Effect And Mechanism Of RASSF4 Protein On The Differentiation And Fusion Of Skeletal Muscle Myoblasts

Research Background:Skeletal muscle is the muscle tissue attached to the bone.It is one of the three muscle tissues of the human body and participates in body movement and energy metabolism.The development and repair after injury of skeletal muscle are closely related to myoblasts.Skeletal muscle is developed from myoblasts.Myoblasts differentiate and fuse to form multinucleated syncytium muscle fibers.Muscle fibers are arranged in bundles,and finally develop into skeletal muscle.Skeletal muscle has significant regeneration ability.When skeletal muscle is injured,satellite cells are activated into myoblasts,which differentiate and fuse to repair skeletal muscle.When skeletal muscle cannot develop normally or be repaired after injury,the body will get diseases,such as congenital myopathy,myasthenia gravis and progressive muscular dystrophy.Therefore,the development and repair after injury of skeletal muscle are of great significance to the body,and it is very important to study the differentiation and fusion mechanism of myoblasts.It was found that the m RNA and protein expression levels of RASSF4 changed during the differentiation of human primary myoblasts in vitro,but whether RASSF4 participated in the process of myogenesis in vivo and its mechanism were unclear.RASSF4 is closely related to ARF6: ARF6 promotes myoblast differentiation and fusion by activating PLD and pip5 k to produce PA and PIP2,while the combination of RASSF4 and PIP5 K can also regulate the production of PIP2;In He La cells,RASSF4 interacts with ARF6-GDP to promote ARF6 activation.Whether RASSF4 regulate myoblast differentiation and fusion by acting with ARF6? Whether it have other mechanisms? Therefore,this paper aims to explore whether RASSF4 participates in the differentiation and fusion process of myoblasts in vivo and in vitro and its mechanism,so as to further improve the differentiation and fusion mechanism of myoblasts and provide a theoretical basis for the treatment of myopathy.Objective:1.Whether RASSF4 involved in the differentiation and fusion of skeletal muscle myoblasts.2.Mechanism of RASSF4 regulating the differentiation and fusion of skeletal muscle myoblasts.Methods:1.Western blot and q PCR were used to detect the expression of RASSF4 in the process of skeletal muscle myoblast differentiation and fusion in vitro,skeletal muscle formation and repair after skeletal muscle injury.2.Western blot and Co-IP were used to observe the relationship between RASSF4 and ARF6 in primary myoblast from repaired skeletal muscle after injury.3.RASSF4 truncated plasmid was constructed and transfected into C2C12,for observing the effect of RASSF4 on myoblast differentiation and fusion.Confocal were used to observe the protein localization;The expression levels of myogenin and myosin were detected by Western blot.Results:1.The results of Western blot and q PCR showed that the expression of RASSF4 increased in the process of skeletal muscle myoblast differentiation and fusion in vitro,skeletal muscle formation and repair after skeletal muscle injury.2.In primary myoblast from repaired skeletal muscle after injury,Western blot experiments showed that the expression of RASSF4 and ARF6 increased significantly;co-IP experiments showed that RASSF4 could bind to ARF6 in both uninjured and injured,and preferentially bind in injured.3.The SARAH domain of RASSF4 may contain nuclear expotr-signal;RASSF4 is a shuttle protein;After deleting the SARAH domain,RASSF4 resides in the nucleus and can promote myoblast differentiation and fusion.Conclusions:1.RASSF4 involved in the differentiation and fusion of skeletal muscle myoblasts.2.RASSF4 regulate the fusion of myoblasts through interaction with ARF6.3.SARAH domain determine the localization of RASSF4 and delete SARAH domain could improve the differentiation and fusion of skeletal muscle myoblasts.