The Role Of RASSF4 In Differentiation And Fusion Of Skeletal Myoblasts

Research Background: Skeletal muscle is one of the most important organs of human body,which accounts for 40% of the weight of human body and plays an important role in exercise and energy metabolism.Skeletal muscle is composed of a large number of myofibers,each of which is formed by myoblast differentiation and fusion.The repair after skeletal muscle injury is depend on satellite cells(a kind of muscle stem cells).The activated satellite cells differentiate into myocytes,and then the myocytes differentiate and fuse with each other to form new myofibers.The formation and repair of skeletal muscle is of great significance for the maintenance of life activities,and myoblast fusion is an important part of this process.Therefore,it is very important to study the fusion mechanism of myoblasts for the normal development of skeletal muscle and the repair after injury.In this study,we mainly explore the mechanism of myoblast fusion using established animal muscle injury model and inducing myoblast fusion in vitro.A previous study found that ARF6 promotes myoblast fusion through PLD1 / PIP5K-PIP2.ARF6 exists in two states: GTP-bound activated form and GDPbound non-activated form.We use ARF6-Q67 L and T44 N plasmid mutants respectively to simulate the two states of ARF6 to study the role of ARF6 in various cellular activities and underlying mechanisms.Many studies show that RASSF4 can regulate the connection between SOCE and ERPM through ARF6-PIP5K-PIP2,and RASSF4 is easier to combine with ARF6-GDP.The research shows that RASSF4 can adjust the connection between SOCE and ERPM through ARF6-PIP5K-PIP2.Studies have shown that RASSF4 can promote myogenic differentiation of human skeletal myogenic cells,but underlying mechanism needs further study.Therefore,this study will explore the role of RASSF4 in the differentiation and fusion of skeletal myoblasts,as well as the interactions between RASSF4 and ARF6 in myoblast.Objective: 1.To study the role of RASSF4 in the differentiation and fusion of myoblasts.2.To study the relationship between RASSF4 and ARF6 in myoblast.Methods: 1.Semi-quantitative PCR and q PCR were used to detect the expression of RASSF4 in the process of skeletal muscle formation,repair after skeletal muscle injury,and myoblast differentiation and fusion in vitro.2.Overexpression of RASSF4 or knockdown of RASSF4 in mouse myoblast cell line C2C12 was used to observe the effect of RASSF4 on myoblast differentiation and fusion,which were detected by fusion index(The ratio of the number of nuclei in the myotubes to the total number of nuclei)and the expression levels of myogenin and myosin,detected by immunofluorescence microscopy and Western blot.3.RASSF4-YFP and ARF6-Q67L/T44N-HA were overexpressed in C2C12.The relationship between RASSF4 and ARF6 was observed by Co-IP and immunofluorescence colocalization experiments.4.The expression of ARF6-GTP was observed after RASSF4 was overexpressed in C2C12 of mouse myoblasts.Results: 1.The results of semi-quantitative PCR and q PCR showed that the expression of RASSF4 increased significantly in the process of skeletal muscle formation,repair after skeletal muscle injury and myoblast differentiation and fusion vitro.2.After overexpression of RASSF4 in C2C12 of mouse myoblasts,the fusion index increased,and the expression of myogenin and myosin also increased.After knocking down RASSF4,the fusion index of C2C12 decreased,and the expression of myogenin and myosin also decreased.3.After overexpression of RASSF4-YFP and ARF6-Q67L/T44N-HA in C2C12 of mouse myoblasts,co-IP experiments showed that RASSF4 could bind to ARF6 in both undifferentiated(D0)and differentiated(D2)C2C12,and preferentially bind to ARF6-T44N;The Immunofluorescence staining of RASSF4-YFP / ARF6-T44N-HA C2C12 showed that RASSF4-YFP and ARF6-T44N-HA were colocalized.4.After overexpression of RASSF4 in C2C12,increased endogenous ARF6-GTP expression.Conclusions: 1.RASSF4 can promote the differentiation and fusion of myoblasts.2.RASSF4 can regulate the fusion of myoblasts through the regulation of ARF6 activity.