Effects Of Selenium-Mediated Myoblasts Exosome MiRNA On Proliferation And Differentiation Of Skeletal Muscle Satellite Cells In Chicken

Skeletal muscle is the most important component of muscle tissue,accounting for about30%-40%of the total weight of animals.Skeletal muscle satellite cells（SMSCs）,also known as skeletal muscle stem cells,are key to maintaining the regenerative ability of skeletal muscle tissue.SMSCs are located between muscle fiber membrane and basement membrane in a special tissue microenvironment-ecological niche.The microenvironment is involved in regulating the ability of SMSCs to rest,proliferate,migrate,and repair damaged tissues.As the direct attachment point of SMSCs,muscle fibers play a crucial role in regulating the fate of SMSCs.Exosomes are important mediators of signal transmission between cells,as carriers of lipids,functional proteins,DNA fragments,and small noncoding RNA（snc RNA）.Micro RNA（miRNA）are the main snc RNA in exosome.Therefore,it is of great significance to study the effects of endocrine signaling exosome miRNA on the proliferation and differentiation of SMSCs.Selenium is an essential trace element in organisms.Skeletal muscle,as the main part of selenium storage,is involved in regulating muscle homeostasis.Selenium deficiency can lead to skeletal muscle injury,but the research of SMSCs on skeletal muscle injury repair and its specific mechanism is still blank.In this study,a mature muscle tube model was established in vitro,and the effect of selenium-mediated exosome miRNA secreted by myoblast muscle tubes on the proliferation and differentiation of SMSCs was investigated from the perspective of skeletal muscle microenvironment,and the mechanism of selenium’s involvement in maintaining the physiological function of skeletal muscle was further revealed.In this study,myoblasts were extracted from the breast muscle of chicken embryos aged12 days and cultured with Na₂Se O₃（Se）.The cell viability was detected by CCK-8,and the expression level of proteins related to the proliferation and differentiation of myoblasts was detected by Western bolt technology,aimed to determine the optimal Se culture concentration for promoting the development of myoblasts.The muscle tube model of cultured myoblasts was established in vitro and cultured in a medium without exosomes for 24 h.The cell supernatant was collected to prepare a conditioned medium（CM）,CM was used to cultivate SMSCs.CCK-8 was used to detect SMSCs activity and the expression levels of proteins related to the proliferation and differentiation of SMSCs were detected by Western bolt to determine the effects of CM on the development of SMSCs.Exosome were isolated from myotube medium by ultra-fast centrifugation,and exosomes were identified by transmission electron microscopy,nanoparticle particle size tracking technology,and Western bolt,and exosomes were sequenced by PE150 sequencing.The main test results are as follows:（1）Chicken embryo myoblasts cultured in vitro differentiated into mature muscle tubes for 96 h.Meanwhile,0.1μM Se cultured myoblasts significantly increased cell viability for 24h and 48 h（P<0.05）.After 24 h culture,0.01μM-0.1μM Se significantly promoted the expression of Myf5 and Cyclin D1 proteins in myoblasts.At 72 h,0.01μM Se and 1μM Se promoted the expression of Myo D and Myo G proteins in myoblasts（P<0.01）,and 0.1μM Se was selected as the concentration for follow-up treatment.（2）Myoblast myotube CM inhibited SMSCs development.Compared with CON group,the activity of SMSCs cultured by Se treatment myotube CM significantly inhibited for 24 h and 48 h（P<0.01）.Compared with the CON group,the expressions of SMSCs proliferation and differentiation-related proteins Myf5,Cyclin D1,Myo D,Myf6,Myo G,and My HC were significantly inhibited by Se treatment myotube CM（P<0.01）.At the same time,the exosome inhibitor GW4869 significantly restored the activity of SMSCs and the expression of proteins related to proliferation and differentiation.The results showed that myoblast myotubule exosome were involved in Se regulation of SMSCs development.（3）The results of exosome morphology analysis,specific marker protein analysis,and nanoparticle size tracking analysis showed that the nanoparticles obtained by ultracentrifugation were bilayer lipid vesicles with typical“saucer-like”structure.TSG101,CD63,and HSP70 proteins were expressed specifically in lipid vesicles,but Calnexin protein was not expressed.Most lipid vesicles of the particle sizes are distributed in the range of 50-300 nm,showing a unimodal distribution,and the peak value is distributed at 159 nm,which accords with the typical characteristics of exosomes.（4）Exosome small RNA sequencing results showed that 571 miRNAs were detected in exosome.DESeq2 software was used to analyze the differential expression of exosomes in CON group and Se group,respectively.The miRNA expression profile in the Se group was significantly different compared to CON group.By setting the difference multiple and significance threshold,4 down-regulated miRNAs and 4 up-regulated miRNAs were screened.The target genes of differentially expressed miRNAs were analyzed by bioinformatics methods,and MAPK,m TOR,and calcium signaling pathways were found to be important signaling pathways involved in Se-regulated SMSCs proliferation and differentiation through myotube exosome miRNA.In summary,this study confirmed that miRNA of mature myoblasts myotube exosomes participated in Se inhibition of SMSCs activity,proliferation,and differentiation in the skeletal muscle microenvironment.