| Muscle is one of the earliest formed tissue during the embryonic development oforganization,which is a complex biological process (myogenic determination，the fusion ofmyoblast into multinucleated myotubes and the next differentiation into muscle cells withterminal morphology and contractility), and regulated by many myogenic regulatory factors.The proliferation and differentiation can regulate and repaire skeletal muscle injury or atrophy.It is very important for the study on proliferation, differentiation of myoblast and the satellitecell of mature organisms.According to consulting the data and referring NCBI microarray database, we found thatthe expression of Sirt2was higher in muscle tissue, and the expression abundance haschanged during the process of proliferation, differentiation and atrophy. Thus wehypothesized that Sirt2would play a role on muscle development. In order to validate thebiological function comprehend the regulation mechanisms of Sirt2, firstly, we detected thetime-sequential expression of Sirt2during myoblast differentiation, and then evaluated theeffect of Sirt2on myoblast proliferation, differentiation, myotube formation and some relatedsignaling pathway factors from morphology to gene expression levels by constructing theexpression and interference vector of Sirt2to treat C2C12myoblast and using flow cytometry,immunofluorescence, Real–time PCR and Western blot. Next, in order to furtherconsummate cell trial in vitro and establish a base for function study of Sirt2, we also detectedthe effect of Sirt2on the regeneration and repair after muscular injury and myoatrophy byinjecting the Sirt2adenovirusin into the constructed muscle injury model and myoatrophymodel and using frozen section, immunofluorescence and Western blot.The specific results are as follows:1. Overexpression of Sirt2in C2C12upregulated the expression of CyclinD1and CDK2,promoted the proliferation of myoblast, activiated the ERK1/2signaling pathway and affectedcell cycle progression2. The expression level of Sirt2showed the first decline after rising trend during muscledifferentiation, and was high in myotubes; Sirt2was found to inhibit myoblast differentiationin early differentiation phase and ruduce mRNA and protein expression of MyoG and MyHC by using Sirt2over-expression vector to treat C2C12myoblasts. By using Sirt2interferencevector, opposite result was observed, the phosphorylation of FoxO1was increased and thetranscription activity of FoxO1was inhibited.3. By building muscle tibialis anterior muscle injury model with BaCl2and intramuscularinjected Sirt2ovexpression adenovirus, the expression of Pax7and Desmin was significantlyincreased, the number of inflammatory cells and phagocytosis enhancement in injured tissuewere increased, the clearance of damaged fibers was accelerated. It was suggested that Sirt2be helpful to repair the muscle injury.4. In myoblast differentiation stage, Sirt2interference inhibited myotube formation,increased atrophy related gene expression of MuRF-1, decreased mTOR expression; Sirt2overexpression obtains the opposite result, PI3K/AKT and p38MAPK signaling pathway wasactivated in this process.5. By construction mouse denervated atrophy model, the tibialis anterior muscle andgastrocnemius muscle were obvious atrophied; The anterior tibial muscle wet weight andmuscle fiber diameter was significantly higher in Sirt2treated group, and the expression ofMuRF-1was decreased, satellite cells was more than the control group.In summary, Sirt2could promote myoblast proliferation through the ERK1/2signalingpathway, and inhibite the early differentiation of muscle cells by affecting FoxO1transcription activity; In later stage of myoblast differentiation, Sirt2could promote myotubeformation through the PI3K/AKT and p38MAPK signaling pathway and also promote therepair after skeletal muscle injury and alleviate denervation muscle atrophy. |
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