The Effects And Molecular Mechanism Of MiR-374b And MiR-299 On Myoblast Differentiation

Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation. This process is controlled by a set of cell signals and transcription factors. microRNA is a kind of ~22nt-long non-coding small RNAs, which abundantly exist in plants and anaminals and conserved in species. These small RNAs act as key transcriptional or post-transcriptional regulators of gene expression. In recent years, more papers showed that microRNAs involve in the regulationof myogenesis. Myf6 is one member of the MRFs(Myogenic Regulatory Factors) family, which orchestrate the expression of many muscle specific genes though recognize and bind to the promotor region. In this paper, we identified microRNAs that directly target Myf6 3’UTR and studied the effect of miR-374 b and mi R-299 on myoblasts differentiation. The main works and results wereas follows: 1. Identification of microRNAs regulatingMyf6geneTo identify microRNAs regulating Myf6, online prediction software miRanda was used to predict miR-374 b, miR-376 c, miR-377, miR-499 and miR-669 f as candidates.We constructedwild type Dual luciferase report vector,which contains the predicted target sites of cadidate microRNAs,and mutational type Dual luciferase report vectors, which have 8bp mutation in the predicted target sites of miR-299 or miR-374 b. Through luciferase reportexperiments, we assured that miR-374 bdirectly target Myf6 gene.We also found that miR-374 b negatively regulates endogenousMyf6 at both mRNA and protein level. 2. The function of miR-374 b during myogenesisQ-PCR was performedto analyze the miR-374 b tissue expression profile and the expression pattern during the differentiation of C2C12 myoblasts. miR-374 bwasuniversally expressed in nine tissues(heart, liver, spleen, lung, kidney, stomach, small intestine, fat and muscle) of 2-month old C57BL6 mice. miR-374 babundanceincreasesfirst and then decrease during myogenesis of C2C12 cells(at0h, 24 h, 72 h and 96 h of differentiation).We studied the effect of miR-374 b on C2C12 cells myogenesis by function gain and loss methods. When over-expression mi R-374 b, the differentiation marker geneMyoG mRNA level was significiently elevate, while MyoD andMyf5 mRNA levels were not changed,but we observed myosin heavychain(MyHC) protein level decreaseby western blot and myotube fusion rate decrease by immunofluorescence(IF). On the contrary, when we introduced miR-374 b inhibitor into C2C12 cells,both MyHC protein level and myotube fusion rate increase. As a result, miR-374 b has the negative effect on C2C12 myogenesis. Moreover, cell proliferation assay showed that miR-374 b promotes C2C12 cell proliferation.We also found that miR-374 b expression level is higher in gastrocnemius(mixture fiber) than that in soleus(slow fiber). After over-expression miR-374 b, both Myh7(slow fiber) and Myh4(fast fiber) expression level decreased, withlarger decreasing amountof Myh7gene; When we inhibit endogenous mi R-374 b level, the opposite result was obtained.As a result, mi R-374 b inhibits slow myofiber formation more effectively than fastmyofiber. 3. The function of miR-299 during myogenesisQ-PCR was taken out to analyze miR-299 tissue expression profile and the tendency of miR-299 level during differentiation of C2C12 myoblast. miR-299 was universally expressed in nine tissues(heart, liver, spleen, lung, kidney, stomach, small intestine, fat and muscle) of 2-month old C57BL6 mice. miR-299 expression abundance increases during myogenesis of C2C12 cells(0h, 24 h, 72 h and 96 h of differentiation).Though over-expression and inhibit experiment, we studied the effect of miR-299 on C2C12 cells myogenesis. When over-expressionmi R-299 by mimics, the differentiation marker genes MyoG,MyoD and Myf5 mRNAlevel increase while MyoG,MyoD and Myf5 mRNA level decrease when we introduced miR-299 inhibitor to C2C12 cells. As a result, miR-299 has a positive effect on C2C12 myogenesis.In this study, we for the first time identified miR-374 b and miR-299 as regulators of myoblasts differentiation. These results added the knowledge about the microRNA regulation network of muscle development and laid thetheory basis for agricultural industry and clinic treatment of muscle disease.