Effect And Mechanism Of MYOC In Regulating Differentiation Of Mouse C2C12 Myoblasts

Skeletal muscle is one of the most important tissues in animals.The research on the regulation of skeletal muscle growth,development and differentiation has always been the focus of attention.The mechanism of skeletal muscle differentiation has been widely reported,which involves various signaling pathways and the regulation of signaling molecules.Our work focuses on the mechanism of skeletal muscle differentiation,mainly on the regulation of muscle by glycoproteins in extracellular matrix.Previous studies have shown that secretory glycoprotein MYOC expression is increased during the differentiation of mouse C2C12 myoblast cells.In the past,MYOC has been reported as a regulator of mesenchymal stem cells(MSC s)osteoblast differentiation,but the regulation of MYOC on mouse C2C12 myoblast differentiation has not been reported yet.Therefore,we believe it is necessary to study the role and mechanism of MYOC in regulating mouse C2C12 myoblast differentiation.In this study,we chose mouse C2C12 myoblast cell as the research object.The main research contents were as follows: The expression of MYOC was detected by western blotting and immunofluorescence during the differentiation of C2C12 cells,and laser confoca l microscopy was used to locate MYOC.The overexpression vector of MYOC was constructed,and the effects on the differentiation of C2C12 cells were detected by overexpressing or inhibiting the expression of MYOC gene by transfecting the overexpression vector of MYOC into C2C12 cells and interfering fragments of siRNA.The proteins that may bind to MYOC in the differentiation process of C2C12 cells were analyzed by immunoprecipitation combined with ESI mass spectrometry sequencing.The membrane protein CAV1,which is closely related to the differentiation of C2C12 cells,was screened out for further study and the binding relationship between the two was verified.The changes of CAV1 expression in different stages of C2C12 cell differentiation were detected by the same method and the localization of CAV1 during the differentiation of C2C12 cells was carried out.C2C12 cells were transfected with siRNA interference fragment designed for CAV1 to inhibit the expression of CAV1,and the effect on C2C12 cell differen tiation was observed.The expression of MYOC was overexpressed or inhibited by transfection of MYOC overexpression vector and siRNA interference fragments to detect the influence of TGF-? pathway.The regulation effect of CAV1 on TGF-? pathway was verified by inhibiting the expression of CAV1 by transfection of siRNA interference fragments.Co-immunoprecipitation method was adopted to simultaneously transfect MYOC overexpression vector and siRNA interference fragment designed for CAV1,to interfere with the expression of CAV1 while overexpressing MYOC,and to detect whether the effects of MYOC on TGF-? pathway and differentiation of C2C12 cells were changed.(1)Western blotting results showed that the expression level of MYOC gradually increased with the differentiation of C2C12 cells.The expression level was the lowest at the 0th day of differentiation,reached the plateau stage at the 3rd day of differentiation,and gradually stabilized,and reached the highest expression level at the 7th day.Immunofluorescence results also showed that the fluorescence staining intensity of MYOC gradually increased during the differentiation of myotubule,suggesting that MYOC was involved in regulating the differentiation of C2C12 cells.Meanwhile,confocal laser microscopy showed that MYOC was mainly localized on the cell membrane in differentiated C2C12 cells.(2)When MYOC gene was overexpressed or inhibited,Western blotting showed that the expression levels of differentiation marker genes MYH2 and MYOG were increased or decreased accordingly.Immunofluorescence results showed that when MYOC was overexpressed,the fluorescence intensity of differentiation marker Desmin increased,and the myotube fusion rate of C2C12 cells increased from 33.24% to 58.28%.When MYOC expression was inhibited,the fluorescence intensity of Desmin decreased.The myotube fusion rate decreased from 36.29% to21.15%,indicating that MYOC could promote the differentiation of C2C12 cells.(3)CAV1 was selected as the object of subsequent studies by immunoprecipitation combined with ESI mass spectrometry,and the results of Co-immunoprecipitation showed that MYOC and CAV1 could bind.The results of laser confocal co-localization showed that both of them shared a common expression position in differentiated C2C12 cells,suggesting that they could bind during the differentiation of mouse C2C12 cells.(4)Western blotting and immunofluorescence results showed that the expression of CAV1 increased with the increase of differentiation degree of C2C12 cells,and the expression level was the lowest on the 0th day and the highest on the 7th day.The correlation between CAV1 and differentiation of C2C12 cells was verified.At the same time,CAV1 was main ly expressed in the cell membrane and cytoplasm of differentiated C2C12 cells under laser confocal microscope.(5)When the expression of CAV1 was inhibited,the differentiation of C2C12 cells was disturbed,the expressions of MYH2 and MYOG were decreased simultaneously,the fluorescence intensity of Desmin was decreased,and the fusion rate of myoduct was decreased by 21.48%compared with the control group,indicating that CAV1 could positively regulate the differentiation of C2C12 cells.(6)When CAV1 was inhibited,the active form of Smad2,the intracytoplasmic marker of TGF-? pathway,in mouse C2C12 myoblast cells was decreased simultaneously,and the active form of p-Smad4,the nuclear marker of TGF-? pathway,was also decreased.The positive regulation effect of CAV1 on TGF-? pathway was verified.(7)Western blotting results showed that when MYOC was overexpressed or inhibited,p-Smad2 and p-Smad4 in mouse C2C12 myoblast cells were simultaneously increased or decreased,indicating that MYOC could also positively regulate TGF-? pathway.(8)The co-transfection results showed that,compared with the overexpression of MYOC gene alone,CAV1 was inhibited under the condition of overexpression of MYOC.Through Western blotting and immunofluorescence results,we could clearly observe that the ability of MYOC to activate TGF-? pathway was blocked.The levels that promote differentiation of C2C12 cells were also disturbed.These results indicate that MYOC promotes C2C12 cell differentiation by regulating TGF-?-mediated signaling pathway through CAV1.In this study,we demonstrated that MYOC can positively activate the TGF-? pathway through CAV1 and thus promote C2C12 cell differentiation.The results not only provide a new idea for the regulation of myoblast differentiation,but also lay a foundation for the research on the mechanism of drug development and the treatment of muscle-related diseases.