Mechanism Of SPARCL1 Regulating C2C12 Cell Differentiation

The differentiation of myoblasts in animals plays a very important role in t he development of skeletal muscles.The differentiation of myoblasts is a very complicated process;which regulated by a variety of cell signaling molecules and pathways.It is known that extracellular matrix(ECM)can participate in the regulation of cell morphology,cell movement,cell differentiation and other biological processes to play important roles in cell life activities.As an extracellular matrix protein,SPARCL1 is known to be involved in the differentiation of mouse myoblast C2C12.However,the specific mechanism of SPARCL1 regulating the differentiation of C2C12 cells is unclear.Therefore,the biological mechanism of SPARCL1 impacted on C2C12 cell differentiation was demonsteated in this study.The results showed that SPARCL1 bound with BMP7 to regulate C2C12 cell differentiation through BMP /TGF-? signaling pathway.Research method,content and conclusion:(1)CRISPR/Cas9 technology and si RNA interference technology were used to activate and inhibit the expression of SPARCL1 gene respectively.Western blotting results showed that the activation or inhibition of SPARCL1 can up-regulate or down-regulate the differentiation marker molecules Desmin and myogenin,Myo G)expression level;immunofluorescence results showed that activation or inhibition of SPARCL1 also up-regulated or down-regulated the myotube fusion rate of cells.The above results indicated that SPARCL1 can affect the differentiation of C2C12 cells.(2)In this study,bupivacaine hydrochloride injection experiment was used to establish an animal model for repairing muscle injury in mice.HE staining results showed that the muscle damage repair model induced by bupivacaine hydrochloride was successfully established.The results of SPARCL1 immunofluorescence showed that the expression leve l of SPARCL1 was the highest when the injury was the most severe(D3),and the expression level of SPARCL1 decreased when the injury repair was completed(D14);Western blotting results showed that the protein expression level of SPARCL1 had similar results,that is,the expression level was higher at(D3),but was down-regulated to a lower level at(D14),suggesting that SPARCL1 may be involved in the repair process of muscle damage in mice.(3)Co-Immunoprecipitation(Co-IP)test results showed that SPARCL1 and BMP7 combined with each other during the differentiation of C2C12 cells.(4)Western blotting and immunofluorescence were used to detect the expression of BMP7 during the differentiation of C2C12 cells.It was found that the expression of BMP7 prote in showed a gradual increase trend;BMP7 immunofluorescence results showed that the expression level of BMP7 in differentiated myotubes also gradually up-regulated with the increase in cell differentiation days.This indicates that BMP7 may be involved in the differentiation process of C2C12 cells.(5)CRISPR/Cas9 and si RNA technology were used to activate and inhibit the expression of BMP7 gene respectively.Western blotting showed that the activation or inhibition of BMP7 can affect the expression level of Myo G and Desmin;the results of immunofluorescence showed that the rate of myotube fusion increased after activation of BMP7.Myotube fusion rate decreased after inhibiting BMP7,indicating that BMP7 can affect the differentiation of C2C12 cells.(6)CRISPR/Cas9 technology and si RNA interference technology were used to activate and inhibit the expression of SPARCL1 gene respectively.Western blotting was used to detect the effect of SPARCL1 on BMP7 gene and BMP / TGF-? signaling pathway.It was found that the activation or inhibition of SPARCL1 could up-regulate or down-regulate the expression level of BMP7,and the expression level of P-SMAD4,an active molecule of BMP / TGF-? pathway,also increased or decreased.Furthermore,the expression levels of cell differentiation molecules Myo G and Desmin were also up-regulated or down-regulated by SPARCL1 activation or inhibition,indicating that SPARCL1 may affect the differentiation of C2C12 cells by regulating the activity of BMP7 and BMP / TGF-? signaling pathway.(7)In order to clarify whether SPARCL1 regulates the BMP / TGF-? signaling pathway through BMP7 and affects the differentiation of C2C12 cells,SPARCL1 was activated and BMP7 was inhibited at the same time.Western blotting results sh owed that due to the inhibition of BMP7,the activation of SPARCL1 did not increase the activity of the BMP / TGF-? pathway,nor did it increase the expression levels of Myo G and Desmin;immunofluorescence results showed that the activation of SPARCL1 did not increase the myotube fusion rate due to the inhibition of BMP7.This indicates that SPARCL1 can affect the differentiation of C2C12 cells through BMP7-mediated BMP / TGF-? signaling pathway.In summary,SPARCL1 can affect the differentiation of C2C12 c ells through BMP7-mediated BMP / TGF-? signaling pathway.This discovery will be helpful to deeply understand the molecular mechanism of SPARCL1 in regulating muscle development and provide new research ideas for the treatment of muscle injury diseases.