Abstract
Circular RNAs (circRNAs) represent a class of covalently closed single-stranded RNA molecules that are emerging as essential regulators of various biological processes. The circRNA circHipk2 originates from exon 2 of the Hipk2 gene in mice and was reported to be involved in acute promyelocytic leukemia and myocardial injury. However, the functions and mechanisms of circHipk2 in myogenesis are largely unknown. Here, to deepen our knowledge about the role of circHipk2, we studied the expression and function of circHipk2 during skeletal myogenesis. We found that circHipk2 was mostly distributed in the cytoplasm, and dynamically and differentially expressed in various myogenesis systems in vitro and in vivo. Functionally, overexpression of circHipk2 inhibited myoblast proliferation and promoted myotube formation in C2C12 cells, whereas the opposite effects were observed after circHipk2 knockdown. Mechanistically, circHipk2 could directly bind to ribosomal protein Rpl7, an essential 60S preribosomal assembly factor, to inhibit ribosome translation. In addition, we verified that transcription factor Sp1 directly bound to the promoter of circHipk2 and affected the expression of Hipk2 and circHipk2 in C2C12 myoblasts. Collectively, these findings identify circHipk2 as a candidate circRNA regulating ribosome biogenesis and myogenesis proliferation and differentiation.
Highlights
Myogenesis plays important roles in skeletal muscle regeneration and growth [1,2]
The results revealed that circHipk2 inhibited myoblast proliferation and promoted differentiation by targeting ribosomal protein L7 (Rpl7), a ribosomal protein that is a component of the 60S subunit
We investigated the role of circHipk2 on myoblast differentiation, and found circHipk2 prevented the expression of the myoblast determination factors at both mRNA (Figure 2e) and protein levels (Figure 2f), whereas overexpression of circHipk2 increased the expression of these differentiation markers (Figure S2D)
Summary
Myogenesis plays important roles in skeletal muscle regeneration and growth [1,2] It is a multistep process, including myoblast proliferation, myocyte differentiation, fusion of multinuclear myotubes with the central nucleus, and further muscle fiber maturation [3,4]. Many studies have revealed the crucial functions of circRNAs in myogenesis as a miRNA sponge [14,15]. The Hipk gene is involved in cell cycle regulation, and functions alternatively as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression [25,26]. Our recent study suggested that overexpression of transcription factor Sp1 promoted differentiation and repressed proliferation in C2C12 myoblasts [27]. Our findings indicated that circHipk may exert regulatory functions in skeletal muscle development
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