Investigation The Role Of Lrtm1 In Myogenic Differentiation

ObjectiveSkeletal muscle injury repairing is a hot issue in sports medicine research.Skeletal muscle has a strong ability to regenerate which can deal with minor damage,but severe damage can lead to extensive and irreversible fibrosis,scarring and loss of muscle function.How to improve the ability of regeneration,repair after skeletal muscle injury and promote the early recovery of patients is still a difficult point of research.Therefore,studying the mechanism of skeletal muscle regeneration can provide a theoretical basis for the repairing of skeletal muscle injury and contribute to the treatment of clinical skeletal muscle injury.This study mainly uses CRISPR/Cas9 technology to construct a C2C12 cell line that stably knocks out the LRTM1 gene and tudies the mechanism of LRTM1 on the myogenic differentiation of C2C12 cells.MethodsThe LRTM1 gene in C2C12 cells was stably knocked out using the CRISPR/Cas9 system,and the monoclonal cells knocked out of LRTM1 were selected to construct a C2C12 cell line stably knocking out LRTM1;Induction of LRTM1 knocked out cell line into myogenic differentiation,Western blot and RT-PCR detection of myogenic differentiation markers Myosin and MyoG expression;CCK8 assaydetects cell proliferation after knockout of LRTM1;Western blot analysis of p-ERK levels in C2C12 cells after knockout of LRTM1 and expression of CyclinD1/CDK4 downstream of ERK;Western blot analysis p-MEK,p-RAF and p-SHC levels in MAPK/ERK pathway after knockout of LRTM1.ResultsSequencing results showed stable knockdown of the LRTM1 gene in the C2C12 cell line;Knockout of LRTM1 significantly inhibited cell myogenic differentiation,and the expression of myogenic differentiation markers Myosin and MyoG was significantly reduced;CCK8 results showed that knockdown of LRTM1 promoted cell proliferation,and Western blot results showed that p-ERK and ERK downstream CyclinD1/CDK4 increased expression after knockout of LRTM1;The levels of p-MEK,p-RAF and p-SHC in the MAPK/ERK pathway are increased after knockout of LRTM1.Conclusion1.The C2C12 cell line stably knocking out the LRTM1 gene was successfully constructed.2.Knockout of LRTM1 inhibits myogenic differentiation of C2C12 cells and promotes cell proliferation.3.The reason for inhibition of myogenic differentiation after LRTM1 knockdown may be that the increase of CyclinD1/CDK4 expression inhibits the transcriptional activity of the myogenic transcription factor MyoD.4.Increased expression of CyclinD1/CDK4 is due to increased p-ERK levels5.Knockout of LRTM1 can activate the MAPK/ERK pathway,and LRTM1 may act to inhibit the MAPK/ERK pathway.