MRTF-A Regulates PAX7-mediated Muscle Regeneration And Its Mechanism

Background: Muscle atrophy is a disease that seriously endangers human health.There are no effective treatments.Muscle stem cell-mediated muscle regeneration is considered to be an important pathway for muscle tissue function reconstruction.Paired box 7,PAX7 is a marker gene for muscle stem cells,muscle satellite cells,during satellite cell proliferation and self-renewal.Played a vital role.However,the molecular mechanism by which PAX7 is involved in satellite cell regulation is not well understood.Our previous study found that MRTF-A is highly expressed in satellite cells.Based on this study,we aimed to investigate the molecular mechanism of MRTF-A regulating PAX7 expression,which in turn affects muscle cell proliferation and self-renewal,and is a clinical prevention and treatment of muscle atrophy.Provide new ideas.Methods: Satellite cells were extracted from mouse tissues and identified by flow cytometry and immunofluorescence.The slow overexpression/knockdown MRTF-A myoblast C2C12 cell line was screened by lentiviral technique and detected by RT-qPCR and Western blot.Expression of MRTF-A and PAX7 and downstream proliferation and differentiation related genes in satellite cells and C2C12 cell line stably overexpressing/knockout MRTF-A;detection of overexpression by immunofluorescence,MTT,EdU,flow cytometry,etc.The effect of knockdown of MRTF-A on proliferation,apoptosis and self-renewal of mouse myoblasts;detection of MRTF-A and PAX7 and other related marker genes during proliferation and differentiation by RT-qPCR and Western blot;Injecting cardiotoxin(CTX)to establish a mouse muscle injury repair model,taking different days of muscle tissue for H&E staining,detecting the degree of injury,and detecting MRTF-A and PAX7 and other related markers by RT-qPCR,Western blot and immunohistochemistry.Gene expression;ChIP,EMSA,dual luciferase to determine how MRTF-A regulates PAX7.RESULTS: The expression of MRTF-A and PAX7 was detected in the proliferation and differentiation stages of primary muscle satellite cells and C2C12 cells.The results showed that the expression levels of both were gradually up-regulated during the proliferative phase and gradually decreased during the differentiation phase,indicating MRTF-A and PAX7 may be involved in the proliferation and differentiation of muscle cells.Further in vivo experiments confirmed that during the repair of muscle damage in mice,the expression levels of MRTF-A and PAX7 were significantly up-regulated with the increase of injury,and as the repair was gradually completed,the two returned to normal levels,indicating MRTF-A and PAX7 is involved in muscle regeneration.Overexpression/interference with MRTF-A can promote/inhibit proliferation and self-renewal of muscle cells,and overexpression or interference with MRTF-A does not trigger apoptosis of muscle cells.MRTF-A affects the expression of related genes and downstream genes of PAX7 during muscle regeneration by regulating PAX7.Most importantly,the regulation of PAX7 by MRTF-A was achieved by direct binding to the CarG-box1 site in the promoter region upstream of the PAX7 gene.Conclusion: MRTF-A affects the transcriptional regulation of PAX7 gene by binding to the promoter region of PAX7 gene,which affects the expression level of PAX7 and its downstream genes,and finally regulates the proliferation and self-renewal process of muscle cells.