| The growth and development of skeletal muscle is a very complex process,including the differentiation of muscle-derived stem cells into mononuclear myoblasts,then fusion into multinucleated myotubes,and finally the formation of mature muscle fibers.The growth and development of skeletal muscle is regulated by many differentiation-related genes,such as myogenic regulatory factor family(MRFs),myocyte enhancer factor 2(MEF2),myostatin,(MSTN)and muscle-specific micro RNA.Recently,a secretory protein FNDC4,is a member of the type III fibronectin module containing protein family.Type III fibronectin module contains a total of five proteins(FNDC1-5).Among them,FNDC4 has the highest homology with FNDC5.Shima Nazem et al used sh RNA's method to construct a FNDC5-knocked m ESCs cell line in 2017.The results showed that the decreased expression of FNDC5 inhibited the differentiation of mouse cardiomyocytes.However,the role of FNDC4 in mouse muscle differentiation has not been studied.Therefore,this study used C2C12 cell line cultured in vitro as a model to explore the role of FNDC4 in mouse skeletal muscle differentiation and its possible molecular mechanism,so as to provide experimental basis for revealing the molecular mechanism of skeletal muscle development.The main contents of this paper are as follows: firstly,the expression and localization of FNDC4 in mouse C2C12 cells were detected by Western blotting and immunofluorescence,and the expression of FNDC4 in the process of muscle injury and repair was detected by Western blotting and immunohistochemical staining.The overexpression or inhibition of FNDC4 gene was carried out by adding FNDC4 protein,overexpression vector of FNDC4 gene and si RNA interference fragment in vitro,and the effect of FNDC4 on the differentiation of mouse C2C12 cells was studied.The effect of FNDC4 on the localization of ?-catenin cells was detected by Western blotting.Furthermore,immunoprecipitation and protein spectrum sequencing were used to analyze the proteins that may bind to FNDC4.It was found that low-density lipoprotein receptor-related protein 6 receptor related protein 6(LRP6)might bind to FNDC4,and the interaction between FNDC4 and LRP6 was further verified by co-immunoprecipitation experiment.C2C12 mouse cells were treated with purified FNDC4 protein and XAV939 to activate and inhibit Wnt/ ?-catenin signal pathway,respectively.Western blotting and immunofluorescence staining were used to observe the effects of mouse muscle cell differentiation and ?-catenin cell localization.FNDC4 protein and Dickkopfassociated protein 1(DKK1),an inhibitory protein of LRP6,were added to activate and inhibit LRP6 function.Western blotting and immunofluorescence staining were used to observe the effects of mouse muscle cell differentiation and ?-catenin cell localization.The experimental results show that:(1)During the differentiation of mouse C2C12 cells,the expression of FNDC4 increased gradually.At the same time,the expression of FNDC4 was significantly correlated with the expression of myogenin(Myogenin,Myo G),a marker of muscle differentiation.Immunofluorescence assay also showed that FNDC4 protein was abundant in fused myotubes.In the process of muscle injury repair in mice,the expression level of FNDC4 increased gradually,reached the highest on the 5th day after injury,and then showed a downward trend.There were two processes of proliferation and differentiation in the repair process of the injured site in mice.The results of,Western blotting and immunofluorescence showed that the PAX7 gene which could maintain muscle satellite cells was first expressed,and the expression increased significantly from 1 to 5 days after injury,and then decreased;the expression of muscle differentiation-related gene Myo G increased gradually during the repair process,and then decreased,and the expression of FNDC4 was significantly correlated with the expression of Myo G.FNDC4 may be involved in the differentiation of myoblasts in injured muscles of mice.(2)The results of Western blotting showed that the expression of myogenin(myogenin,Myo G)increased after overexpression of FNDC4,while the expression of Myo G decreased after interfering with FNDC4.Immunofluorescence results showed that the rate of myotube fusion increased after overexpression of FNDC4 and decreased after interfering with FNDC4,and the effect of adding FNDC4 protein in vitro was consistent with that of overexpressed FNDC4,indicating that the expression of FNDC4 could affect the differentiation of mouse C2C12 cells.(3)The results of Western blotting and immunofluorescence showed that the localization of ?-catenin in nucleus decreased after interfering with FNDC4,indicating that FNDC4 may affect the differentiation of mouse C2C12 cells by affecting Wnt/ ?-catenin signal pathway.(4)Protein sequencing showed that FNDC4 might interact with LRP6,and coimmunoprecipitation showed that FNDC4 could bind with LRP6.(5)The addition of FNDC4 in vitro and inhibition of Wnt/ ?-catenin signal pathway,Western blotting and immunofluorescence showed that FNDC4 affected the differentiation of mouse C2C12 cells through Wnt/ ?-catenin signal pathway.(6)The addition of FNDC4 protein and LRP6 competitive inhibitor DKK1 protein,Western blotting and immunofluorescence results showed that FNDC4 acted on LRP6 to activate Wnt/ ?-catenin signal pathway and then affect the differentiation of mouse C2C12 cells.The present study demonstrated that FNDC4 affects C2C12 cell differentiation and participates in the repair of muscle injury in mice.Moreover,FNDC4 may activate the Wnt/?-catenin signaling pathway through LRP6 phosphorylation,thereby activating the transcription of C2C12 differentiation-related genes via increased nuclear translocation of ?-catenin.Overall,our findings provide a foundation for further exploration of the role of FNDC4 as well as insights into the mechanisms of muscle differentiation and potential treatment of muscle injury. |
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