Dexamethasone Enhances The Induction Of Muscle Atrophy Through MiR-322

Dexamethasone(Dex),as an effective synthetic glucocorticoid,is commonly used as a therapeutic agent due to its potent antiinflammatory,antishock,and immunosuppressive functions.However,Dex also causes passive effects,such as skeletal muscle atrophy.The treatment of muscle atrophy is still a problem to be solved urgently,and new and effective treatment methods are needed.As a hot topic in recent years,the research on microRNAs(miRNAs)is likely to provide a new solution for the treatment of Dex-induced muscle atrophy.miRNAs are small endogenous noncoding RNA molecules that negatively regulate gene expression by targeting sequences in the 3?-untranslated region(3?-UTR)of mRNAs.In previous reports,miRNA microarray was used to investigate the differential expressions of miRNAs in C2C12 myotubes treated by Dex or PBS.Experimental results showed that miR-322 expression in Dex-treated C2C12 myotubes was increased significantly.In this study,C2C12 myotubes were differentiated from C2C12 myoblasts and then were treated with Dex.Immunofluorescent staining for myosin heavy chain(MyHC)was used to show the shape and diameter of C2C12 myotubes.The results showed that Dex reduced the diameter of the C2C12 myotubes(p<0.01).Dex elevated the expression of Atrogin-1 and MuRF-1,the marker genes of muscle atrophy,at 24 h after Dex treatment(p<0.05).This suggested that Dex-induced atrophy in C2C12 myotubes.The expression of miR-322 was investigated in Dex-induced C2C12 myotubes.The results showed that Dex increased the expression of miR-322(p<0.05).To investigate the role of miR-322 in C2C12 myotube atrophy,the expression of miR-322 in the C2C12 myotube was increased or decreased.Our results showed that miR-322 overexpression reduced the Dex-induced myotubes diameter,accompanied with increased expressions of Atrogin-1 and MuRF-1.However,miR-322 inhibitor showed the opposite effects in Dex-treated C2C12 myotubes.Thus,miR-322 aggravated Dex-induced atrophy in C2C12 myotubes.To investigate the mechanism of miR-322 promoting Dex-induced muscle atrophy,IGF1 R and INSR of mice were predicted as putative target genes of miR-322 using the bioinformatic tool TargetScan.We then cloned the 3?-UTRs of IGF1 R and INSR,which included the binding sites of miR-322,into separate plasmids.Luciferase assays showed that miR-322 reduced the luciferase activity(p<0.05).Transfecting with miR-322 mimic into C2C12 myotubes resulted in decreased protein levels of IGF1R(p<0.01)and INSR(p<0.01).Conversely,transfecting with miR-322 inhibitor resulted in increased expressions of IGF1R(p<0.01)and INSR(p<0.05).To further assess whether IGF1 R and INSR mediate atrophy in Dex-induced C2C12 myotubes.The results showed that either knock-down of IGF1 R or INSR induced atrophy in C2C12 myotubes(p<0.05).These results indicated that IGF1 R and INSR played key roles in miR-322 contributing to the atrophy in Dex-induced C2C12 myotubes.The role of miR-322 in Dex-induced muscle atrophy has also been demonstrated in vivo.Gastrocnemius weight/body weight(GW/BW)and grip strength were decreased by miR-322 agomir and increased by miR-322 antagomir(p<0.05).Hematoxylin-eosin staining(HE staining)showed that muscle fiber diameter decreased by miR-322 agomir.However,miR-322 antagomir showed the opposite effect to miR-322 agomir in the gastrocnemius muscles of mice(p<0.05).Meanwhile,the expressions of atrogenes Atrogin-1 and MuRF-1 were increased by miR-322 agomir and inhibited by miR-322 antagomir(p<0.01).These data indicated that miR-322 contributed to Dex-induced muscle atrophy,whereas miR-322 inhibition attenuated Dex-induced muscle atrophy in vivo and in vitro.This provided a theoretical basis for the treatment of muscle atrophy and the safe use of Dex.