The Role Of MiR-192 In Proliferation And Myogenic Differentiation Of Sheep Skeletal Muscle Satellite Cells

Muscle development is an important field in developmetal biology. Recently, some studies found that miRNA involved in muscle development. Sheep are important farm animals worldwide, producing high quality meat with the ability to adapt to different climatic and grazing conditions. An improved understanding of the molecular mechanisms of ovine muscle development(myogenesis) may increase meat productivity in the sheep industry. Our study employed myogenic differentiation of sheep skeletal muscle satellite cells to mimic muscle development in vitro.Here, we demonstrated that miR-192 was down-regulated during the myogenic differentiation of satellite cells and mouse C2C12 myoblasts. We found that miR-192 repressed satellite cells differentiation. Dual-luciferase reporter assay confirmed that RB1 was the direct target of miR-192 during satellite cells myogenic differentiation. Knockdown of endogenous RB1 by siRNA promoted proliferation but inhibited myogenic differentiation of satellite cells. The following is the main resuts of our study:(1) We collected fetal sheep leg muscle samples and then carefully separated satellite cells according to previous study. The satellite cells were proliferated in presence of serum(growth medium(GM)) and induced to terminally differentiated myotube when serum was withdrawn from the GM(differentiation medium(DM)). They formed significant myotubes after they were induced to differentiation. Moreover, the myogenin, an early myogenic marker, and myosin heavy chain(MHC), a late myogenic marker, were both normally expressed during satellite cells differentiation.(2) We found that the expression level of miR-192 was significantly higher in skeletal muscle of sheep fetuses than in adult. The expression of miR-192 in two developmental stages of sheep skeletal muscle was confirmed by qPCR. The results showed that miR-192 was up-regulated in skeletal muscle of sheep fetuses at 90 days post coitus, and was down-regulated in adult at 2.5 years postnatal.(3) MiR-192 levels declined on days 1 and 3 differentiation, which was a period of myotube formation. Similarly, miR-192 was also down-regulated on days 3 differentiation of C2C12 myoblasts. The results showed that miR-192 was down-regulated during satellite cells and C2C12 myoblasts myogenic differentiation.(4) The introduction of miR-192 decreased the protein levels of myogenin and MHC by days 2 and 3 of differentiation. These changes also were confirmed by significant decrease in the mRNA expression levels of myogenin and MHC. Moreover, overexpression of miR-192 dampened myotube formation, as confirmed by decreased differentiation index. We also introduced the 2’-O-methyl antisense oligonucleotides against miR-192 and single-stranded negative control into satellite cells. Inhibition of miR-192 enhanced the protein and mRNA expression of myogenin and MHC, respectively, and this was accompanied by enhanced myotube formation. Next, we explored the role of miR-192 in mouse C2C12 myoblasts differentiation. Similarly, overexpression of miR-192 inhibited C2C12 myoblasts differentiation, while inhibition of miR-192 promoted their differentiation. We concluded that miR-192 played a negative role in the myogenic differentiation of sheep skeletal muscle satellite cells and mouse C2C12 myoblasts. Next, we examined the role of miR-192 on proliferation of sheep SCs using 5-Ethynyl-2’-deoxyuridine(EdU) cell proliferation assay. We found that overexpression and inhibition of miR-192 increased and decreased the proportion of EdU-positive cells, respectively, indicating that miR-192 promoted proliferation of SCs. Moreover, the MTT assay further confirmed that miR-192 enhanced SCs proliferation.(5) MiR-192 significantly reduced the reporter activity of the wild-type RB1 3’UTR luciferase reporter and no reduced activity was observed with the mutant RB1 3’UTR luciferase reporter. These results demonstrated that miR-192 directly targeted the 3’UTR of RB1. Furthermore, the introduction of miR-192 repressed endogenous RB1 mRNA and protein in sheep satellite cells. Together, these results suggest that the protein and mRNA expression of RB1 were directly inhibited by miR-192. And knockdown of RB1 significantly repressed the satellite cell myogenic differentiation in culture.Together, our study uncovered that miR-192 targeting RB1 inhibits myogenic differentiation but promotes proliferation of sheep skeletal muscle satellite cells.