| MicroRNAs(miRNAs)are a class of endogenous small non-coding RNAs composed of ~ 22 nucleotides.In recent years,miRNAs involved in various processes,including the animal development,disease and tumorigenesis.Development of skeletal muscle is a multistep process,including satellite cells activation and proliferation,myoblasts differentiation,multinucleate myotubes formation.The different developmental stages of skeletal muscle was regulated by many miRNAs,such as miR-1,miR-133,miR-181,mi R-206,miR-17-92,miR-34,miR-127,and miR-432 et al.Therefore,miRNA plays an important role in the skeletal muscle development.MiR-106a-5p is a member of mi R-106a~363 cluster,but its function on myogenisis is not yet clear.This study aims to determined the function and potential mechanism of miR-106a-5p in C2C12 myogenisis.In this study,we utilized the C2C12 mouse skeletal muscle cell line as a model system.Given that miR-106a-5p was dramatically down-regulated in myogenic differentiation,mi R-106a-5p agomir was used to analysis its function and potential mechanism on C2C12 myogenisis.we predicted the potential target genes of miR-106a-5p through bioinformatics analysis software,and verified by dual luciferase reporter assay.The main results are as follows:1.MiR-106a-5p Inhibits the AKT Signaling Pathway to Suppress C2C12 Cell DifferentiationOverexpression of miR-106a-5p inhibited C2C12 myoblast differentiation,differentiation index,the number of multinucleated myotubes was significantly lower(P < 0.05),In addition,myotubes had narrow and short size;Myogenic marker genes MyoD,MyoG and MyHC were significantly decreased(P < 0.05);fusion related gene Myomarker and Myomixer were significantly reduced(P < 0.05).In addition,AKT phosphorylation was inhibited(P < 0.05).IGF1,a activator of AKT pathway,promoted C2C12 myogenic differentiation(P < 0.05),and restored the inhibitory effect of miR-106a-5p on C2C12 myogenic differentiation(P < 0.05).2.MiR-106a-5p Promotes C2C12 Cell ProliferationAfter miR-106a-5p being transfected,the expression level of cyclin CyclinD1,CyclinE and CyclinB increased significantly(P < 0.05),proliferating cell nuclear antigen(PCNA)increased significantly(P < 0.05),the ability of DNA replication strengthed,and accelerated the G1 to S phase transition(P < 0.05)..3.MiR-106a-5p Promotes Atrophy of C2C12 MyotubesMiR-106a-5p was decreased in Dex induced myotube atrophy and aging model mouse(6-month old).MiR-106a-5p was transfected into fully differentiated C2C12 myotubes(about 5 days of differentiation).Overexpression miR-106a-5p induced myotube atrophy,reduced myotube diameter significantly(P < 0.05);atrophy related gene MAFbx was significantly elevated(P < 0.05),but MURF1 mRNA had no obvious change(P > 0.05),and both protein levels were significantly up-regulated(P < 0.05).In addition,miR-106a-5p blocked the AKT signaling pathway in myotube atrophy process.4.E2F3,SP1,and PIK3R1 are the targets of miR-106a-5p in C2C12 myogenesisOverexpression of miR-106a-5 no effects on the expressions of E2F3 and SP1 mRNA,but significantly decreased the E2F3 and SP-1 protein levels in differentiation(P < 0.05);In addition,mi R-106a-5p significantly down-regulated the expression of PI3K(p85α)protein during differentiation and post-differentiation(P < 0.05).The psi-Check2-E2F3,psi-Check2-SP1,and psi-Check2-PIK3R1 dual luciferase reporter vector were constructed and found that the luciferase activity of their wild type vector(P < 0.05).In summary,miR-106a-5p targeted PIK3R1 to block PI3K-AKT signaling pathway during C2C12 differentiation and post differentiation,inhibited myogenic differentiation,and induced myotube atrophy.In addition,miR-106a-5p could promote C2C12 proliferation,and E2F3 and SP-1 are targets gene of miR-106a-5p in myogenic differentiation. |
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