Effect Of ECM2 Expression On Bovine Muscle-derived Satellite Cell Differentiation

The growth and development of muscles are affected by both genetic and environmental factors.The extracellular matrix not only provides support for the cells,but also plays an important role in regulating cell proliferation,migration,and differentiation.Recent studies have found that the secreted protein acidic and rich in cysteine(SPARC)family in the extracellular matrix(ECM)has a strong role in regulating muscle differentiation.And SPARC has a strong expression in the developed tissue and cells in proliferation and differentiation.ECM2(the extracelluar matrix 2)is a member of the SPARC family.The results of the high-throughput sequencing in our laboratory showed that the expression of ECM2 on the third day of MDSCs differentiation was significantly higher than that on the first day.Therefore,this experiment used bovine MDSCs isolated from fetus as experimental materials to investigate the effects of ECM2 gene on the differentiation of bovine MDSCs.In this research,the expression of ECM2 gene during the differentiation process of bovine MDSCs was studied.Activation or inhibition the expression of ECM2 by using CRISPR-d Case9 system have the effects on the differentiation of bovine MDSCs,and then we explored the role of ECM2 in regulating the PI3 K signaling pathway.The results are as follows:1.To investigate the expression pattern of ECM2 during the differentiation of bovine MDSCs, the total proteins and the culture were collected at 0,1,3,5,7 and 9 days of differentiation.Western blot and immunofluorescence techniques were used to detect the expression of ECM2 protein in bovine MDSCs during differentiation.The results showed that,the expression of ECM2 in cells gradually increased with the differentiation of bovine MDSCs,and the expression of the ECM2 tended to be stable after 5 days of differentiation.At the same time,the content of ECM2 in the culture was also detected.The content of ECM2 in the culture also gradually increased and reached its peak at 3-5 days of differentiation.2.Using CRISPR-d Cas9 system,three activation vectors and three inhibition vectors of ECM2 were constructed respectively.They were transfected into bovine MDSCs and then differentiated for 48 hours.The proteins in the cells were collected and detected by Western blot.The best activation and inhibition vectors were selected.The results showed that p SPg RNA-N1 was the best activation vector,and p SPg RNA-N2 was the best inhibition vector.3.The effect of activating ECM2 gene on the differentiation of bovine MDSCs was explored.The p SPg RNA-N1 vector that activate ECM2 gene were transfected into bovine MDSCs and then MDSCs were induced to differentiate.Immunofluorescence staining technique was used to observe the changes of myotube morphology and the rate of myotube fusion.The results showed that the shape of myotubes became thicker and longer,and the myotube fusion rate increased significantly.Western blot was used to detect the expression of muscle differentiation-related genes MYH3 and MYOG.As a result,it was found that the expression levels of MYH3 and MYOG in the cells were significantly increased after activation of the ECM2 gene.It shows that activation of ECM2 gene can promote the differentiation of bovine MDSCs.4.To investigate the effect of inhibiting the expression of ECM2 gene on the differentiation of bovine MDSCs,the p SPg RNA-N2 vector that can inhibite the expression of ECM2 gene were transfected into bovine MDSCs and MDSCs were induced to differentiate.Immunofluorescence staining techniques were used to observe the changes in myotube morphology and the rate of myotube fusion.The results showed that the shape of the myotubes was shorter and the myotube fusion rate was significantly decreased.Western blot was used to detect the expression of muscle differentiation-related genes MYH3 and MYOG.As a result,it was found that the expression levels of MYH3 and MYOG in the cells were significantly reduced after inhibiting the expression of ECM2.It shows that inhibiting the expression of ECM2 will inhibit the differentiation of bovine MDSCs.5.To explore the molecular mechanism of ECM2 action,firstly,the relationship between ECM2 and PI3 K signal pathway was studied.After activation and inhibition the expression of ECM2 by CRISPR-d Cas9 system,the protein level and phosphorylation level of PI3 K or Akt were observed.The results showed that phosphorylation levels of PI3 K and Akt decreased after activation of the ECM2 gene,and phosphorylation levels of PI3 K and Akt increased after inhibiting the expression of ECM2.It shows that ECM2 gene can inhibit PI3 K signaling pathway.Next,to explore the effect of PI3 K signaling pathway on the differentiation of bovine MDSCs,PI3 K signaling pathway was inhibited by LY294002(PI3K signaling pathway inhibitor),and detected the expression levels of bovine MDSCs differentiation markers MYH3 and MYOG.The results showed that the expression levels of MYH3 and MYOG both increased.The above results indicate that ECM2 gene promotes the differentiation of bovine MDSCs by inhibiting the PI3 K signaling pathway.This experiment confirmed the effects of ECM2 gene on the differentiation of bovine MDSCs for the first time,it demonstrated that ECM2 gene can promote the differentiation of bovine MDSCs,further more,it pointed out that ECM2 gene can promote the differentiation of bovine MDSCs by inhibiting PI3 K signaling pathway.It provides a theoretical basis for genetic breeding of livestock cattle.