| PartⅠiTRAQ-coupled2D LC–MS/MS analysis on differentiallyexpressed proteins in denervated tibialis anterior muscle of RattusnorvegicusObjective:The purposes of this study were to examine the differentially expressed proteins indenervated tibialis anterior (TA) muscle at different time point after sciatic nervetransection in rats by quantitative proteomics technology. The bioinformatics analysiswas performed to screen out the "key" protein involved in denervation-induced muscleatrophy. Exploring the biological function of the protein contributed to reveal themolecular mechanism involved in denervation-induced muscle atrophy and to providethe scientific basis for the prevention of denervation-induced muscle atrophy.Methods:1. The animal models of rat transacted sciatic nerves were established. Twenty-fourSprague–Dawley (SD) rats were randomly divided into4groups (n=6) to undergooperation (for3operated groups) or sham operation (for a control group) to the sciaticnerve.2. The total protein was extracted from muscles and quantitified using RC DCprotein assay kit. Proteins precipitated with acetone were dissolved in dissolution bufferand sequentially reduced, alkylated, digested, labeled with the iTRAQ tag, purified andfractionated using an off-line strong-cation exchange column. Following, the fractionwas analyzed by Nano Aquity UPLC system connected to an LTQ Orbitrap XL massspectrometer. 3. SEQUEST soft was used to identify and quantify the peptides and proteins.4. The differentially expressed proteins were classified according to annotationsfrom Swiss-Prot/TrEMBL database and GO database. The differentially expressedproteins were analyzed by GO, KEGG signal pathway and STRING interaction analysis.Screen the "key" proteins involved in denervated muscle atrophy.5. Some differentially expressed proteins were validated by western blot.6. The model of myotube atrophy was constructed in vitro and the biologicalfunctions of key protein were explored.Results:1. Isobaric tags for relative and absolute quantitation (iTRAQ) coupled withtwo-dimensional liquid chromatography-tandem mass spectrometry were performed todetect a total of260proteins that were differentially expressed in the rat tibialis anteriormuscle at different times after rat sciatic nerve transection. The dysregulated proteinsmainly included metabolic enzymes, chaperones, signal molecules, structural proteins,extracellular matrix-related proteins, energy metabolism-related proteins, andubiquitin–proteasome pathway-related proteins et al. The proteins involved in metabolicenzymes were the most.2. Four expression change patterns were noted:54proteins were down-regulatedwith time;17proteins displayed up-regulation at1-or4-week post-nerve transection,and then gradual down-regulation. The two types of change pattern-related proteinsmainly involved in energy metabolism-related proteins, structural proteins,ribonucleoprotein, and etc.101proteins were upregulated with time;88proteinsdisplayed down-regulation at1-or4-week post-nerve transection, then gradualupregulation. The last two types of change pattern-related proteins mainly included thefollowing proteins: chaperones, metabolic enzymes, extracellular matrix-relatedproteins, structural proteins, signaling molecules and ubiquitin–proteasomepathway-related proteins.3. GO analysis of our selected dataset revealed that the differentially expressedproteins during denervation participated in several biological processes includingglycolysis, tricarboxylic acid cycle and cellular respiration. 4. KEGG analysis of our selected dataset suggested the involvement of severalpathways including citrate cycle, glycolysis/gluconeogenesis and neurotrophin signalingpathway.5. The results from interactions for the differentially expressed proteins in theSTRING protein protein interaction database indicated that TRAF6, which was notdetected in our study, might be upregulated in denervated atrophic skeletal muscle andplay important role in denervation induced atrophy.6. In this study, four representative proteins (CRYAB and PEBP1were included inthe proteins displayed high level expression. PYGM and KCRS were included in theproteins displayed low level expression), were chosen to be validated by western blot.These data agreed with the expression changes shown by the iTRAQ analysis. At thesame time, the expression of TRAF6was validated by western blot, which agreed withthe predicted expression change.7. TRAF6was upregulated in denervation induced muscle atrophy. Theknockdown of TRAF6could rescue dexamethasone-induced skeletal muscle atrophy.Conclusions:1. A total of260differentially expressed proteins were detected in the deverationinduced muscle atrophy by iTRAQ coupled with2D LC MS/MS. The dysregulatedproteins mainly included metabolic enzymes, structural proteins, signal molecules andchaperones et al.2. The bioimformatics analysis indicated the altered expression of energymetabolism-related proteins could play crucial role in deveration induced muscleatrophy.3. TRAF6was upregulated in denervation-induced muscle atrophy. Theknockdown of TRAF6could rescue dexamethasone-induced myotube atrophy in vitro. PartⅡ the mechanisms and effects of TRAF6indenervation-induced muscle atrophyObjective:The purposes of this study were to explore the mechanisms and effects of TRAF6in denervation-induced muscle atrophy.（1）Knockdown of TRAF6could rescue denervation-induced muscle atrophyMethods:1. The animal models of rat sciatic nerves transaction were established. Sixteen ratswere randomly divided into2groups (n=8)(one operated group injected withTRAF6-siRNA or control group injected with control-siRNA). Transfection withTRAF6-siRNA or control-siRNA was initiated on the same day. Injection in TA muscleswas performed every three days for2weeks. All animals were killed14days after thebeginning of injection.2. The mass of muscles was used to analyze the wet weitht ratio of TA. The crosssection area of TA muscle fiber was accessed by Masson trichrome staining.3. The expression of TRAF6and its downstream signaling molecules, MAFBx andMuRF1, were examined in TA muscles from operated group or control group.Results:1. The weight ratio or the cross-sectional area (CSA) of the TA muscles injectedwith TRAF6-siRNA was significantly larger than that injected with control-siRNA(P<0.05).2. The qPCR and Western blot analysis demonstrated that the mRNA and proteinexpressions of TRAF6, MAFBx, and MuRF1were significantly lower in the TAmuscles injected with TRAF6-siRNA than that in the TA muscles injected withcontrol-siRNA (P<0.05).（2）miR-351interacts with TRAF63’UTRMethods:1. The expression of miR-351was determined during the denervation-induced muscle atrophy by realtime RT-PCR. The relationship was analyzed between theexpression of miR-351and the expression of TRAF6during denervation-inducedmuscle atrophy.2. Luciferase reporter gene vectors containing TRAF63’UTR and TRAF63’-UTRmutant were constructed.3. TRAF63’UTR or TRAF63’-UTR mutant and miR-351mimic or miRNA mimicNegative Control cotransfected into HEK293cells. Then the activity of luciferasereporter gene was tested.Results:1. miR-351displayed a gradual decrease during denervation-induced muscleatrophy. There was a negative correlation between the expression of miR-351and theexpression of TRAF6during denervation-induced muscle atrophy.2. Luciferase reporter gene vectors containing TRAF63’UTR and TRAF63’-UTRmutant were successfully constructed.3. The results from the activity of luciferase reporter gene analysis demonstratedthat miR-351could inhibit the activity of luciferase reporter gene containing TRAF63’UTR, and that miR-351did not significant change in the activity of luciferase reportergene containing TRAF63’UTR mutant.（3） miR-351could rescue denervation-induced muscle atrophyMethods:1. The animal models of rat sciatic nerves transection were established. Sixteen ratswere randomly divided into2groups (n=8)(one operated group injected with miR-351agamir or control group injected with miRNA agamir negative control). Injection withmiR-351agamir or control group injected with miRNA agamir negative control wasinitiated on the same day. Injection in TA muscles was performed every three days for2weeks. All animals were killed14days after the beginning of injection.2. The mass of muscles was used to analyze the wet weight ratio of TA. The crosssection area of TA muscle fiber was accessed by Masson trichrome staining.3. The expression of miR-351was determined by realtime RT-PCR. The expression of TRAF6and its downstream signaling molecules, MAFBx and MuRF1,were examined in TA muscles from operated group or control group with western blot.Results:1. The weight ratio or the CSA of the TA muscles injected with miR-351agamirwere significantly larger than that injected with miRNA agamir negative control(P<0.05).2. The qPCR and Western blot analysis demonstrated that the mRNA and proteinexpressions of TRAF6, MAFBx, and MuRF1were significantly lower in the TAmuscles injected with miR-351agamir than that in the TA muscles injected with miRNAagamir negative contro (P<0.05).Conclusion:1. Knockdown of TRAF6could rescue denervation-induced muscle atrophy.2. There was a negative correlation between the expression of miR-351and theexpression of TRAF6in denervated muscle atrophy and an interaction betweenmiR-351and TRAF63’UT.3. miR-351inhibited denervated muscle atrophy by targeting TRAF6. |
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