of p27 were verified using luciferase reporters in HEK293T cells. To investigate whether preventing the reduction in p27 protein rescues the reduction in lipid accumulation, p27 was overexpressed in MSCs with reduced miR-221 and miR-222. This did not lead to a significant change in lipid accumulation in MSCs, suggesting that miR-221 and miR-222 do not act solely through an effect on this protein.
