Osteoglycin inhibition by microRNA miR-155 impairs myogenesis

Abstract

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.

High-throughput analysis of the RNA-induced silencing complex in myotonic dystrophy type 1 patients identifies the dysregulation of miR-29c and its target ASB2

“…miR-155 impairs myogenesis 31 , 32 ; moreover, its targets TSPAN13 and LRP1B are also targeted by miR-222 and miR-381, respectively, suggesting a potential cooperative action between these miRNAs.…”

Comparative analysis of miR-155 tissue expression profiles of different breeds of chicken

“…3c). Since miR-155 can inhibit myoblast differentiation and skeletal muscle formation [10, 11], whether the low expression of miR-155 is related to the rapid growth of broilers needs to be further studied in the future.…”

“…Interestingly, the emerging evidences of miR-155 role in the development of skeletal muscle and adipogenesis have been noticed recently. On the one hand, miR-155 can repress the expression of MEF2A (a member of the myogenic enhancer factor 2 family of transcription factors) and osteoglycin (Ogn, an important component of the skeletal muscle secretome), and inhibit proliferation and differentiation of myoblast cells [10, 11]. Moreover, miR-155 also regulates the expression of Olfactomedin-like 3 (OLFML3), which may affect prenatal skeletal muscle development in pig [12].…”

MicroRNA-mRNA Co-sequencing Identifies Transcriptional and Post-transcriptional Regulatory Networks Underlying Muscle Wasting in Cancer Cachexia

“…Our transcriptome data from C2C12 muscle cells treated with TNF-α/IFN-γ further support this fact by presenting not only a reduction in the expression of collagen genes but also in other genes involved in ECM assembly, including Ogn. Our group had previously shown that Ogn affects the proliferation and differentiation of C2C12 muscle cells [85]. A previous study has also investigated the potential contributory factors outside the fiber within the muscle microenvironment in humans and animals and found that Pax7 responds to NF-κB by impairing the regenerative capacity of myogenic cells to drive muscle wasting in cancer cachexia [80].…”