Analysis Of The Cell Biological Behavior During The Phase Of Serpine1 Promotes C2C12 Cell Myogenic Differentiation

Objective:The time-dependent expression of Serpine1 mRNA during myoblast-induced differentiation of C2C12 myoblasts was observed.Combined with the known functions of Serpine1,this hypothesis is proposed that Serpine1 plays an important role in the myogenic differentiation of C2C12 myoblasts and influences cell proliferation and migration.Methods:（1）Myogenic differentiation medium containing 2%horse serum effectively promotes the myogenic differentiation of C2C12 myoblasts:according to the experimental design,a part of C2C12 myoblasts was cultured in myogenic differentiation medium containing 2%horse serum respectively for 0d,1d,2d,3d,4d,5d,6d,7d（n=3）,collected cells at the corresponding time point,subjected to carry on cell flow cytometry experiments to detect the cell cycle,and confirmed that the myogenic differentiation medium containing 2%horse serum effectively blocked the cell cycle in the G₁/S phase;the other part of C2C12 myoblasts were cultured in myogenic differentiation medium containing 2%horse serum respectively for 0d,2d,4d,6d,8d,and 10d（n=3）,then the cells were collected at the corresponding time points and detected by RT-qPCR about the mRNA expression level of myogenic regulatory factors including Myf5,Myod,Myog and Myh3,confirmed the myogenic differentiation medium containing 2%horse serum effectively promotes the myogenic differentiation of C2C12 myoblasts.（2）Serpine1 and myogenic differentiation of C2C12 myoblast cells:Firstly,C2C12 myoblasts were cultured in the myogenic differentiation medium containing 2%horse serum for 0d,2d,4d,6d,8d,and 10d（n=3）,respectively,then in terms of the experimental design,the expression level of Serpine1 mRNA expression level was detected by RT-qPCR,and analyzed the time-dependent expression of Serpine1 mRNA during myogenic differentiation.Further,Negative control cells group（NC group）and Serpine1 knockdown cells group（Serpine1 knock down group）were obtained by si RNA interference;Control cells group（Ctrl group）and Serpine1 over-expression cells group（Serpine1 over-expression group）were obtained by lentivirus infection which contained Serpine1-assembled plasmids.In order to investigate the effect of Serpine1 on myogenic differentiation of C2C12 myoblasts.The above groups of cells were cultured in the myogenic differentiation medium for 0d,1d,2d,3d,4d,5d,6d,and 7d（8d）（n=3）,and cells at different time points were collected,RT-qPCR was explored to detect the mRNA expression levels of Serpine1 and myogenic regulatory factors including Myf5,Myod,Myog,and Myh3.（3）Serpine1 and C2C12 cell migration:Through wound healing assay,NC cells group,Serpine1 knock down cells group,Ctrl cells group and Serpine1 over-expression cells group were artificially wounded,and to explore the relationship between Serpine1and myogenic differentiation of C2C12 myoblasts,and confirm whether Serpine1 is responsible for the myogenic differentiation of C2C12 myoblasts through its effect on cell migration.（4）Serpine1 and C2C12 cell proliferation:C2C12 cells were cultured in growth medium for 12h,24h,36h,48h,60h and 72h（n=3）.Cells at corresponding time points were collected for RT-qPCR detection to obtain the time-dependent pattern of Serpine1mRNA expression.Then,NC cells group,Serpine1 knock down cells group,Ctrl group cells and Serpine1 over-expression cells group were cultured for 12h,24h,36h,48h,60h and 72h,respectively,CCK8 was detected at corresponding time points to explore the effect of Serpine1 on C2C12 cell proliferation and verify whether Serpine1 affects myogenic differentiation of C2C12 myoblasts by affecting cell proliferation.Results:（1）Myogenic differentiation medium containing 2%horse serum effectively promotes the myogenic differentiation of C2C12 myoblasts:flow cytometry was applied to detect the cell cycle,the results displayed that myogenic differentiation medium containing 2%horse serum effectively blocked the cell cycle in the G₁/S phase;RT-qPCR detected the mRNA expression levels of myogenic regulators including Myf5,Myod,Myog and Myh3,and the results showed that compared with myogenic differentiation 0 d,the mRNA expression of MRFs were increased with the progression of myogenic differentiation time,especially reached the highest level at the 6-8th day.（2）Serpine1 promotes C2C12 myoblasts myogenic differentiation:RT-qPCR results indicated that the mRNA expression level of Serpine1 decreased with the progression of C2C12 myoblast myogenic differentiation time.which speculated that Serpine1 played an important role in the myogenic differentiation of C2C12 myoblasts.The observation of knockdown or overexpression of Serpine1 showed that the mRNA expression level of the myogenic regulator factors in the Serpine1 knock down group was down-regulated,meanwhile,in the Serpine1 over-expression group was significantly upregulated.Suggestive that Serpine1 promotes myogenic differentiation of C2C12 myoblasts.（3）Serpine1 inhibits cell migration:The Wound healing experiment was used to observe the migration motility of C2C12 myoblasts.Through knocking down or overexpressing Serpine1,the migration motility of cells were observed,compared with the NC group during the experimental observation period,the results showed that the wound healing area of Serpine1 knock down group cell were larger,indicating that the cell migration rate was higher;and the wound healing area of the Serpine1 over-expression group cell showed smaller when compared with the Ctrl group cell during the experimental observation period,indicating that the cell migration rate was lower.Suggesting that Serpine1 may be involved in inhibiting cell migration.（4）Seemingly,Serpine1 has no effect on C2C12 myoblasts proliferation in vitro:RT-qPCR results showed that the mRNA expression level of Serpine1 is regularly down-regulated with the progress of proliferation time,suggesting that Serpine1 may be involved in regulating C2C12 myoblast proliferation.However,CCK8 detected the cell viability of cells in the NC group,Serpine1 knock down group,Ctrl group,and Serpine1 over-expression group,and the results showed that knockdown or overexpression Serpine1 did not affect the proliferation of C2C12 myoblasts.Conclusion:（1）Myogenic differentiation medium containing 2%horse serum effectively promotes the myogenic differentiation of C2C12 myoblasts.（2）Serpine1 promotes myogenic differentiation of C2C12 myoblasts.（3）Serpine1 inhibits cell migration.（4）Serpine1 may be not affect the proliferative ability of C2C12 myoblasts.In conclusion,Serpine1 regulates the myogenic differentiation of C2C12myoblasts,suggesting that Serpine1 can effectively improve skeletal muscle regeneration and repair,but does not rely on cell migration or proliferation to exert its effect.