The Effect Of Muscle-specific MiRNAs On The Myoblast Differentiation And Muscular Dystrophy

Objective:To study the relationship of muscle-specific miRNAs with muscular dystrophy, the expression changes of muscle-specific miRNAs in the myoblast proliferation and differentiation, and in muscular dystrophy patients were tested. On the basis of cytological experiments and bioinformatics website, the effect of miRNA over-expression on myoblast differentiation and target gene was detected. It lay the foundation for speculating the effect of miRNAs on muscular dystrophy. Method:1. Mice myoblast C2C12was cultured and then the expression of muscle-specific miRNAs (-1,-133a,-206) in the myoblast proliferation and differentiation was detected by quantitative real-time PCR analysis.2. Firstly, muscular dystrophy cases was selected by clinical feature, hematoxylin-eosin staining, creatase histochemisty and specific staining, then detect the expression level of Muscle-specific miRNAs (-1、-133a、-206) in the muscular dystrophy patients tissue and muscle tissue nonspecific changes by quantitative real-time RT–PCR analysis.3. Through the above experimental, screen of the muscle-specific miRNA has more acts on myoblast differentiation and muscular dystrophy, bioinformatics software is applied to forecast the mRNA target.4. Transfect myoblast C2C12with miRNA mimic, MTT assay was employed for C2C12cells proliferation assay. Views of C2C12transfected with miRNA mimic under inverted microscope, at the same time detect the expression level of mRNA target by quantitative real-time RT–PCR and Western blot analysis.Results1. The expression level of muscle-specific miRNAs (-1、-133a、-206) inproliferation is lower to differentiation. As the extension of myoblast differentiation time, the expression level of muscle specific miRNA(miR-1、-133a、-206) increased（the value of P is0.0002、0.005、0.0001）,among which miR-1is the most highlight.2. Compared to muscle tissues nonspecific changes, the expression level of miR-1、-133a、-206is higher in8cases of muscular dystrophy patients,miR-206is significantly upregulated.(P=0.04).3. Pax7,Pax3,Cx43and Hmgb3were chosed as target genes of miR-206in the myoblast differentiation and the pathophysiological process of muscular dystrophy according to miRDB, miRNA.org,Target ScanHuman6.2and Pictar bioinformatics software forecast.4. The expression levels of Pax7,Pax3,Cx43and Hmgb3gene in C2C12cell transfected with miR-206mimic were significantly decreased by quantitative real-time RT–PCR. Western blot showed that the expression level of Pax7,Pax3,Cx43and Hmgb3decreased in C2C12cell transfected with miR-206mimic.5. MTT experiments showed that miR-206overexpression suppress C2C12cell proliferation. Compare to negative control,the ration of inhibition is（21.35±0.0068）%(P=0.004)ConclusionsThe expression level of miR-1, miR-133a and miR-206increases in myoblast differentiation and muscular dystrophy patients. MiR–206might be more important to the pathophysiological process of muscular dystrophy and differentiation of myoblast by targeting several genes.