Abstract
MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression in post-transcriptional fashion, and emerging studies support their importance in regulating many biological processes, including myogenic differentiation and muscle development. miR-29 is a promoting factor during myogenesis but its full spectrum of impact on muscle cells has yet to be explored. Here we describe an analysis of miR-29 affected transcriptome in C2C12 muscle cells using a high throughput RNA-sequencing platform. The results reveal that miR-29 not only functions to promote myogenic differentiation but also suppresses the transdifferentiation of myoblasts into myofibroblasts. miR-29 inhibits the fibrogenic differentiation through down-regulating both extracellular matrix genes and cell adhesion genes. We further demonstrate that miR-29 is under negative regulation by TGF-beta (TGF-β)–Smad3 signaling via dual mechanisms of both inhibiting MyoD binding and enhancing Yin Yang 1 (YY1)-recruited Polycomb association. Together, these results identify miR-29 as a pleiotropic molecule in both myogenic and fibrogenic differentiation of muscle cells.
Highlights
MicroRNAs are non-coding single-stranded RNAs of 21–25 nucleotides and constitute a novel class of gene regulators that are found in a variety of eukaryotic organisms. miRNAs negatively regulate their targets at the post-transcriptional level through binding to their 39 UTRs [1,2].Mounting evidences support the importance of miRNAs in skeletal muscle development and muscle related diseases
Subsequent Gene Ontology (GO) analysis with upregulated list of genes revealed that the top ranked lists of enriched GO categories include ‘‘contractile fiber’’, ‘‘contractile fiber part’’, ‘‘sarcomere’’, ‘‘myofibril’’, ‘‘I band’’, ‘‘Z disc’’ (Table S3), which is in agreement with the previously identified roles of miR-29 in accelerating muscle regeneration
C2C12 myoblasts were transfected with siRNA oligos against Smad2, Smad3 or Smad7, using scrambled siRNAs (SCR) as a control. 48 hr posttransfection, the expressions were examined by Western blotting using a-Tubulin as a loading control. (B) C2C12 myoblasts, transfected with the indicated siRNA oligos, were incubated with TGF-b in DM for 48 hrs at which time the expressions of miR-29 were measured. (C) miR-29-promoter-luc reporter activities in C2C12 myoblasts transfected with the above siRNA oligos and treated with TGF-b in DM for 48 hrs. (D) miR-29 expressions in primary myoblasts isolated from Smad3+/+, Smad3+/2 or Smad32/2 mice. (E) miR-29 expressions in primary myoblasts from Smad7+/+ or Smad72/2 mice. (F) Smad7+/+ or Smad72/2 mice were injected with Cardiotoxin (CTX) into TA muscles to induce muscle regeneration
Summary
MicroRNAs (miRNAs) are non-coding single-stranded RNAs of 21–25 nucleotides and constitute a novel class of gene regulators that are found in a variety of eukaryotic organisms. miRNAs negatively regulate their targets at the post-transcriptional level through binding to their 39 UTRs [1,2].Mounting evidences support the importance of miRNAs in skeletal muscle development and muscle related diseases. The process of skeletal muscle cell differentiation is orchestrated by transcription factors MyoD, Myf, myogenin, MRF4, and Mef. The process of skeletal muscle cell differentiation is orchestrated by transcription factors MyoD, Myf, myogenin, MRF4, and Mef2 These factors activate muscle genes to coordinate myoblasts to terminally withdraw from cell cycle and subsequently fuse into multinucleated myotubes [3]. MyoD replaces the silencing complex causing the derepression of miR-29 transcriptional expression. We further demonstrated that this regulatory circuit is disrupted in Rhabdomyosarcoma which may contribute to the development of this tumor. These findings suggest that miR-29 involved circuitries are critical regulator of gene expression in skeletal muscle cells. It is our interest to explore the full spectrum of the influence by miR-29 in these cells and discover other targets under the control of miR-29
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