Regulation of multiple target genes by miR-1 and miR-206 is pivotal for C2C12 myoblast differentiation

Abstract

SummaryMicroRNAs are short non-coding RNAs involved in post-transcriptional regulation of multiple messenger RNA targets. The miR-1/ miR-206 family is expressed during skeletal muscle differentiation and is an integral component of myogenesis. To better understand miR-1/miR-206 function during myoblast differentiation we identified novel target mRNAs by microarray and characterized their function in C2C12 myoblasts. Candidate targets from the screen were experimentally validated together with target genes that were predicted by three different algorithms. Some targets characterised have a known function in skeletal muscle development and/or differentiation and include Meox2, RARB, Fzd7, MAP4K3, CLCN3 and NFAT5, others are potentially novel regulators of myogenesis, such as the chromatin remodelling factors Smarcd2 and Smarcb1 or the anti-apoptotic protein SH3BGRL3. The expression profiles of confirmed target genes were examined during C2C12 cell myogenesis. We found that inhibition of endogenous miR-1 and miR-206 by antimiRs blocked the downregulation of most targets in differentiating cells, thus indicating that microRNA activity and target interaction is required for muscle differentiation. Finally, we show that sustained expression of validated miR-1 and/or miR-206 targets resulted in increased proliferation and inhibition of C2C12 cell myogenesis. In many cases the expression of genes related to non-muscle cell fates, such as chondrogenesis, was activated. This indicates that the concerted downregulation of multiple microRNA targets is not only crucial to the skeletal muscle differentiation program but also serves to prevent alternative cell fate choices.

Mechanisms of impaired differentiation in rhabdomyosarcoma

“…Interestingly, this regulation was limited to the JR1 eRMS cell line, as similar repression of Pax3 was not observed in the more commonly studied Rh30 aRMS cells . Other miR‐206 targets that interfere with myogenic differentiation by regulating myoblast proliferation and survival were also recently described . Previous studies have shown that Pax3 is expressed in myogenic precursors, and analogously to Pax7, functions as an inhibitor of terminal differentiation .…”

Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor‐mediated effects on myogenic microRNA expression

“…Myogenic miRNAs are critical regulators of SC differentiation and growth, have important roles in muscle remodeling, and their dysregulation has been implicated in diseases of cardiac and skeletal muscle. Numerous downstream miR‐1/miR‐206 targets have been identified in C2C12 cells, but the specific role of miR‐1/miR‐206 family members in myoblast differentiation remains to be determined (52).…”

miR‐181a regulates p62/SQSTM1, parkin, and protein DJ‐1 promoting mitochondrial dynamics in skeletal muscle aging

“…C2C12 myoblasts were maintained in DMEM (Sigma‐Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma‐Aldrich; Goljanek‐Whysall et al, 2012). For cholesterol‐linked miR‐181a mimic, antagomiR‐181a, or antagomiR scrambled, 50% confluent C2C2 myoblasts were treated at 100 nM final concentration for 48 hr previous to treatment with 10 µM CCCP or 100 nM bafilomycin A (both Sigma‐Aldrich).…”