| The pig is not only an important agricultural animal,but also a significant model animal for exploring the molecular mechanisms of skeletal muscle growth and development and other human-related diseases.Skeletal muscle formation is an extremely important step in animal growth and development,in which a muscle-specific transmembrane protein,TMEM8c(also known as Myomaker),significantly promotes myobalst fusion,and the results have been validated in many species.However,the role of Myomaker in the fusion of pig myoblast cells and the molecular regulation mechanism are still unclear.Therefore,in this study,real-time polymerase chain reaction(RT-PCR)was used to detect the expression of Myomaker in the growth and development of pig skeletal muscle and myoblasts fusion process.The effect of Myomaker overexpression on the fusion of myoblast cells was studied by immunofluorescence.Through 3’RACE(3’Rapid Amplification of c DNA Ends)technology,we gained Myomaker 3’UTR sequence,and in combination with the dual luciferase reporting system,we screened the target miRNA of Myomaker and verified the effect of target miRNA on myoblast fusion.Finally,the expression of Myomaker in skeletal muscle regeneration was studied by building a model of skeletal muscle injury in pigs.The main results of this study are as follows:(1)The RT-PCR results showed that the expression level of Myomaker in muscle tissues was significantly higher than that in other tissues(heart,liver,spleen,lung,kidney and skin)(P < 0.01),and the expression level of Myomaker in embryo was significantly higher than that after birth(P < 0.01).Western blot results were consistent with RT-PCR results.Primary muscle satellite cells(MSCs)were successfully isolated from the longest dorsal muscle of the Danish Landrace and induced differentiation.The results showed that Myomaker expression increased during cell differentiation and peaked on the fourth day of differentiation.(2)The results of Myomaker function study showed that compared with the control group,Myomaker overexpression promoted the formation of multinuclear myotube,and myotube fusion index significantly increased(P < 0.05),indicating that Myomaker could also promote pig myoblast fusion.(3)Through MEGA6.0 software,we built Myomaker amino acids evolutionary tree,and use the 3’RACE technology successfully amplified in pig Myomaker 3?UTR.(4)Targeted binding of miR-205 to Myomaker 3’UTR was confirmed by Targetscan,RNAhybrid and dual luciferase reporting system.Functional verification of miR-205 showed that compared with the control group,the overexpression of miR-205 significantly inhibited the expression of Myomaker and the fusion of myoblasts(P <0.05)In addition,Myo G(Myogenic differentiation marker)expression was also inhibited(P < 0.05).(5)The skeletal muscle injury model of pigs was successfully constructed by injecting 0.5% Bupivacaine into the dorsal longest muscle of Danish Landrace.The results showed that the expression of Myomaker was activated in the process of skeletal muscle injury,and its expression reached its peak on the fifth day of injury.A large number of new muscle fibers appeared,indicating that Myomaker played an important role in the regeneration process of muscle injury.In addition,some myogenic transcription factors such as Myo D,Myo G and Myf5,have similar expression patterns as Myomaker.In conclusion,this study revealed that Myomaker plays an important role in the process of pig muscle cell fusion and skeletal muscle regeneration,and miR-205 regulates Myomaker expression and myoblasts fusion by targeting the 3’UTR of Myomaker. |
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