Nur77 Promotes Cell Adhesion And Myogenesis Through Regulating Zeb1 Transcript Levels In Myoblasts

Objective:Skeletal muscle is one of the largest organs of the human body,accounting for approximately 40% of total body weight.In addition to supporting exercise and breathing,it also plays an essential role in physiological activities such as body metabolism and endocrine system.Therefore,the loss of skeletal muscle mass could inevitably lead to decline in functional activity,resulting in a decline in the quality of life and survival rate,especially in the elderly.Further exploration on the mechanism of skeletal muscle myogenesis has attracted more attention.Skeletal muscle development,also termed as myogenesis,that occurs both during embryonic development as well as regeneration and repair after injury.Skeletal muscle is a highly differentiated tissue composed of bundles of muscle fibers that generate contraction,force and maintaining movement.Muscle fibers are formed by the fusion of monocyte precursor cells,called myoblasts and satellite cells,during development.Satellite cells are activated and differentiated into muscle fiber stem cells during regeneration after muscle injury.Differentiation of skeletal myoblasts into myofibers is a multistep process involving withdrawal from the cell cycle,adoption of a musclespecific transcriptional program,cell elongation,and cell-cell fusion.Myoblast fusion is one of the key steps in skeletal muscle development.During myoblast fusion,a variety of cell separation times occur,including migration,adhesion,elongation,cell recognition,and alignment.In order to fuse,the stretched myoblasts must adhere to the cell surface and recognize the transmembrane proteins correctly.A variety of proteins that regulate the formation and recognition of cell-cell contact are involved in this process,including n-and m-cadherin,caleolin-3,N-and V-cadherin,and classic integrins.The role of these proteins in myoblast differentiation has also been demonstrated in knockout mice,as well as in vitro studies of primary cells.For example,N-cadherin is expressed throughout the myogenic process.N-cadherin can promote the differentiation of myoblasts and activate the expression of muscle specific proteins in myoblasts.M-cadherin was detected in embryonic precursor cells and developing myoblasts.In adult skeletal muscle,m-cadherin is expressed in both stationary satellite cells and regenerated skeletal muscle.Integrin ? 1 has been shown to be involved in myoblast fusion and assembly of myofibroblast cytoskeleton.Integrin ? 3 has also been shown to promote myoblast differentiation in vitro.Therefore,cell adhesion is essential for myoblast fusion.Nuclear receptor 4A 1(Nur77,Nr4a1)is an important member of the nuclear orphan receptor 4A subfamily and the most in-depth research.It has been reported to be involved in the regulation of diverse biological and physiological processes and metabolism in different tissue,such as development,proliferation,apoptosis and energy metabolism.Most studies focus on the ?-adrenergic receptor-mediated signaling pathways and the activation of Nur77 in skeletal muscle development,myogenesis and Glut4-mediated glucose uptake.Global knockout of Nur77 inhibits skeletal muscle development and impairs glucose metabolism.In contrast to the ablation of Nur77 function,in vivo Nur77 overexpression significantly improves skeletal muscle energy metabolism,leading to the mitochondrial biogenesis and induction of oxidative metabolism.It has also been reported that Nur77 could increase the myoblasts fusion by promoting the expression of myogenic specific factors(Myo D and Myo G).The above results suggest that Nur77 is an important factor involved in skeletal muscle generation.However,the research on Nur77 in myoblasts has not been in-depth as well as the mechanism by how Nur77 regulates myoblasts adhesion in myotubes is not reported.This study aims to establish Nur77 overexpression animal and cell models to observe the effects of Nur77 on skeletal muscle mass,muscle function and myoblast differentiation in vivo and in vitro,and try to clarify the mechanism of Nur77 regulating myogenesis from the perspective of myoblast adhesion.Methods:Part ?: Effect of Nur77 on the function of mouse skeletal muscle1.RNA expression profiles of skeletal muscle atrophy and myogenesis were downloaded from NCBI GEO Database.Myoblast differentiation transcription regulators gene set was downloaded from Uniprot KB.The analysis of raw data was completed by code of Python and screen out important myogenic regulatory factors.2.Ten 2-month-old Male C57BL/6 mice were randomly divided into control group(WT)and Nur77 overexpression group(Nur77-AAV),5 in each group.The control virus and Nur77 overexpression AAV were injected into the bilateral gastrocnemius muscle to construct Nur77 overexpression mouse model.3.After 4 weeks' injection,mice grasping force was measured by a grip strength meter.The gastrocnemius muscle was obtained and weighed,then frozen in liquid and stored in a-80? freezer for use.For histological analysis,the sections of gastrocnemius muscle were stained with H&E.Part ?: Effect of Nur77 on the adhesion of skeletal muscle cells1.The mouse myoblast cell line(C2C12)were cultured and transfected with Control lentivirus and Nur77 overexpression lentivirus.After transfection,the stable transfected cell lines were selected with Puromycin,and verified by Western blot and qRT-PCR.2.The mouse myoblast cell line(C2C12)was cultured in vitro,and the shRNA control lentivirus and Nur77 shRNA lentivirus were transfected.After the transfection was completed,puromycin selection was performed to construct a low expression control(Control)and Nur77 low expression(sh-Nur77)stable Transform the cell line and verify the cell model by Western blot and qRT-PCR.3.Using phalloidin to stain the cytoskeleton to observe the morphological changes of myoblasts.4.qRT-PCR and Western blot were used to detect the mRNA and protein expression of adhesion factors in Control group and Nur77 overexpression group myoblasts,Control group and sh-Nur77 group myoblasts and Control group and Nur77-AAV overexpression mouse gastrocnemius muscle tissue.5.Immunofluorescence technology was used to detect the protein level expression and localization of adhesion factor of Control and Nur77 overexpression myoblasts,Control and Nur77 shRNA myoblast,and Control group and Nur77-AAV group mice.Part ?: Study on the mechanism of Nur77 affecting myoblast adhesion and skeletal muscle formation1.The upstream genes that regulate cell adhesion factors expression were predicted from the Ch EA3 transcription factor prediction site.2.The compilation analysis of raw data were downloaded from NCBI GEO database.3.qRT-PCR and Western blot were used to detect mRNA and protein levels of Zeb1 in Nur77 overexpression and Nur77 knockout myoblast.4.In myoblasts,the Zeb1 low-expressing cell model was constructed by transfection control(Control)and Zeb1 small interferingRNA(si Zeb1),and the cell model was verified by Western blot and qRT-PCR.5.qRT-PCR was used to detect the transcription level expression of cell adhesion factors of si Zeb1 and control myoblasts.Western blot was used to detect the protein expression of cell adhesion factors(N-cadherin).6.Transfect Zeb1 overexpression plasmid and control plasmid into Nur77 low-expressing myoblasts(sh-Nur77)and control myoblasts.Western blot was used to detect cell adhesion factor(N-cadherin)protein expression.Phalloidin cells Skeleton dyeing was used to observe its morphological changes7.The JASPAR website was used to predict the binding site of Nur77 binding sequence element in the Zeb1 promoter region.8.Ch IP and luciferase reporter experiments were used to detect the regulatory effect of Nur77 on Zeb1 transcription activity.9.Zeb1 small interferingRNA and Control small interferingRNA were transfected into Nur77 overexpression and its control myoblasts and cultured under differentiation condition for 5 days.The myotube formation was observed under bright field microscope.Results:Part ?: Effect of Nur77 on the function of mouse skeletal muscle1.205 differential genes were screened from theRNA-seq and microarray databases of skeletal muscle atrophy and skeletal muscle development.These differential genes were intersected with the transcription factor gene set of skeletal muscle differentiation,and Nur77 was selected as an important skeletal muscle regulator.2.Compared with Control group mice,Nur77-AAV group mice showed higher grip.The gastrocnemius muscle weight was increased after Nur77-AAV treatment.The cross sectional area of skeletal muscle in Nur77-AAV group was significantly larger than the WT group.Part ?: Effect of Nur77 on the adhesion of skeletal muscle cells1.Nur77-overexpressing myoblasts showed a phenotype of large cell size and polygonal macrophage-like shape,as well as more cell-cell junction area between myoblasts through phalloidin staining.2.Smaller sell size,spindle-shaped,and decreased myoblast contact area were observed compared to Control group myoblasts.3.The mRNA expression of cadherin and integrin family members was significantly increased in the Nur77 overexpression myoblasts.N-cadherin was significantly increased in the Nur77 overexpression myoblasts.More N-cadherin accumulated at the cell junction area in the Nur77 overexpression group compared to the control group.4.The mRNA expression of cadherin and integrin family members was significantly decreased in the sh-Nur77 myoblasts.N-cadherin was significantly decreased in the shNur77 myoblasts.Less N-cadherin accumulated at the cell junction area in the sh-Nur77 group compared to the control group.5.In gastrocnemius muscle after Nur77-AAV injection,the mRNA expression of cadherins and integrins were increased and protein expression of N-cadherin was also unregulated.Part ?: Study on the mechanism of Nur77 affecting myoblast adhesion and skeletal muscle formation1.The Pearson correlation coefficient of Nur77 and Zeb1 mRNA was the highest in the overlap set of significantly different genes.2.mRNA and protein levels of Zeb1 were increased upon Nur77 overexpression and decreased upon Nur77 knockout C2C12.3.Deletion of Zeb1 significantly reduced the mRNA levels of cell adhesion molecules.The protein level of N-cadherin was also decreased.4.Transfection of exogenous Zeb1 overexpression plasmid into Nur77 low expression myoblasts can rescue the decrease of N-cadherin expression and the decrease of contact between myoblasts caused by the low expression of Nur77.5.The JASPAR analysis of the Nur77 binding sites identified a consensus motif and 3Nur77 binding sites located with to the TSS at the Zeb1 promoter.6.Ch IP-q PCR and luciferase reporter assay showed that Nur77 could directly bind to the Zeb1 promoter region in C2C12.7.In Nur77 overexpression myoblasts,the number of myotubes was significantly decreased in Zeb1 interference group compared with the control group.Conclusion:Part ?: Effect of Nur77 on the function of mouse skeletal muscle1.Nur77 acts as an important transcription factor in skeletal muscle.2.The overexpression of Nur77 shows a phenotype of skeletal muscle hypertrophy.Part ?: Effect of Nur77 on the adhesion of skeletal muscle cells1.Nur77 can promote the connection between cells and increase the expression of cell adhesion factors in myoblasts and mice.Part ?: Study on the mechanism of Nur77 affecting myoblast adhesion and skeletal muscle formation1.In myoblasts,Zeb1 could regulate the expression of cell adhesion molecules.2.Nur77 as the transcription factor of Zeb1 to regulate myoblasts adhesion and myogenesis.