Abstract
MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle. Using miRNA sequencing and bioinformatics analysis, we previously found that the miRNA miR-664-5p is significantly differentially expressed in longissimus dorsi muscles of Rongchang pigs. However, the molecular mechanism by which miR-664-5p regulates myogenesis remains unclear. In this study, using flow cytometry, 5-ethynyl-2'-deoxyuridine staining, and cell count and immunofluorescent assays, we found that cell-transfected miR-664-5p mimics greatly promoted proliferation of C2C12 mouse myoblasts by increasing the proportion of cells in the S- and G2-phases and up-regulating the expression of cell cycle genes. Moreover, miR-664-5p inhibited myoblast differentiation by down-regulating myogenic gene expression. In contrast, miR-664-5p inhibitor repressed myoblast proliferation and promoted myoblast differentiation. Mechanistically, using dual-luciferase reporter gene experiments, we demonstrated that miR-664-5p directly targets the 3'-UTR of serum response factor (SRF) and Wnt1 mRNAs. We also observed that miR-664-5p inhibits both mRNA and protein levels of SRF and Wnt1 during myoblast proliferation and myogenic differentiation, respectively. Furthermore, the activating effect of miR-664-5p on myoblast proliferation was attenuated by SRF overexpression, and miR-664-5p repressed myogenic differentiation by diminishing the accumulation of nuclear β-catenin. Of note, miR-664-5p's inhibitory effect on myogenic differentiation was abrogated by treatment with Wnt1 protein, the key activator of the Wnt/β-catenin signaling pathway. Collectively, our findings suggest that miR-664-5p controls SRF and canonical Wnt/β-catenin signaling pathways in myogenesis.
Highlights
MicroRNAs are noncoding RNAs that regulate gene expression at the post-transcriptional level and are involved in the regulation of the formation, maintenance, and function of skeletal muscle
Sequence alignment of mature miR-664-5p among multiple species, including mice and pig, showed that miR-664-5p was poorly conserved in the seed sequence (Fig. S1B), the similar expression profiles of miR-664-5p in myogenesis between mice and pigs implied that miR-664-5p played a crucial role in skeletal muscle development
We further performed GO term analysis for miR-664-5p target genes and found that miR-664-5p may be involved in skeletal muscle development, especially muscle cell proliferation and differentiation (Fig. 1E)
Summary
Our previous study demonstrated that miR-664-5p showed a differential expression in different growth stages (Fig. S1A), and we detected miR-664-5p expression in the skeletal muscle of 2-, 8-, 26-, and 52-week-old mice. The results indicated that the over- G and H) and down-regulated the levels of cell cycle marker expression of SRF attenuated miR-664-5p–induced effects on genes, including cyclin E and cyclin D1, but it significantly myoblast proliferation by decreasing EdU-positive cells (Fig. 4, increased the levels of p27 (Fig. 4, I–K). The results showed that the Wnt treatment restored miR-664-5p–induced inhibitory effects on myogenic differentiation by increasing the MyHC-positive cells, differentiation index, and multinucleated myotube fusion index, and it up-regulated the expression of MyoG and MyHC (Fig. 7, A–D). All of these results demonstrated that Wnt was a direct target gene of miR-664-5p in differentiating cells. Mation through targeting SRF and the Wnt– catenin signaling pathway (Fig. 8G)
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