title: Insulin suppresses distal-less homeobox 5 expression through the up-regulation of microRNA-124 in 3T3-L1 cells.
abstract: Distal-less homeobox 5 (Dlx5) is a pro-osteogenic but anti-adipogenic transcription factor that regulates lineage commitment in mesenchymal stem cells. Although the expression of Dlx5 is known to be decreased by adipogenic stimuli, the mechanism of Dlx5 down-regulation has not yet been clarified. MicroRNAs (miRNAs) are small regulatory RNAs that post-transcriptionally regulate many biological functions, including cell differentiation. In this study, we examined whether miRNAs are involved in down-regulation of Dlx5 following adipogenic stimuli. We screened candidate miRNAs that have a direct target site in the Dlx5 3'UTR using computational prediction programs, selected seven miRNA candidates with the highest binding score and observed their expression levels in 3T3-L1 murine pre-adipocytes. Among the miRNAs examined, only miR-124 was significantly up-regulated by 24-h incubation in adipogenic medium. Among the four components of adipogenic stimuli (1-methy-3-isobutyl xanthine, insulin, indomethacin and dexamethasone), insulin exhibited the highest stimulatory effect on miR-124 expression. Insulin significantly increased the expression of miR-124 precursors including pri-miR-124-1, pri-miR124-2 and pri-miR-124-3. LY294002, an inhibitor of phosphatidylinositol-3-kinase, prevented the regulatory effect of insulin on the expression levels of miR-124 and Dlx5. Over-expression of a miR-124 mimic decreased the expression of Dlx5 while increasing adipogenic differentiation in 3T3-L1 cells. Blocking miR-124 with anti-miR-124, a hairpin inhibitor of miR-124, increased the expression level of Dlx5 and suppressed adipogenic differentiation. When reporter assays were performed with a reporter construct containing the Dlx5 3'UTR sequence downstream of a luciferase gene, miR-124 mimic suppressed, but anti-miR-124 enhanced, luciferase activity in an miR-124 binding site-dependent manner. These results suggest that insulin-induced miR-124 plays a pivotal role in post-transcriptional regulation of Dlx5 during adipogenic differentiation and that miR-124 exerts pro-adipogenic effects by targeting Dlx5, at least in part.
authors: Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, South Korea.
authors: Abdul S
authors: AS
authors: Qadir
authors: Kyung Mi
authors: KM
authors: Woo
authors: Hyun-Mo
authors: HM
authors: Ryoo
authors: Jeong-Hwa
authors: JH
authors: Baek
mesh_terms: D020413:3' Untranslated Regions; D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D050156:Adipogenesis; D000818:Animals; D001665:Binding Sites; D002867:Chromones / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D020413:3' Untranslated Regions; D002867:Chromones; C501120:Dlx5 protein, mouse; D018398:Homeodomain Proteins; D007328:Insulin; D035683:MicroRNAs; C540079:Mirn124 microRNA, mouse; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012322:RNA Precursors; D034741:RNA, Small Interfering; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
keywords: 1-methy-3-isobutyl xanthine; Adipocyte; Dlx5; IBMX; Insulin; LPL; a hairpin inhibitor for miR-124; a non-targeting control hairpin inhibitor; a small RNA mimic of mature miR-124; anti-miR-124; anti-miR-Control; control-mimic; lipoprotein lipase; miR-124; miR-124-mimic; miRNA; miRNA mimics negative control; microRNA