Molecular Mechanism Of CircRBBP7 Promoting Myoblast Proliferation And Differentiation And Repairing Mouse Muscle Damage

Understanding the development and growth regulation mechanism of skeletal muscle is the focus of animal genetics and medicine.CircRNA has been found to play a key role in muscle development through transcription and posttranscription translation of the regulator gene.However,the potential regulatory mechanism of circRNA on skeletal muscle development has not been fully elucidated.In this study,we identified a new circRNA circRBBP7,and systematically studied its regulatory effects on muscle development by constructing cell and muscle injury models.Research has found that circRBBP7 is a potential regulatory factor affecting muscle cell development,which can serve as a candidate marker gene for beef cattle genetic breeding and has the potential to be applied in the treatment of muscle injury related diseases.The specific research results are as follows:1.Research on the circular structure detection and encoding ability of circRBBP7.Using RNase R for the digestion of circRBBP7,it was found that circRBBP7 has a stable and nondegradable circular RNA structure.Sanger sequencing confirmed that circRBBP7 has a circular junction region.Further preparation of rabbit derived polyclonal antibodies against circRBBBP7 was carried out,and Western blot analysis revealed that circRBBP7 can encode a protein of approximately 13 k Da.Myoblast immunofluorescence and FISH results showed that circRBBP7 was present in the nucleus and cytoplasm.Explanation: circRBBBP7 is an encoded circRNA.2.Study on the effect of circRBBP7 on the proliferation and differentiation of bovine myoblasts and its molecular regulatory mechanism.Overexpression of circRBBP7 in bovine myoblast cells can significantly promote the expression of marker genes for myoblast proliferation and differentiation;CCK8 and Ed U detection of circRBBP7 can promote myoblast proliferation;The immunofluorescence results of differentiated cells showed that circRBBP7 can improve the efficiency of myoblast differentiation into myotubes.Through RNA seq sequencing,it was found that overexpression of circRBBP7 significantly upregulated the expression of myoblast development related genes such as MDM2.KEGG enrichment analysis showed that differentially expressed genes(DEG)were mainly enriched in the p53 and PI3K-Akt signaling pathways.In addition,overexpression of circRBBP7 downregulated genes is related to slow muscle formation during differentiation.Co-IP mass spectrometry analysis revealed that circRBBP7 binds specifically to VEGFR-1.The results indicate that overexpression of circRBBP7 can upregulate MDM2 expression and promote p53 degradation by targeting VEGFR-1,followed by increased expression levels of CDK2,Cyclin D1,and Myo D,ultimately promoting proliferation and differentiation.3.Application of circRBBP7 in muscle injury repair in C57 mice.Constructing a muscle injury model in C57BL/6 mice,it was found that overexpression of circRBBP7 in the injured tissue resulted in tighter and more complete muscle fibers.On the 14 th day of muscle injury repair,the expression level of Myo D in the injury repair muscle tissue overexpressing circRBBP7 was100 times higher than that in the control group.And through homologous fluorescence double labeling,the muscle tissue on the 14 th day of muscle injury repair was detected.It was found that in the muscle tissue transfected with circRBBP7 in vivo,the number of fast muscle fibers was significantly higher than that of slow muscle fibers.This indicates that circRBBP7 can promote the repair and regeneration of skeletal muscle injuries.In summary,this study found that circRBBP7 can encode peptides that target VEGFR-1.The expression of MDM2 is significantly regulated by circRBBP7 and VEGFR-1.MDM2 is a key regulatory gene in the p53 signaling pathway,affecting the stability of p53.The downstream genes CDK2 and Cyclin D1 of the p53 signaling pathway are key genes regulating muscle development.Therefore,we speculate that circRBBP7 is involved in the MDM2/p53 signaling pathway,Furthermore,it promotes the proliferation and differentiation of bovine myoblasts.Meanwhile,circRBBP7 can significantly promote the repair and regeneration of skeletal muscle damage in C57BL/6 mice.This study provides a reference for the application of molecular regulation technology in livestock genetic breeding and provides a theoretical basis for promoting the development and utilization of modern biotechnology in highquality livestock germplasm resources.