Effects Of SFPQ Protein On The Differentiation And Fusion Of Skeletal Muscle Myoblasts

Background: Skeletal muscle accounts for 40% of human body weight,plays an important role in exercise and energy metabolism,and is one of the most important tissues of the human body.Skeletal muscle is composed of a large number of muscle fibers,and each multinucleated muscle fiber is formed by the differentiation and fusion of mononuclear myoblasts.The repair of skeletal muscle after injury is also accomplished by the activation,proliferation,differentiation and fusion of muscle satellite cells(a type of muscle stem cell)to form new myofibers.Skeletal muscle development and regeneration are of great significance to the maintenance of life activities,and the fusion of myoblasts is an important part of this process.The differentiation and fusion of myoblasts is a complex dynamic process,which is regulated by many cellular molecules and proteins,but the mechanisms are not fully understood.SFPQ is a DNA/RNA binding protein involved in m RNA splicing,maturation and translation.Studies have found that SFPQ co-localizes with DUX4(the causative gene of facial scapulohumeral muscular dystrophy)in myoblasts.Overexpression of DUX4 causes ectopic expression of SFPQ.The expression level of SFPQ m RNA decreases during the differentiation and fusion of myoblasts.And before the fusion of myoblasts,SFPQ expresses on the cell membrane and disappears after the fusion,but its destination is still unclear.Another study has found that knocking out SFPQ in skeletal muscle can cause progressive muscle mass loss and metabolic myopathy in mice,which are manifested as decreased skeletal muscle mass,impaired motor function,severe growth defects,and premature death.It shows that SFPQ is involved in the development of skeletal muscle.But this study only knocked out SFPQ in fast-twitch fibers,and did not explore the effect of knocking out SFPQ on the differentiation and fusion of myoblasts.However,the role of SFPQ in this process is unknown.Objectives: 1.Explore the role of SFPQ in the differentiation and fusion of myoblasts.2.Observe the localization changes of SFPQ during the differentiation and fusion of myoblasts.Methods: 1.RT-q PCR and Western blot analysis of SFPQ m RNA and protein expression during myoblast differentiation and fusion,skeletal muscle development,and regeneration(intramuscular injection of cardiac toxin).2.Use immunofluorescence or CRISPR/Cas9 technology to insert GFP into the Nterminus of myoblast SFPQ to observe the translocation of SFPQ during myoblasts differentiation and fusion.3.Use CRISPR/Cas9 to knock out SFPQ in myoblasts to study its role in myoblast differentiation and fusion.Results: 1.During the differentiation and fusion of myoblasts,the expression of SFPQ m RNA decreases in vitro,and the expression of protein first increases and then decreases;in the process of skeletal muscle development and damage repair in mice after birth,both m RNA and protein expression first increase and then decrease.2.Before myoblast differentiation and fusion,SFPQ protein is expressed in the cell membrane,cytoplasm and nucleus,and is translocated from the membrane and cytoplasm to the nucleus at the early stage of differentiation and fusion,and from the nucleus to the cytoplasm at the late stage of differentiation and fusion.3.The CRISPR/Cas9 gene editing method has been established in C2C12 cells,and a myoblast clone with 75% knockout efficiency of SFPQ protein has been screened.Conclusions: 1.The expression of SFPQ protein increases during the active phase of myoblast differentiation and fusion,and it participates in the process of myoblast differentiation and fusion.2.The localization of SFPQ protein changes during the differentiation and fusion of myoblasts.