The Regulation Function Of MiR-1/206 Tageting Pax-7 During The Process Of Retinoic Acid Suppressing C2C12 Cells' Differentiation

Backgrounds:MicroRNAs(miRNAs)are endogenous small RNAs,forming complex regulatory networks with their target genes,adjusting the level of translation,affecting the gene expression of transcription factors,signal molecules.Muscle associated micro RNAs(MyomiRs)are mi RNAs which specifically expressing in muscle tissue,it has been considered that MyomiRs play a significant role in the process of muscle development.Muscle tissue development determines the number of muscle fibers,size and fiber type,the process above involves the interaction and mutual cooperation between multiple genes,and that is been considered to be an extremely complex multi-stage process.The process of differentiation is very important to the decision of the muscle cells' fate and to the final formation of the muscle tissue,among the variety genes regulating the process above,MRF and MEF2 family are the most important transcription factors,two important members Myo D and Myf5 are thought to have the function of deciding during the early forming of myoblast,and ensuring the cells into the myogenic process.Among the variety regulatory factors of Myo D/Myf5,Pax7,one important member of the family of paired box(Pax),can directly target on Myo D/Myf5,maintain muscle cell proliferation and inhibiting its early differentiation,thus to be considered to have negative control effect on skeletal muscle development.It has been reported there surely exist direct targeting relation between two main members of MyomiRs,mi R-1,mi R-206 and Pax7 factor,Myo D/Myf5 factor,and subsequently form a complete feedback loop,hence regulate the process of muscle progenitor cells changing from proliferation to differentiation.A slice influence on the expression level or control function of one member in the targeting relationship would cause abnormal cell differentiation,leading to the occurrence of dysplasia,its specific mechanism is considered to be related to the environmental factors and genetic factors.Retinoic acid(Retinoic acid,RA),a intermediate metabolite of vitamin A,has been widely used in a variety of skin diseases and tumor therapies,hence it is has become one of the common environmental factors of palatal dysplasia.Studies have shown that RA induced mouse embryos cleft palate model often accompany with a tongue muscle dysplasia,to be specific,tongue muscle fiber line and expression of factors regulating the myogenic process(Myogenic Regulatory Factors,MRFs)are abnormal.We speculated that the in this process,the targeted relationship between mi R-1/206 and Pax7,Myo D/Myf5 may be a key regulatory mechanism.Preliminary results ofourgroup confirmed that inthe RA induce cleft palate with tongue tissue dysplasiamice model,the correlation between mi R-1/206,Pax7 and Myo D/Myf5 still exists,butin the case of RA inducing abnormal myogenic differentiation,the deep mechanism of the regulation function of this correlation is still not clear.Aim: By detecting the relative expression level of mi R-1/206,Pax7 and MRFs of C2C12 cells during the process ofinduced myogenic differentiation in vitro,to researchthe effect of RA on MyomiRs and on the myogenic differentiation;By detecting the relative expression level of Pax7 and MRFstoresearchthe function of over-expressed mi R-1/206 in the case of RA influenced C2C12 cells myogenic differentiation,giving possible evidence of the mechanismof the important regulating function of MyomiRs in the process of abnormal differentiation.Methods: 1.A certain amount of RA was added duringthe myogenic differentiation of C2C12,the expression levels of MyomiRs,MRFs and Pax7 were detected by real-time quantitative PCR 2.By using liposome transfection technique,over-expressed the mi R-1/206 of C2C12 cells,detectedthe morphological changes in the case of RA influenced C2C12 cells myogenic differentiation.The expression levels of MyomiRs,MRFs and Pax7 were detected after over-expressing mi R-1/206.Results:1.During the myogenic differentiation of C2C12 cells,the density volume,and the forming populations of multi-core myotubes of cells increased,cell morphology elongated;with the presence of RA,the arrangement of cells were disordered,the cell volume and the forming populations of multi-core myotubes formation decreased,the process of myogenic differentiationwas restrained.With the presence of RA,during the process of myogenic differentiation of C2C12 cells,the relative expression of mi R-1/206 was less than which in the normal group(p < 0.01).With the presence of RA during the process of myogenic differentiation of C2C12 cells,the relative expression of MRFs was less than which in the normal group(p < 0.01);the relative expression of Pax7 on day 1,day 2 were significantly higher than normal group(p < 0.01).2.After over-expressing mi R-1/206,compared with empty plasmid transfection group,the length of cell,forming populations of multi-core myotubes,andcell size increased;With the presence of RA,after over-expressing,forming populations of multi-core myotubes also increased.After over-expressing mi R-1,on the second day of differentiation,the relative expression of Myf5 of C2C12 cell line was significantly higher than that in NC group(p<0.01);the relative expression of Pax7 was significantly lower than that in NC group(p<0.01);With the presence of RA,the relative expression of Myf5 was still significantly higher than that in NC group(p<0.01).After over-expressing mi R-206,on the second day of differentiation,the relative expression of Myo D was significantly higher than that in NC group(p<0.01);the relative expression of Myf5 was significantly higher than that in NC group(p<0.05);the relative expression of Pax7 was significantly lower than that in NC group(p<0.01);With the presence of RA,the relative expression of Myf5 was still significantly higher than that in NC group(p<0.01);the relative expression of Pax7 was significantly lower than that in NC group(p<0.01).Conclusions:Excessive RA had a significant role in suppressing the progress of differentiation of C2C12,and during this process,the relative expression of MyomiRs,namely mi R-1/206,were detected to be reduced,consequently the relative expression of Pax7 was affected,and therefore the expression of Myo D/Myf5 was down-regulated.The targeting above was the possible mechanism of RA suppressing C2C12 myogenic differentiating.Moreover,over-expressing MyomiRs can partly rescue the differtiation delaying of C2C12 caused by RA,and that was considered to be achieved through the targeting relation stated above.