Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul 110-749, South Korea.
Exp Cell Res. 2013 Aug 15;319(14):2125-34. doi: 10.1016/j.yexcr.2013.04.020. Epub 2013 May 3.
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.
远端同源盒 5（Dlx5）是一种促成骨但抗成脂的转录因子，它调节间充质干细胞的谱系决定。尽管已知 Dlx5 的表达会被成脂刺激物下调，但 Dlx5 下调的机制尚未阐明。miRNAs（miRNA）是一种小的调节 RNA，可在后转录水平上调节许多生物学功能，包括细胞分化。在这项研究中，我们研究了 miRNA 是否参与了成脂刺激后 Dlx5 的下调。我们使用计算预测程序筛选了在 Dlx5 3'UTR 上有直接靶位点的候选 miRNA，选择了结合得分最高的 7 个 miRNA 候选物，并观察了它们在 3T3-L1 鼠前脂肪细胞中的表达水平。在所检查的 miRNA 中，只有 miR-124 在 24 小时孵育于成脂培养基中时显著上调。在 4 种成脂刺激物（1-甲基-3-异丁基黄嘌呤、胰岛素、吲哚美辛和地塞米松）中，胰岛素对 miR-124 表达的刺激作用最高。胰岛素显著增加了 miR-124 前体的表达，包括 pri-miR-124-1、pri-miR124-2 和 pri-miR-124-3。PI3K 抑制剂 LY294002 阻止了胰岛素对 miR-124 表达水平的调节作用。miR-124 模拟物的过表达降低了 Dlx5 的表达，同时增加了 3T3-L1 细胞的成脂分化。用 miR-124 的发夹抑制剂 anti-miR-124 阻断 miR-124，增加了 Dlx5 的表达水平并抑制了成脂分化。当在包含下游 lucif erase 基因的 Dlx5 3'UTR 序列的报告基因构建体上进行报告基因测定时，miR-124 模拟物以依赖 miR-124 结合位点的方式抑制，但 anti-miR-124 增强了 luciferase 活性。这些结果表明，胰岛素诱导的 miR-124 在成脂分化过程中对 Dlx5 的转录后调节中起关键作用，并且 miR-124 通过靶向 Dlx5 至少部分发挥促脂生成作用。
