Osteoglycin inhibition by microRNA miR-155 impairs myogenesis

Skeletal myogenesis is a regulated process in which mononucleated cells, the myoblasts, undergo proliferation and differentiation. Upon differentiation, the cells align with each other, and subsequently fuse to form terminally differentiated multinucleated myotubes. Previous reports have identified the protein osteoglycin (Ogn) as an important component of the skeletal muscle secretome, which is expressed differentially during muscle development. However, the posttranscriptional regulation of Ogn by microRNAs during myogenesis is unknown. Bioinformatic analysis showed that miR-155 potentially targeted the Ogn transcript at the 3´-untranslated region (3´ UTR). In this study, we tested the hypothesis that miR-155 inhibits the expression of the Ogn to regulate skeletal myogenesis. C2C12 myoblast cells were cultured and miR-155 overexpression or Ogn knockdown was induced by transfection with miR-155 mimic, siRNA-Ogn, and negative controls with lipofectamine for 15 hours. Near confluence (80–90%), myoblasts were induced to differentiate myotubes in a differentiation medium. Luciferase assay was used to confirm the interaction between miR-155 and Ogn 3’UTR. RT-qPCR and Western blot analyses were used to confirm that the differential expression of miR-155 correlates with the differential expression of myogenic molecular markers (Myh2, MyoD, and MyoG) and inhibits Ogn protein and gene expression in myoblasts and myotubes. Myoblast migration and proliferation were assessed using Wound Healing and MTT assays. Our results show that miR-155 interacts with the 3’UTR Ogn region and decrease the levels of Ogn in myotubes. The overexpression of miR-155 increased MyoG expression, decreased myoblasts wound closure rate, and decreased Myh2 expression in myotubes. Moreover, Ogn knockdown reduced the expression levels of MyoD, MyoG, and Myh2 in myotubes. These results reveal a novel pathway in which miR-155 inhibits Ogn expression to regulate proliferation and differentiation of C2C12 myoblast cells.

骨骼肌发生（Skeletal myogenesis）是一类受精密调控的生物学过程，其间单核细胞——成肌细胞（myoblasts）将经历增殖与分化两个阶段。当启动分化程序后，成肌细胞会彼此对齐黏附，随后相互融合形成终末分化的多核肌管（multinucleated myotubes）。既往研究已鉴定出骨糖蛋白（osteoglycin, Ogn）为骨骼肌分泌组的重要组成成分，其在肌肉发育进程中呈现差异性表达模式。然而，在肌发生过程中，microRNAs对Ogn的转录后调控机制尚未明确。生物信息学分析结果显示，miR-155可靶向结合Ogn转录本的3'非翻译区（3´-untranslated region, 3´ UTR）。本研究旨在验证“miR-155通过抑制Ogn的表达以调控骨骼肌发生”这一科学假说。我们培养了C2C12成肌细胞，通过脂质体转染试剂（Lipofectamine）分别介导miR-155模拟物、Ogn小干扰RNA（siRNA-Ogn）及其阴性对照转入细胞，以实现miR-155过表达或Ogn敲低，转染时长为15小时。当细胞达到80%–90%的近汇合密度时，使用分化培养基诱导成肌细胞分化为肌管。采用双荧光素酶报告基因实验验证miR-155与Ogn 3'UTR的相互作用；通过实时定量聚合酶链反应（RT-qPCR）与蛋白质印迹（Western blot）分析，证实miR-155的差异表达与成肌分子标志物（Myh2、MyoD及MyoG）的表达水平显著相关，且可抑制成肌细胞与肌管中Ogn的蛋白及基因表达。此外，我们采用划痕愈合实验与MTT实验评估了成肌细胞的迁移与增殖能力。本研究结果表明：miR-155可直接结合Ogn的3'UTR区域，并降低肌管内Ogn的表达水平；miR-155过表达可上调MyoG的表达水平，降低成肌细胞的划痕愈合率，同时下调肌管中Myh2的表达；Ogn敲低则可降低肌管内MyoD、MyoG及Myh2的表达水平。综上，本研究揭示了一条全新的调控通路：miR-155通过靶向抑制Ogn的表达，进而调控C2C12成肌细胞的增殖与分化过程。