Grünhagen Johannes, Bhushan Raghu, Degenkolbe Elisa, Jäger Marten, Knaus Petra, Mundlos Stefan, Robinson Peter N, Ott Claus-Eric
Institute for Medical Genetics and Human Genetics, Charité - Universitätsmedizin Berlin, Berlin, Germany; Research Group Development and Disease, Max Planck Institute for Molecular Genetics, Berlin, Germany.
J Bone Miner Res. 2015 May;30(5):796-808. doi: 10.1002/jbmr.2412.
MicroRNAs play important roles during cell reprogramming and differentiation. In this study, we identified the miR-497∼195 cluster, a member of the miR-15 family, as strongly upregulated with age of postnatal bone development in vivo and late differentiation stages of primary osteoblasts cultured in vitro. Early expression of miR-195-5p inhibits differentiation and mineralization. Microarray analyses along with quantitative PCR demonstrate that miR-195-5p alters the gene regulatory network of osteoblast differentiation and impairs the induction of bone morphogenetic protein (BMP) responsive genes. Applying reporter gene and Western blot assays, we show that miR-195-5p interferes with the BMP/Smad-pathway in a dose-dependent manner. Systematically comparing the changes in mRNA levels in response to miR-195-5p overexpression with the changes observed in the natural course of osteoblast differentiation, we demonstrate that microRNAs of the miR-15 family affect several target genes involved in BMP signaling. Predicted targets including Furin, a protease that cleaves pro-forms, genes encoding receptors such as Acvr2a, Bmp1a, Dies1, and Tgfbr3, molecules within the cascade like Smad5, transcriptional regulators like Ski and Zfp423 as well as Mapk3 and Smurf1 were validated by quantitative PCR. Taken together, our data strongly suggest that miR-497∼195 cluster microRNAs act as intracellular antagonists of BMP signaling in bone cells.
微小RNA在细胞重编程和分化过程中发挥着重要作用。在本研究中，我们鉴定出miR-497∼195簇，它是miR-15家族的成员之一，在体内随着出生后骨骼发育的年龄增长以及体外培养的原代成骨细胞的晚期分化阶段而强烈上调。miR-195-5p的早期表达会抑制分化和矿化。微阵列分析以及定量PCR表明，miR-195-5p改变了成骨细胞分化的基因调控网络，并损害了骨形态发生蛋白（BMP）反应性基因的诱导。通过应用报告基因和蛋白质印迹分析，我们表明miR-195-5p以剂量依赖性方式干扰BMP/Smad信号通路。系统地比较miR-195-5p过表达后mRNA水平的变化与成骨细胞分化自然过程中观察到的变化，我们证明miR-15家族的微小RNA影响了几个参与BMP信号传导的靶基因。预测的靶标包括弗林蛋白酶（一种切割前体形式的蛋白酶）、编码受体（如Acvr2a、Bmp1a、Dies1和Tgfbr3）的基因、级联反应中的分子（如Smad5）、转录调节因子（如Ski和Zfp423）以及Mapk3和Smurf1，通过定量PCR对这些靶标进行了验证。综上所述，我们的数据强烈表明miR-497∼195簇微小RNA在骨细胞中作为BMP信号传导的细胞内拮抗剂发挥作用。
