Down-regulation Of Wnt5a Gene Affects Proliferation And Differentiation Of C2C12Myogeneic Cells

Background:Wnt family are signaling molecules regulating different developmental processes, such as proliferation, asymmetric division, patterning and cell fate determination which play an important role in muscle development process.Wnt5a is an important member of the Wnt signaling family, but it is still lack of the study of regulation of myogenic cell development. C2C12myogenic cell line is a immortalized cell line from C3H mice skeletal muscle satellite cell. It often be used as the object of study of the skeletal muscle cell development.Objective: To investigate the influence of Wnt5a on proliferation, cell cycle anddifferentiation after down-regulation of Wnt5a gene on C2C12myogeneic cells.Methods:1. Three siRNAs (siRNA-1, siRNA-2, siRNA-3) and unrelatedsequence (Negative control, NC) were designed according to the gene sequence ofmouse Wnt5a in Gene Bank. C2C12cells were transfected using Lipofectamine2000,and then the most effective siRNA sequences used for subsequent experiments wasselected according to the results of SYBR Green Real-time RT-PCR.2. C2C12cellswere transfected using Lipofectamine2000, and the cells were divided intoexperimental group (siRNA group) and the negative control group (NC group). The cellproliferation was evaluated by CCK-8, and the cell cycle was evaluated by flowcytometry.3. C2C12cells become muscle-induced differentiation after switching todifferentiation medium containing2%horse serum. The expression levels of myogenicregulatory factor family members (Myf5, myogenin and MRF4) and Wnt5a after celldifferentiation were evaluated by using SYBR Green Real-time RT-PCR. Theexpression level of Myosin in C2C12cells differentiation process and themorphological changes of cells were evaluated by Immunocytochemistry.Results:1. Compared with NC group and other siRNA groups, the expression of Wnt5a in C2C12cells has been greatly decreased (96.78%) by using siRN A-1.2. After the expression of Wnt5a has been decreased, the results of CCK-8showed that no significant change of proliferation was detected in C2C12cells,so it has no significant difference (P>0.05). The results detected by flow cytometry showed that the cells arrested in each phase were not significantly increasedafter the expression of Wnt5a has been decreased in C2C12cells, so it has nosignificant difference (P>0.05).3. After the expression of Wnt5a has been decreased, in the differentiation day0of C2C12cells, the expression level of Wnt5adecreased significantly, so it has a significant difference (P<0.05). In the differentiation day2of C2C12cells, the expression level of Myf5and MRF4decreasedsignificantly, so it has a significant difference (P<0.05). Immunocytochemical method shows that Myosin express in the multinucleated myotube cells, and the cytoplasm stain strongly positive. Compare with siRNA group and NC group, thereis no significant difference in cell morphology.Conclusions: Down-regulation of Wnt5a gene in vitro has no significant effect onthe proliferation and cell cycle of C2C12cells. While muscle-induced differentiation ofC2C12cell lines, the expression level of Myf5and MRF4decreased significantly,suggesting that Wnt5a may regulate the differentiation of skeletal musle cells.