Effects Of Guanylacetic Acid On Myogenic Differentiation And Muscle Growth Through The Regulation Of MiRNA

Recent studies have shown that guanidinoacetic acid?GAA?,as an amino acid derivative of endogenous animal tissues?including muscles and nerves?,can enhance muscle growth performance.MicroRNA?miRNA?is a very conservative non-coding protein RNA,which directly degrades target gene RNA or inhibits its translation.As a post-transcriptional regulator,miRNA plays a key role in nutrition-mediated myocyte formation.However,the effects of GAA on myogenic differentiation,skeletal muscle growth and the potential regulatory mechanism of miRNAs in this process have not been clarified.In this study,different concentrations of GAA were used to treat C2C12 cells,and the expression levels of related genes were measured to study the effects of GAA on the proliferation,differentiation and myotube growth of C2C12 cells.RNA-seq and qRT-PCR were used to identify the differentially expressed miRNAs between GAA treatment group and control group.MiR-133a-3p and miR-1a-3p were used as candidate regulatory factors of GAA affecting the function of C2C12 cells,and further functional verification experiments were carried out at the cellular level.Bioinformatics was used to predict the target genes of miRNA-133a-3p and miRNA-1a-3p,and dual-luciferase reporting experiments was used to verify the accuracy of the target genes.Different concentrations of GAA were used to feed mice to verify the accuracy of target genes in vivo.The purpose of this study is to explore the new mechanism of GAA in muscle development.The results are as follows:?1?The higher the dose of GAA,the stronger the differentiation ability of C2C12myoblasts.C2C12 cells could use GAA to increase creatine concentration.After GAA treatment,the proliferation rate of C2C12 cells decreased,the expression levels of CCND1and CDK4,related to promote cell proliferation,were down-regulated,and the expression level of P21,related to inhibit cell proliferation,was up-regulated.GAA treatment promoted the expression of MyoD and MyoG genes and increased the rate of myotubal fusion.After GAA feeding,the protein level of MyHC in gastrocnemius muscle and the cross-sectional area of gastrocnemius muscle were increased.?2?GAA regulated muscle formation through microRNAs.The primary reads?NC1,NC2,GAA1,GAA2?of 11.42M¹4.76M were obtained by small RNA sequencing.The analysis showed that 8 kinds of miRNAs?including miR-133a-3p and miR-1a-3p?differentially expressed after GAA treatment.The target gene function enrichment analysis of differentially expressed miRNAs showed that these genes were involved in cell differentiation(469 genes,P=3.64x10^-63),Wnt signaling pathway(31 genes,P=1.15x10^-4),PI3K-Akt signaling pathway(63 genes,P=9.45x10^-4),and AMPK signaling pathway?26 genes,P=0.012?.?3?GAA treatment reduced the expression of microRNA-133a-3p and microRNA-1a-3p,and promoted muscle development The diameter of myotube was increased about 40%and the expression of MyHC protein was increased in GAA-treated mice.GAA reduced the expression of miR-133a-3p and miR-1a-3p in gastrocnemius muscle of mice,and the MyHC protein level was also increased.Overexpression/inhibition of miR-133a-3p and miR-1a-3p resulted in a significant increase in the thickness of the myotubes transfected with miRNA inhibitor,and the co-transfection of miR-133a-3p and miR-1a-3p inhibitors had synergistic effects.qRT-PCR and Western blot analysis showed that the expression of MyHC protein was increased significantly after miRNA inhibitors transfected the myotubes?especially the co-transfection of miR-133a-3p and miR-1a-3p inhibitors?.?4?miR-133a-3p and miR-1a-3p affected muscle growth by regulating Akt/mTOR/S6K signaling pathway through targeting Insr and EIF4E.Bioinformatics analysis and doul-luciferase report experiment showed that Insr and EIF4E were target genes of miR-133a-3p and miR-1a-3p,respectively.After GAA treatment or transfection of microRNA inhibitors,the phosphorylation level of Akt/mTOR/S6K signaling pathway were increased significantly in cell level.In conclusion,GAA promoted myoblast differentiation and skeletal muscle growth through activation of Akt/mTOR/S6K signaling pathway induced by miR-133a-3p and miR-1a-3p.