| The growth and development of skeletal muscle is a very complex biological process.A large number of studies show that MSTN negatively regulates the proliferation and differentiation of skeletal muscle.At present,most of the studies on the mechanism of MSTN regulating muscle development are focused on the interaction between MSTN and target protein.Therefore,obtaining other mechanisms of action is helpful to further understand the process of MSTN regulating muscle development.1.The muscle of MSTN-/-Luxi cattle and wild-type Luxi cattle were analyzed by using Label Free and LS-MS/MS technology.135 ubiquitinated peptides were identified,belonging to 66proteins.Compared with wild-type cattle,there are 17 ubiquitinated peptides in MSTN-/-cattle,belonging to 17 proteins,4 of which are up-regulated and 13 are down regulated.Bioinformatics analysis showed that the ubiquitinated differential protein was significantly enriched in the process of muscle growth and development,energy metabolism and ubiquitin protein hydrolysis.String analysis showed that the ubiquitinated differential protein was mainly enriched in the process of muscle growth and development.As ubiquitin proteolysis system may play an important role in MSTN mediated muscle development,ubiquitin activator and ubiquitin binding enzyme play a key role in ubiquitin proteolysis system.Combined with related references of muscle atrophy and development,ubiquitin activator UBA1,ubiquitin binding enzyme UBE2L3 and ubiquitin binding enzyme screened from ubiquitin differential protein were selected UBE2N,Myo D1 and Myo G,the myomodulators of MRFs family,were selected to analyze the interaction of string protein with MSTN.The results showed that MSTN,UBA1,UBE2N,UBE2L3,Myo D1 and Myo G had interaction relationship.It is suggested that MSTN may play a regulatory role in muscle development and differentiation through the key ubiquitinases in the ubiquitination pathway.2.Because it is difficult to study the muscle development in cattle,in order to study the effect of MSTN on the development and differentiation of cattle muscle,the following experiments all use the animal skeletal muscle satellite cells established in the laboratory to induce myogenic differentiation in vitro.Firstly,the MSTN inhibitory expression model of bovine skeletal muscle satellite cells was successfully constructed by using si-MSTN.The number of Ed U positive cells in bovine skeletal muscle satellite cells was detected by Ed U cell proliferation experiment,and the effect of MSTN on the proliferation of bovine skeletal muscle satellite cells was studied.The expression of My HC in m RNA and protein level was detected by q RT-PCR and Western blot,and the myotubes formation of bovine skeletal muscle satellite cells was further detected by immunofluorescence technology to study the effect of MSTN on myogenesis of bovine skeletal muscle satellite cells.It was found that interfering with MSTN expression can promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells.3.To further study the relationship between MSTN and ubiquitin proteasome pathway.Firstly,Western blot was used to detect the ubiquitination level of proteins in bovine skeletal muscle satellite cells.It was found that interfering with the expression of MSTN can promote the ubiquitination of proteins in bovine skeletal muscle satellite cells.Therefore,1.25μmol/L proteasome inhibitor MG132 was selected to treat bovine skeletal muscle satellite cells for 24 hours.By blocking the degradation of ubiquitin protein by proteasome,the ubiquitin level of protein in bovine skeletal muscle satellite cells was increased.The number of Ed U positive cells in bovine skeletal muscle satellite cells was detected by Ed U cell proliferation assay,and the cell differentiation marker My HC was detected by Western blot at the protein level,the effects of ubiquitination on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells were studied.It is speculated that MSTN affects the development and differentiation of bovine muscle through ubiquitin proteasome pathway.4.Ubiquitin activating enzyme is the starting point of ubiquitin enzyme-linked reaction,and ubiquitin activating enzyme UBA1 is responsible for 99%ubiquitin activation in cells.Meanwhile,UBA1 is a ubiquitin modified protein in MSTN-/-bovine muscle ubiquitin proteomic analysis.In Chapter 1,it is speculated that MSTN may play a regulatory role in muscle development and differentiation through the ubiquitin related enzymes in the ubiquitin pathway.So in this study,we first used MSTN inhibition expression model to detect the expression level of UBA1 in bovine skeletal muscle satellite cells by Western blot,and found that interfering MSTN expression can promote the expression of UBA1 in bovine skeletal muscle satellite cells.Furthermore,si-UBA1was successfully used to construct the inhibition expression model of bovine skeletal muscle satellite cells by UBA1.By interfering with UBA1,we further analyzed the regulation mechanism of MSTN on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells through ubiquitin proteasome pathway.The number of Ed U positive cells in bovine skeletal muscle satellite cells was detected by Ed U cell proliferation experiment,and the effect of uba1 on the proliferation of bovine skeletal muscle satellite cells was studied.q RT-PCR and Western blot were used to detect the expression of myogenic regulatory factor Myo D1 and differentiation marker My HC in m RNA and protein levels of bovine skeletal muscle satellite cells.Meanwhile,immunofluorescence technology was used to further detect the myotubes formation of bovine skeletal muscle satellite cells and to study the effect of UBA1 on myogenic differentiation of bovine skeletal muscle satellite cells.It is suggested that the effect of MSTN on myogenic differentiation of bovine is through ubiquitin activator UBA1 in ubiquitin proteasome pathway,which further regulates Myo D1 expression.The change of Myo D1 expression level further regulates myogenic differentiation of bovine skeletal muscle satellite cells.To sum up,this study analyzed the ubiquitination proteomics of MSTN-/-cattle.Bioinformatics analysis found that the ubiquitination differential protein was related to muscle development.In this paper,we used bovine skeletal muscle satellite cells to induce myogenic differentiation model in vitro,and found that interfering with MSTN expression can up regulate protein ubiquitination level,while ubiquitination can promote differentiation;at the same time,interfering with MSTN expression can up regulate the expression of UBA1,while interfering with UBA1 expression can down regulate the expression of Myo D1 and My HC,the changes of Myo D1 expression level further regulated the myogenic differentiation of bovine skeletal muscle satellite cells. |
