β2 Adrenoceptor Signaling Modulates Myoblast Differentiation via miR-374b-5p/GSK3β and miR-326-3p/Tnfrsf11a Axes

Myoblast differentiation is a crucial step of skeletal muscle regeneration and is impaired in the absence of the β2 adrenoceptor (ADRβ2). Since miR-374b and miR-326 regulate cell differentiation, this study investigated if ADRβ2 regulates myoblast differentiation via miR-374b-5p and miR-326-3p. Primary myoblasts from ADRβ2 knockout (β2KO) mice were cultured in proliferation and differentiation media. In silico, cellular and molecular analyses were performed to evaluate NF-κB signaling activation and validate mRNAs as miR-374b-5p and miR-326-3p targets in β2KO differentiating myoblasts. Differentiating myoblasts from β2KO mice had decreased mRNA levels of Nr4a1, Myog, Myh3, Cdkn1c, and Tnni2 and expression of miR-374b-5p and miR-326-3p. Overexpression of these miRs increased fusion index and gene expression of myogenic markers of these cells. In silico analyses indicated that GSK-3β and receptor activator of NF-kB (Tnfrsf11a) had potential binding sites for miR-374b-5p and miR-326-3p, respectively. The dual-luciferase reporter assay showed that GSK-3β and Tnfrsf11a were targets of miR-374b-5p and miR-326-3p, respectively. Regarding miR-326-3p, there was a reduction of NF-κB signaling and NF-κB mRNA levels in differentiating myoblasts from β2KO mice, and an upregulation of Tnfrsf11a and a downregulation of Tnfrsf11. In summary, ADRβ2 modulates myoblast differentiation via miR-374b-5p/GSK-3β and miR-326-3p/Tnfrsf11a axes.