| Skeletal muscle accounts for approximately 50% of an animal’s carcass and analyzing the molecular genetic mechanism that regulates its growth and development is the key to improving animal meat performance.The number of muscle fibers has been determined after birth,and muscular growth is primarily characterized by an increase in muscle fibre size.Micro RNAs(miRNAs)are small non-coding RNAs of about 22 nt in length,which have been widely reported to play an important role in the development and regeneration of human and animal skeletal muscle,mainly regulating the growth of chicken skeletal muscle by targeting genes involved in signaling pathways.The miR-181-5p family was involved in a variety of organismal activities and mammalian skeletal muscle cells,but the mechanisms by which the miR-181-5p family regulates the growth and development of chicken skeletal muscle remain largely unknown.In this study,we used chicken skeletal muscle satellite cells(SMSCs)as the main material to study two members of the miR-181-5p family in chickens,miR-181a-5p and miR-181b-5p,and used quantitative real-time fluorescence PCR(RTqPCR),Western Blot,immunofluorescence,dual-luciferase reporter analysis and other assays and methods to investigate the miR-181-5p family.The techniques and methods were used to functionally validate the role of the miR-181-5p family in the differentiation of SMSCs and to investigate its regulatory mechanism in myogenesis,and the main findings were obtained as follows:1.By comparing the skeletal muscle of broilers and layers by detecting muscle development related indexes,it was found that the body weight,breast muscle weight and the rate of increase of broilers aged 0-6 weeks were significantly higher than those of laying broilers(P<0.05),and the cross-sectional area of breast muscle fibres was significantly larger in broilers than in layers at 3 weeks of age.In addition,the expression levels of Myo D1,Myo G,Myf5,and My HC genes,which regulate muscle differentiation,were significantly higher in the pectoral muscle of broilers than those of layers(P<0.01).2.Through the query of miRbase database,it was found that chicken miR-181a-5p and miR-181b-5p are located near the PTPRC gene on chromosome 8 of the chicken genome(GRCg7b)and are produced at the 5’ end of the miR-181 family precursor miRNAs miR-181 a and miR-181 b.Analysis of the expression levels of miR-181a-5p and miR-181b-5p in different tissues of chickens revealed that they were highly expressed in skeletal muscle and significantly higher in broiler breast muscle than in layers(P<0.01).3.In cultured chicken skeletal muscle satellite cells in vitro,RT-qPCR and Western Blot assays revealed that overexpression of miR-181a-5p and miR-181b-5p both promoted the m RNA and protein expression levels of Myo D1,Myo G,and My HC(P<0.05),but the effect was reversed after interference treatment of the miR-181-5p family(P<0.01);immunofluorescence staining showed that miR-181-5p family induced myotube formation in skeletal muscle satellite cells.These results suggest that the miR-181-5p family promotes skeletal muscle satellite cell differentiation.4.Bioinformatics analysis predicted that growth transforming factor beta receptor 1(TGFBR1)was the target gene of miR-181a-5p and miR-181b-5p.Dual luciferase reporter gene validation showed that miR-181a-5p and miR-181b-5p bind targetingly to the 3’UTR region of TGFBR1.Overexpression or interference with miR-181a-5p and miR-181b-5p in cultured chicken skeletal muscle satellite cells in vitro resulted in a corresponding decrease or increase in m RNA and protein expression levels of TGFBR1(p<0.01),indicating that the miR-181-5p family inhibits TGFBR1 expression.5.Functional validation of the TGFBR1 gene revealed that interference with TGFBR1 in cultured chicken skeletal muscle satellite cells in vitro resulted in an increase in m RNA expression levels as well as protein expression levels(P<0.01)of genes related to cell differentiation(Myo D1,Myo G and My HC),and immunofluorescence results showed that myotubular differentiation was inhibited,indicating that the TGFBR1 gene negatively regulates the differentiation process of skeletal muscle satellite cells.Further analysis revealed that the protein expression level of SMAD2/3 downstream of TGF-β signaling was largely unchanged after disturbing TGFBR1 in cells(P>0.05),while the protein expression of phosphorylated SMAD2/3 was significantly reduced(P<0.05),indicating that TGFBR1 could activate the TGF-β signaling pathway.6.Western Blot detection of SMAD2/3 phosphorylated protein expression levels after miR-181a-5p and miR-181b-5p overexpression and interference revealed that knockdown of the miR-181-5p family promoted phosphorylated SMAD2/3 protein expression(P<0.05),whereas overexpression of the miR-181-5p family resulted in a decrease in SMAD2/ 3phosphorylation(P<0.05),indicating that the miR-181-5p family plays a negative regulatory role in the TGF-β signaling pathway.In conclusion,the results of this study suggested that the miR-181-5p family blocked the TGF-β signaling pathway by targeting and silencing the expression of TGFBR1,thereby promoting the differentiation process of chicken skeletal muscle satellite cells.The above findings can be used as a reference for further research into the molecular mechanisms of miRNAs in chicken skeletal muscle growth and development,as well as a theoretical foundation for performing molecular breeding experiments to improve chicken muscle growth performance. |
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