| Background: Skeletal muscle is the largest organ in the human body,and mature skeletal muscle is formed by multinucleated muscle fibers.The formation of muscle fibers depends on the proliferation,differentiation and fusion of stem cells during development and after injury.Physiological and sequential aging leads to the aging of stem cells and impairs the ability of regeneration.Microtubule is the central component of eukaryotic cytoskeleton,which can regulate cell life cycle and cell morphology.The organization and number of intracellular microtubules depend on the proper regulation of microtubule nucleation,and the microtubule nucleation of α/β-tubulin subunits is mediated by γ-tubulin complex.Myoblasts are a kind of stem cells.Stem cell quiescence is known to be related to the inhibition of G0/G1 during the cell cycle transition.Studies have shown that mesoderm/mesenchymal homeobox 1(Meox1)triggers a change in stem cell fate by preventing G2 phase.However,it is not clear whether Meox1 is involved in myoblast differentiation,myotube fusion and microtubule nucleation.Objective: To explore the roles and possible molecular mechanisms of Meox1 in regulating skeletal muscle My OGenic cell differentiation and microtubule nucleation.Methods:(1)Using skeletal muscle C2C12 myoblasts as experimental materials,10% fetal bovine serum proliferation culture environment and 2% horse serum differentiation induction culture conditions were established,respectively.(2)RT-PCR and Western blotting were used to detect the expression of Meox1 during the proliferation and differentiation of C2C12 myoblasts,and RT-PCR was used to detect the expression of NFAT during the proliferation and differentiation of C2C12 myoblasts.(3)The effect of Meox1 on the differentiation and myotube formation of C2C12 myoblasts was detected by immunofluorescence.(4)C2C12 My OGenic cells were treated with Meox1 overexpression or knockdown mediated by adenovirus,and changes in the expression of myocyte generating factor(My OG)and myocyte enhancing factor(MEF2C)were analyzed using immunofluorescence staining.(5)The PKA inhibitor H-89 was treated 24 hours after Meox1 overexpression,and My HC immunofluorescence staining was performed 6 days after differentiation to evaluate changes in C2C12 My OGenic cell differentiation and myotubular fusion;the PKA agonist 8-Bro-c AMP was added into culture medium 24 hours after Meox1 knockdown by sh RNA,and My HC immunofluorescence staining was performed 6 days after differentiation to evaluate whether its inhibitory effect could be reversed.(6)After C2C12 myoblasts overexpressed or knocked down Meox1,the expression characteristics of tubulin(α/β/γ-Tubulin),pericentric protein(pericentrin,PC)and actin(F-Actin)during the proliferation and differentiation of C2C12 myoblasts were analyzed.Results:(1)Meox1expressions were obviouosly increased during both proliferative and differentiation of C2C12 My OGenic cells.(2)At two days of differentiation,the number of myotubes fused with less than three myoblasts were significantly reduced in the Ad-sh Meox1 group;the number of myotubes fused with 3-5 myoblasts were increased in the Ad-Meox1 group,while the difference was not significant in the Ad-sh Meox1 group compared with the control group.At four days of differentiation,the number of myotubes fused with less than three myoblasts was significantly reduced in the Ad-sh Meox1 group compared with the control group.The numbers of myotubes with less than 3 myoblast cells fusion were reduced in the Ad-Meox1 group,the number of myotubes with 3-5 myoblast cells fusion in the Ad-Meox1 group were more than in the control group(Ad-Null),and the number of myotubes in the Ad-sh Meox1 group was less than in the control group(AdNull);the number of myotubes with 5+ myoblast cells fusion was more than the control group(Ad-Null),and the number of myotubes in the Ad-sh Meox1 group were less than the control group(Ad-Null);at four days of differentiation of C2C12 My OGenic cells,the number of myotubes with 5+ myoblast cells fusion was higher in both the control and Ad-Meox1 groups,and the difference was not significant;however,compared to the control group,the number of myotubes with 5+ myoblast cells fusion in the Adsh Meox1 group were significantly reduced in the Ad-sh Meox1 group compared to the control group.(3)The regulation of myoblast differentiation by Meox1 is related to PKA signal pathway.;compared to control group,differentiating C2C12 myoblasts treated with Adsh Meox1 had markedly reduced NFATc1 expression levels and increased NFATc2 expression levels;In the proliferation of C2C12 myoblasts with treatment of Adsh Meox1,NFATc1 expression levels were increased;NFATc2 and NFATc4 expression levels were decreased.(4)In the process of myoblast differentiation,tubulin was scattered in the low Meox1 group compared with the control group,and could not nucleate microtubules.Overexpression of Meox1 in myoblasts promoted PC and γ-tubulin mediated microtubule nucleation,and then PKA blocker partially abolished this effect;when Meox1 was knocked down,microtubule nucleation was reduced,and microtubule nucleation was restored after PKA activator was given.Conclusion :(1)Meox1 regulates My OGenic cell differentiation by affecting My OG/MEF2C;(2)Meox1 regulates My OGenic cell differentiation by affecting NFAT;(3)Meox1-mediated myoblast differentiation is related to microtubule nucleation. |
