Mechanism Of MiR-125b-5p Targeting TRAF6 Relieves Denervation-induced Muscle Atrophy

Background: Skeletal muscle atrophy is a debilitating consequence of peripheral nerve damage.The loss of muscle mass upon denervation is introduced by the fact that the balance of protein anabolism and catabolism is broken.Excessive proteolysis pathway during skeletal muscle atrophy involves activation of the ubiquitin-proteasome system(UPS)and the autophagy-lysosomal system(ALS).Tumor necrosis factor(α)receptor adaptor protein 6(TRAF6),a key E3 ubiquitin ligase,regulates skeletal muscle differentiation,development,atrophy,and regeneration by activating different downstream signals or factors in various skeletal muscle models.In recent years,emerging evidence suggests that micro RNAs(miRNAs)with involvement in several signaling pathways induced by complicated factors are critical for skeletal muscle physiology and pathology.In early research,we found that that miR-125b-5p may have a potential regulatory effect of TRAF6.Therefore,this study systematically investigated the effects of TRAF6 on UPS and ALS during Hank’s solution-induced C2C12 myotubes atrophy and denervation-induced muscle atrophy,and revealed the specific mechanism by TRAF6 was regulating protein degradation pathways.Furthermore,we elucidate the protective effect and mechanism of miR-125b-5p by targeting TRAF6 in denervated skeletal muscle,adding new content of the molecular regulation mechanism,which to provide the experimental basis for the prevention and treatment of denervation-induced muscle atrophy.PartⅠ TRAF6 relieves protein degradation in denervated muscle atrophyObjectiveThe purpose of this study was to elucidate the important target molecules and the specific pathways regulated by TRAF6 involved in the process of denervated muscle atrophy.Exploring new content for the molecular regulation mechanism of denervated muscle atrophy.Methods1.The C2C12 myotubes atrophy model were established by treating with Hank’s solution.The myotubes were labeled by Myosin heavy chain(MHC)immunofluorescence staining.The expression of TRAF6 of myotube atrophy was detected by the Western Blot,as well as the related proteins expression of UPS and ALS.2.The animal models of rat sciatic nerve disconnection were prepared;The weight ratio of the tibialis anterior(TA)muscle was analyzed at 3 days,7 days,14 days and 28 days after denervation.The cross-sectional area(CSA)of the TA muscle fiber was accessed by immunofluorescence staining of Laminin.Observing and analyzing the changes of the TA longitudinal section ultrastructure.The RT-q PCR and Western Blot were used to detect the TRAF6 expression at different time points after denervation,and to test the related proteins expression of UPS and ALS.3.Constructing a lentiviral vector of TRAF6.The animal models of rat sciatic nerve disconnection were established.TRAF6-sh RNA or control-sh RNA was injected into the ipsilateral TA muscle.The weight ratio of TA muscle were analyzed at 14 days after surgery.The RT-q PCR and Western Blot were used to detect the expression of TRAF6 in experimental group and control group 14 days after denervation.The CSA of TA muscle was analyzed by immunofluorescence staining.Ultrastructural changes of TA muscle observed by transmission electron microscopy.The expression changes of TRAF6 in experimental and control group 14 days after denervation were examined.The best viral interference group was determined and the expression changes of related proteins were also detected.Results1.The diameter of C2C12 myotubes decreased,while the expression of TRAF6,Mu RF1 and MAFbx were higher than the control group.The expression of ALS related proteins were higher than the control group.2.The weight ratio or the CSA of the TA muscle gradually decreases with the prolongation of the denervation time point.3.Observing the ultrastructure of the TA muscle,it can be seen that the normal myofiber structure shows that the myofilament sarcomere has a clear structure,neatly arranged and wide sarcomere.There was no abnormalities in the mitochondria as well as myocyte nucleus in the myocyte organelle.With the prolongation of time after denervation,the filamentous sarcomeres arrangement of TA muscle became more disordered,the morphology of mitochondria became more irregular,the vacuolation became more serious,and the number of mitochondria with vacuole structure increased gradually.4.The expression of TRAF6,Mu RF1 and MAFbx is higher than that of the control group,and the expression of ALS related proteins at different atrophy time points were greater than the control group.5.The weight ratio of the TA muscle and the CSA of muscle fibers in the TRAF6-sh RNA injection group were larger than these in the negative control group.6.Compared with control group,Ultrastructural observation of TA muscle fibers showed that the vacuole state of muscle fibers mitochondria in TRAF6 sh RNA injection group was alleviated,and the length of sarcomeres was slightly lengthened.7.The Mu RF1 and MAFbx expression in the TA muscle of the TRAF6 sh RNA injection group were also significantly inhibited.The expression of ALS related proteins were also significantly lower than that injected with control-sh RNA.Conclusion1.TRAF6 is up-regulated during C2C12 myotubes atrophy and denervation-induced muscle atrophy;which causes the expression of muscle-specific E3 ubiquitin ligases Mu RF1 and MAFbx,as well as activates ALS associated proteins expression.2.Inhibition of TRAF6 attenuates muscle atrophy caused by denervation,which may be achieved by further inhibiting the expression of key proteins of the UPS and ALS associated proteins.PartⅡ miR-125b-5p targeting TRAF6 rescues denervation-induced skeletal muscle atrophyObjective1.The purposes of this study were to investigate whether there is an upstream regulatory factor of TRAF6,and can further regulate denervated muscle atrophy by targeting TRAF6.2.To elucidate that miR-125b-5p can regulate TRAF6 in the process of denervated muscle atrophy.3.To explore the specific molecular mechanism of targeting inhibition of TRAF6 on denervated muscle atrophy by miR-125b-5p.Methods1.The expression of miR-125b-5p was determined during the denervation-induced muscle atrophy and C2C12 myotube atrophy by RT-q PCR.The relationship was analyzed between the expression of miR-125b-5p and TRAF6.2.Construction of a dual luciferase reporter gene system containing the target gene TRAF6 3’-UTR and TRAF6 3’-UTR mutant.TRAF6 3’-UTR wild type or TRAF6 3’-UTR mutant was co-transfected into HEK293 cells with miR-125b-5p mimic or miRNA mimic negative control.Then the activity of luciferase reporter gene was tested.3.The C2C12 myotube atrophy model was prepared: the atrophying myotube was divided into four groups(negative control group,miR-125b-5p mimic group,miR-125b-5p inhibitor group,miR-125b-5p mimic+TRAF6 overexpression group).The expression of MHC was detected by immunofluorescence staining to show the morphological changes of myotube atrophy.The expression of TRAF6,Mu RF1,MAFbx and ALS related proteins were detected by Western blot in the experimental group and control group of C2C12 atrophying myotubes.4.The sciatic nerve transection model were established: 32 SD rats were randomly divided into 4 groups(negative control group,miR-125b-5p agomir group,miR-125b-5p antagomir group,miR-125b-5p agomir+TRAF6 overexpression group).The experimental group and the control group were given multiple injections of the TA muscle on the day of surgery.TA muscle was taken on the 14 th day.The CSA of TA muscle fiber was analyzed by Laminin immunofluorescence staining.The ultrastructural changes of TA muscle in experimental group and control group were observed by transmission electron microscopy.The expression of miR-125b-5p in the TA muscle were detected by RT-q PCR.The expression of TRAF6 and its downstream signaling molecules,Mu RF1 and MAFbx,and ALS related proteins in the TA muscle from the experimental group and the control group were detected by Western blot.Results1.The expression of miR-125b-5p decreased gradually during myotube atrophy and denervated muscle atrophy,and the expression of miR-125b-5p and TRAF6 was negatively correlated during muscle atrophy.2.A double luciferase reporter gene system containing TRAF6 3’-UTR and TRAF6 3’-UTR mutant was constructed successfully.The luciferase reporter gene system analysis revealed that miR-125b-5p significantly inhibited the activity of luciferase reporter gene containing TRAF6 3’UTR,but had no influence in the activity of luciferase reporter gene containing TRAF6 3’UTR mutant.3.The diameter of myotubes in the miR-125b-5p mimic group was larger and in the miR-125b-5p inhibitor group was smaller than that in the negative control group.Compared with the miR-125b-5p mimic group,the diameter of myotubes in the mir-125b-5p mimic + TRAF6 overexpression group decreased significantly.The Western blot analysis demonstrated the expression of TRAF6,Mu RF1,MAFbx and the ALS related proteins in the miRNA-125b-5p mimic group was significantly inhibited,while that in the miRNA-125b-5p inhibitor group was increased.Compared with the miRNA-125b-5p mimic group,all of these proteins were up-regulated.4.Injection of different drugs into denervated TA muscle showed that the CSA of muscle fibers in the miR-125b-5p agomir group was significantly larger,the mitochondrial vacuolar was relieved,and the length of the sarcomere was slightly longer.The CSA of muscle fibers in the miR-125b-5p antagomir group was significantly smaller,and more severe mitochondrial vacuoles,irregular myofilament sarcomere and smaller sarcomere length existed than that in the control group.The CSA of the TA muscles in the miR-125b-5p agomir + TRAF6 overexpression group was significantly smaller than that in the miR-125b-5p agomir group,while the phenomenon of autophagy is more serious.5.Compared with the negative control group,the expression of TRAF6 and its downstream signaling molecules,Mu RF1 and MAFbx in TA muscles were significantly inhibited in the miR-125b-5p agomir group,but increased in the miR-125b-5p antagomir group.The expression of TRAF6,Mu RF1 and MAFbx in the miR-125b-5p agomir+TRAF6 overexpression group was up-regulated than the miR-125b-5p agomir group.The expression of ALS related proteins were significantly inhibited in the miR-125b-5p agomir group,but increased in the miR-125b-5p antagomir group than the negative control group.Compared with the miR-125b-5p agomir group,the expression of ALS related proteins in the miR-125b-5p agomir+TRAF6 overexpression group was up-regulated.Conclusions1.The expression of miRNA-125b-5p was down-regulated during denervation-induced muscle atrophy and C2C12 myotube atrophy,which is contrary to the expression pattern of TRAF6.2.There is an interaction between miR-125b-5p and the 3’UTR of TRAF6.3.miR-125b-5p can attenuate denervation-induced muscle atrophy and nutrient deprivation-induced myotube atrophy by targeting TRAF6,and reduce the expression of UPS associated proteins and ALS associated proteins.