CircMGA Depresses Myoblast Proliferation and Promotes Myotube Formation through miR-144-5p/FAP Signal

SIMPLE SUMMARY: Circular RNAs (circRNAs) are a class of RNAs with a circular structure that can regulate genes by acting as microRNA sponges to neutralize microRNA and release mRNA. Poultry muscle growth and development include the increase of myoblast cell numbers before birth and hypertrophy of muscle fibers after birth. In this study, we used a series of molecular and cell biology methods to identify a novel circRNA, named circMGA, that could combine miR-144-5p competitively with FAP and inhibit myoblast proliferation and promote myotube formation. The results of this study have provided new evidence and extended the knowledge about non-coding RNAs in muscle development. ABSTRACT: Circular RNAs are endogenous and abundant in skeletal muscle, and may not only be involved in regulating gene expression in a variety of ways, but also function as important regulators in poultry muscle development. Our previous research found that circMGA was differentially expressed during chicken muscle embryo development; however, as a novel circular RNA, the regulating mechanism of circMGA in myogenesis has never been studied before. In this study, we aimed to investigate the functional roles and related molecular mechanisms of circMGA in chicken primary myoblast cells. CircMGA originated from the exon 13–14 of MGA gene, was differentially expressed during embryo development and myogenesis differentiation, and could inhibit myoblast cell proliferation by repressing cell cycle related genes and promote myotube formation through MyoD and MyHC. Biotin-labeled miRNA pulldown assay and luciferase reporter assay result showed that miR-144-5p could directly target circMGA and FAP, indicating that there could be a competing endogenous RNA mechanism between circMGA and FAP. In function, miR-144-5p showed opposite regulation in myoblast cell with circMGA and FAP, just as expected. circMGA co-transfected with miR-144-5p or si-FAP could effectively eliminate the inhibition of miR-144-5p on myoblast proliferation and differentiation. In conclusion, we found a novel circRNA, named circMGA, which generated from the 13–14 exon of the MGA gene, and could inhibit myoblast proliferation and promote myotube formation by acting as the sponge of miR-144-5p and through miR-144-5p/FAP signal. Moreover, circMGA could effectively eliminate the inhibition of miR-144-5p on myoblast differentiation, thus releasing FAP and promoting myotube formation.