Abstract
The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.
Highlights
Skeletal muscles are involved in a wide range of life activities, including thermoregulation, movement, organ protection, respiration, and buffering
The results on the expression of miR-21-5p in the skeletal muscle and other tissues showed that miR-21-5p was highly expressed in the breast muscle and leg muscle higher than those expressed in the heart, lung, gizzard, and other tissues, whereas the lowest expression was observed in the brain and glandular stomach (Figure 1C)
To investigate the expression pattern of miR-21-5p during the development of skeletal muscle in chickens, we conducted qRT-PCR analysis and the results showed that the expression level of miR-21-5p gradually increased during the development of skeletal muscle in chicken embryos (Figure 1D)
Summary
Skeletal muscles are involved in a wide range of life activities, including thermoregulation, movement, organ protection, respiration, and buffering. In meat-producing animals, the development of skeletal muscle is closely correlated with the quality and quantity of meat products. The body of newly born animals is made up of numerous numbers of muscle fibers that are basically fixed together. In addition to major transcriptional factors, DNA methylation, non-coding RNA (ncRNAs), and signaling pathways play significant regulatory roles in the growth and development of skeletal muscles, including myopathy, remodeling, growth, and repair [3,4,5,6]. MicroRNAs (miRNAs) are functional RNAs containing about 21 nucleotides that are formed from long transcripts after two consecutive splicing processes, which are essential for the development of an organism [7]. It was reported to participate actively in the regulation of gene expression at the post-transcriptional level, resulting in mRNA degradation or translation disruption, by complete or incomplete complementary pairing with the 30
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