Effects Of Retinoic Acid On Proliferation Apoptosis And Myogenic Differentiation Of C2C12Cell Lines In Vitro

Background: All-trans retinoic acid (atRA) is the active metabolite of vitamin A,which regulates a wide range of biological processes including embryonic development,organ genesis, cell proliferation, differentiation, and apoptosis through RA receptors(RARs) and retinoid X receptors (RXRs). The mouse myoblastic cell line C2C12iswidely applied as the model of muscle regeneration attributed to its rapid differentiationin vitro and forming contractile myotubes and producing characteristic muscle proteins.Objective:To detect the changes of cell proliferation of C2C12treated with atRAin vitro; get the maximum concentration of atRA which has no influence on the cellproliferation of C2C12in vitro, and observe the changes of cell apoptosis and myogenicdifferentiation process of C2C12under this condition of atRA.Methods:1. The C2C12cells were exposed to different concentration of atRA,the cell proliferation and cell cycle distribution were determined by CCK-8and flowcytometry respectively and choose the maximum concentration of atRA which has noinfluence on the cell proliferation of C2C12in vitro.2. Cell apoptosis was detected byAnnexin V and PI through the flow cytometry.3. The changes of mRNA levels of theMRFs including Myf5, MyoD, Myogenin, and MRF4in process of myogenicdifferentiation were determined by Real-time RT-PCR and immunocytochemistry wasused to determine the changes of Myosin expression and to observe the morphologicalchanges of C2C12cells.Result:1. The results of the CCK-8shows that if the concentration of atRA invitro upto40μM, it will decrease the proliferation of C2C12, the change is significant(P<0.05). Flow cytometry indicates that when the concentration of atRA is less then1μM, it has no influence on the cell proliferation of C2C12in vitro, but if theconcentration of atRA is more than1μM, it will significantly increase the proportion ofC2C12cells in G0/G1stages (P<0.05). 2. Flow cytometry results show that when1μM atRA was added to the medium ofboth the RA groups and the Control groups, the level of cell apoptosis in RA groups isnot significantly changed contrast with the Control groups (P>0.05).3. mRNA expression changes of MRFs: when1μM atRA was added to themedium of the RA group, Myf5expression level are lower in RA groups than inControl groups, and the change is significant on the second day (P<0.05). MyoD mRNAexpression level in atRA groups is higher than the Control groups at half day, and thechange is significant (P<0.05), but it expressed similarly on the first day and thenascended on the second day, and not significantly (P>0.05). Myogenin expression levelsascended on the days detected in both A groups and Control groups, but RA groups arehigher, and the changes are significant on the second day (P<0.05). The mRNAexpression level of MRF4changed in atRA groups and Control groups, but the changesare not significant (P>0.05).4. Myosin expressed in cytoplasm of the multi-core muscle tubes. Multi-coremuscle tubes appear on the second day of differentiation in both RA groups and Controlgroups. On the sixth day more differentiated muscle cells fused into longer multi-coremuscle tubes, and RA groups are slenderer then Control groups. Myosin expressedgradually enhanced.Conclusions: The cell proliferation and the cell cycle of C2C12are influenced byatRA dose dependently in vitro,1μM atRA has no influence on the apoptosis of C2C12but it plays an evident role in the process of myogenic differentiation of the C2C12.