TCEA3 Promotes Differentiation Of C2C12 Cells Via An Annexin A1-mediated TGF-? Signaling Pathway

Skeletal muscle production is a highly ordered process that involves the proliferation of myoblasts,the formation of myotubes by cell fusion,and the maturation of these myotubes into various muscle fibers.Myoblast differentiation requires interaction between complex cellular processes,including muscle-specific transcriptional and growth factor regulation and control of external signals.These processes together determine the fate of cell differentiation.Previous experimental results indicate that activation of TCEA3 promotes the differentiation of bovine skeletal muscle satellite cells.Therefore,we speculate that TCEA3 plays an important role in the differentiation of mouse myoblasts.TCEA3 is a member of the transcription elongation factor family.It not only promotes transcription and plays other roles in the cytoplasm,but its mechanism of action is unclear.Annexin A1 positively reg ulates myocyte differentiation by promoting the migration of satellite cells.In the present study,we examined the expression,localization and role of TCEA3 and Annexin A1(ANXA1),which is located in the cytoplasm and also promotes cell differentiation,in C2C12 myoblasts,and examined the changes in protein expression and muscle fiber number after overexpression and inhibition of TCEA3 TCEA3 expression increased with an increase in the number of differentiation days and was mainly expressed in the cell cytoplasm,where it promotes differentiation.Co-immunoprecipitation experiments and western blotting analysis revealed that TCEA3 interacts with ANXA1.Our results showed that interactions between TCEA3 and ANXA1 affect cell differentiation by mod ulating the TGF-? pathway.The main research results obtained are as follows:(1)Immunofluorescence technique was used to detect the expression of TCEA3 in C2C12 myoblast differentiation.During the proliferative phase(0 D)and different differentiation phases(1-9 D of differentiation),the expression of TCEA3 gradually increased with the increase of C2C12 myoblast myotubes.Western blotting results showed that the expression of Myo G and TCEA3 proteins increased with the differentiation of C2C12 myoblasts.(2)p EGFP-N2-TCEA3 was transfected into C2C12 myoblasts and observed under fluorescence microscope.The expression of TCEA3 in the nucleus and cytoplasm was detected by nuclear protein separation.The results showed that TCEA3 mainly accumulated in the cytoplasm,with differentiation.Progression,the expression level of TCEA3 in the cytoplasm is on the rise.(3)si RNA TCEA3 overexpression vector and CRISPR/Cas9 construction technology activate and inhibit TCEA3 expression,respectively.Myogenin(Myo G),myotube fusion index,and expression levels increase with overexpression of TCEA3.The opposite res ult was observed after inhibition of TCEA3.These experimental data indicate that overexpression of TCEA3 activates C2C12 myoblast differentiation in vitro.(4)Protein separation and gel staining were performed by immunoprecipitation and sequencing was performed to detect proteins interacting therewith.We detecte d ANXA1,an annexin that acts as a potential binding partner for TCEA3,similar to TCEA3,which is loca ted in the cytoplasm.(5)After transfection of the ANXA1 inhibitory vector,the expression of ANXA1 and MYOG proteins was significantly reduced.Morphologically,inhibition of ANXA1 affects myotube formation.ANXA1 overexpression vector was transfected into C2C12 cells,and Wes tern blot analysis showed that the expression of MYOG increased when ANXA1 expression increased.The results indicate that activation of TCEA3 can activate C2C12 differentiation in vitro.(6)First we demonstrate that the TGF-? signaling pathway promotes C2C12 differentiation.When TGF-? signaling is inhibited,the function of TCEA3 to promote C2C12 differentiation is also inhibited.To test whether TCEA3 acts through ANXA1,we overexpress TCEA3 after ANXA1 inhibition and detect changes in smad2,p-smad2,MYOG,TCEA3 and ANXA1 protein levels.Compared with inhibition of ANXA1 alone,inhibition of ANXA1 overexpressed TCEA3 and detection of p-smad2,MYOG levels did not change significantly.Therefore,we demonstrate that TCEA3 affects TGF-? signaling pathway through ANXA1,thereby affecting C2C12 cell differentiation.Through the above studies,TCEA3 interacts with ANXA1 to affect the differentiation of C2C12 cells by affecting the TGF-? signaling pathway.The results provide a theoretical basis for further understanding of skeletal muscle growth and development mechanisms and gene therapy for muscle lesions.