Full article: Notoginsenoside R1 promotes MC3T3E1 differentiation by upregulating miR23a via MAPK and JAK1/STAT3 pathways

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathways

Abstract

Abstract

GrowingevidencehaveprobedastimulatoryinfluenceofNotoginsenosideR1(NGR1)withosteoblasticprobability.miR-23aplaysacrucialroleinosteoblastdifferentiation.WhereaswhetherthereexistsamiRs-relatedmechanismbywhichNGR1promotespreosteoblastdifferentiationremainsunexplored.Wepre-treatedMC3T3-E1withNGR1toanatomizeRunx-2andOsxexpressionaswellasALPactivity.PhosphorylationofregulatorswasevaluatedbyWesternblot.SB203580andRuxolitinibwereusedtoreducethephosphorylationofregulators.TheeffectsofNGR1onmiR-23awereverifiedbyqRT-PCR.WeanalyzedtheexpressionofRunx-2andOsx,ALPactivityaswellasphosphorylationofregulatorsinMC3T3-E1stimulatedwithNGR1andtransfectedwithmiR-23ainhibitor.WefoundthatNGR1enhancedRunx-2andOsxexpressionaswellasALPactivityinaconcentration-dependentmanner.NGR1mightexhibitanefficaciouspromotiononRunx-2,OsxandALPactivitybyincreasedphosphorylationofMAPK,JAK1,andSTAT3.NGR1resultedinmiR-23aoverexpressionwhichpositivelymodulatedRunx-2andOsxexpressionaswellasALPactivity.OurresultsshowedthatmiR-23ainhibitorreducedthephosphorylationofMAPK,JAK1andSTAT3inMC3T3-E1pre-treatedwithNGR1.Inconclusion,NGR1exhibitedanefficaciouspromotiononpreosteoblastdifferentiationbyup-regulatingmiR-23athroughMAPKandJAK1/STAT3pathways.Highlights:

NGR1inducesMC3T3-E1differentiation;

miR-23aispositivelyregulatedbyNGR1;

NGR1regulatesMAPK/JAK1/STAT3throughmiR-23a.

Keywords:NotoginsenosideR1, microRNA-23a, preosteoblastdifferentiationIntroduction

Bonefracturehealingisacomplexbiologicalprocessthatsequentiallyinvolvesinflammatoryresponse,primarycartilaginouscallusformation,revascularization,calcification,finallybonerepairandremodelling[1].Eveniffracturepatientsareinthestateofclinicaltreatment,therearestillconsiderablecasesofnon-unionanddelayedunion.Todate,avarietyoftreatmenttherapieshaveemerged,forinstance,surgicalintervention,mechanicalforces,pharmacotherapy,celltherapyandmolecularbiologytherapy[2–5],aswellasacombinationofseveralmethods.Bioactiveagentsincombinationwithtargetedtherapeuticshavedrawnresearchers’attention,especiallythroughmodulatingmicroRNAs(miRs)andsignallingpathwaysforacceleratingosteoblastogenesis[6,7].Giventhatpreosteoblastsarebone-formingcells,itprovidesusanewinsightintoacceleratingfracturehealingbystimulatingcellsdifferentiationforboneformation.

NotoginsenosideR1(NGR1)isanaturaltriterpenesaponincompounddetectedinthetraditionalChinesemedicinePanaxnotoginseng[8].ItsmolecularstructureisshowninFigure1.NGR1hasbeenreportedtoexertmultiplepharmacologicaleffectsonthecellbiologicalactivities,forinstance,cardioprotectiveeffectsagainststressinjuries[9],pro-angiogenicactivity[10],andneuroprotection[11].Currently,agrowingnumberofworkshaveprobedastimulatoryinfluenceofNGR1oncellswithosteoblasticprobability,forinstance,preosteoblasts[12]andosteoblasts[13].Accordingly,ithasbeenrevealedthatPanaxnotoginsengsaponinstimulatesosteogenesisprocessincludingosteoblasticproliferation,differentiationandmineralizationindicatedbyenhancedalkalinephosphatase(ALP)activity,mineralizationandosteoblast-relatedmolecules[14].ThemechanismsbywhichNGR1promotesbonegenerationarecentredonseveralsignallingpathways[15],whereasitisstillunexploredthatwhetherthereexistsamiRs-relatedmechanismbywhichNGR1promotespreosteoblastdifferentiation.

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathwaysAllauthorsChunshengWang,HuanweiSunYimingZhonghttps://doi.org/10.1080/21691401.2019.1573189Publishedonline:04March2019

Figure1.MolecularstructureofNGR1.

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Figure1.MolecularstructureofNGR1.

miR-23aaregenerallyinvolvedinangiogenesis[16],haematopoiesis[17],andcellularsenescence[18].Italsoplayscriticalrolesinmodulatingthebalancebetweenosteoblastandadipocytedifferentiationinbonemarrowmesenchymalstemcells[19].Zenget al.revealedthatmiR-23aclusterdirectlyrepressesanegativeregulatoroftransforminggrowthfactor-βPrdm16withthealternativeofsclerostinexpressionbyRNA-sequencinganalysis[20].Intriguingly,miR-23ahaspreviouslybeenrevealedtodirectlytargetandregulateRun-relatedtranscriptionfactor2(Runx-2)forsuppressingNGR1-inducedangiogenesiswhichispositivelycorrelatedwithcancerdevelopment[21].Nevertheless,itisstillundefinedthatwhetherNGR1modulatesmiR-23ainpreosteoblastcells.Hence,wefocusedattentionontheregulatoryeffectsofNGR1onmiR-23a.

NGR1coordinationofthesignallingpathwaysandmiRs,inpreosteoblasts,mightbeconduciveinboneformationforfracturetherapy.Asaconsequence,wehypothesizedthatNGR1wasabletopromotedifferentiationofpreosteoblaststhroughtriggeringsignallingpathwaysbyregulatingmiRsexpression.Toverifyourassumption,weprimarilyassessedtheaccelerationofNGR1onthedifferentiationofMC3T3-E1cells.Subsequently,weanatomizedthealterationofRunx-2andOsterix(Osx)expression,ALPactivity,andphosphorylatedexpressionofregulatoryfactorsinsignallingpathways.

MaterialsandmethodsMC3T3-E1cellscultureandtreatment

ThemousepreosteoblastcellsMC3T3-E1wereobtainedfromtheAmericanTypeCultureCollection(ATCC,Manassas,VA).MC3T3-E1cellsweremaintainedinα-minimumessentialmedium(α-MEM;Gibco,NY)replenishedwithfoetalbovineserum(FBS)(10%;Gibco),penicillin(100units/mL;Invitrogen,Carlsbad,CA),andstreptomycin(100 mg/mL;Invitrogen)inahumidifiedatmospherecontaining5%CO2at37 °C.MC3T3-E1cellswerestimulatedwithNGR1dilutedwithdimethylsulphoxide(DMSO;Sigma,St.Louis,MO)in0–50 μmol/Lfor48 h.Forrepressingmitogen-activatedproteinkinase(MAPK)signallingpathway,MC3T3-E1cellsweretreatedwithSB203580(SB)(10 μmol/L;Sigma).AsforJanuskinase1/signaltransducerandactivatoroftranscription3(JAK1/STAT3)pathway,westimulatedMC3T3-E1cellswithRuxolitinib(RU)(300 nmol/L;Sigma)toblockthissignallingpathway.

Transfection

miR-23ainhibitoranditscorrespondingnegativecontrol(NC)weresynthesizedbyGenePharma(Shanghai,China).MC3T3-E1cellsweretransfectedwithmiR-23ainhibitororNCwithLipofectamine3000(Invitrogen)withreferencetoitsdescription.

Alkalinephosphataseactivityassay

MC3T3-E1cellswerelysedwithM-PERmammalianproteinextractionreagent(0.1 mol/l;Pierce,Appleton,WI)for30 minafterrinsedwithpre-coldphosphate-bufferedsaline(PBS;Sigma)twice.Aftercentrifugation(104 × g,15 min),thesupernatantwasdeterminedwithp-nitrophenylphosphate(pNPP;Sigma)at405 nm.Particularly,thesample(50 μL)wasblendedwith50 μLpNPP(1 mg/mL)dissolvedwith1 mol/ldiethanolaminebuffercontaining0.5 mmol/LMgCl2(pH9.8)(Sigma),andthenincubatedat37 °Cfor15 minonbenchshaker.Forterminationreaction,200 μLNaOH(2 mol/l)wasaddedintothereactionmixture(1:1,v/v).BCA™ProteinAssayKit(Pierce)wasappliedtoquantifytotalproteincontent.Calfintestinalalkalinephosphatase(Sigma)wasappliedforquantificationasastandard.

Westernblottingassay

ForWesternblottingassay,proteinswereinitiallyabstractedfromMC3T3-E1cellswithRIPAlysisbuffer(Beyotime,Shanghai,China).Proteaseinhibitors(Roche,Guangzhou,China)wereappliedintheextractionprocess.ProteinconcentrationswereexaminedwithBCATMProteinAssayKit(Pierce).Theproteinswereseparatedbysodiumdodecylsulphate-polyacrylamidegelelectrophoresis(SDS-PAGE)afterwards.Isolatedproteinsweretransferredtopolyvinylidenedifluoride(PVDF)membranes(Millipore,Billerica,MA).Themembraneswereblockedwithbovineserumalbumin(BSA;Millipore)for2 handincubatedwiththefollowingantibodies:anti-Runx-2(ab23981,1 µg/mL),anti-Osx(ab209484,1:1000)(Abcam,Cambridge,UK),anti-MAPK(9102,1:1000),anti-p-MAPK(91021,1:1000),anti-JAK1(3344,1:1000),anti-p-JAK1(74129,1:1000),anti-STAT3(12640,1:1000),anti-p-STAT3(94994,1:1000)andβ-actin(4967,1:1000)(CellSignalingTechnology,Danvers,MA)at4 °Covernight.Themembraneswereprobedwithsecondaryantibodies(7074,1:5000)(CellSignalingTechnology)for1 hafterwashingthreetimes.MembranesweretransferredintoBio-RadChemiDocTMXRSsystem(Bio-Rad,Shanghai,China)andcoveredwith200 μLImmobilonWesternChemiluminescentHRPSubstrate(Millipore).TheproteinsignallingwasvisualizedandquantifiedwithImageLabTMsoftware(Bio-Rad).β-actinwasappliedforrelativequantificationasaninternalcontrol.

qRT-PCRanalysis

TheexpressionofmiR-23aatRNAlevelwasanalyzedbyquantitativereversetranscriptionPCR(qRT-PCR).TotalRNAwasisolatedfromMC3T3-E1cellswithTrizolreagent(LifeTechnologiesCorporation,Carlsbad,CA).Besides,TaqmanMicroRNAReverseTranscriptionKitandTaqmanUniversalMasterMixII(AppliedBiosystems,FosterCity,CA)wereusedforquantifyingmiR-23a.U6wasusedasahousekeepinggene.

Statisticalanalyses

Eachexperimentwasperformedforthreetimes.Theresultswereexpressedasmean ± standarddeviation(SD).StatisticalanalyseswereconductedwithGraphPadPrismSoftware(GraphPadSoftware,LaJolla,CA).Weperformedunpairedorpairedtwo-tailedStudent’st-testforcomparisonsbetweentwogroups.One-wayanalysisofvariance(ANOVA)wasemployedformultiplecomparisons.Werecognizedthestatisticallysignificantresultswhenpvalueswere.05.

ResultsRunx-2andOsxexpression,aswellasALPactivity,wereenhancedbyNGR1invitro

ForascertainingthesignificanceofincreasedRunx-2andOsxexpressionaswellasALPactivityinthepreosteoblastcellspre-treatedwithNGR1,westimulatedMC3T3-E1cellswithNGR1atdifferentconcentrations(10,30,and50 μmol/L).WedetectedthatRunx-2andOsxproteinlevelsweremarkedlyenhancedinMC3T3-E1cells(p  .05,.01or.001)(Figure2(A,B)).Similarly,ALPactivitywassignificantlyenhancedparalleledwiththatofthecontrolgroup(p  .05orp  .01)(Figure2(C)).Specifically,thesimulatedconcentrationofNGR1waspositivelyrelatedwithRunx-2andOsxexpressionandALPactivity.TheseresultssuggestedthatNGR1promotedpreosteoblastdifferentiationverifiedbyincreasedRunx-2andOsxexpressionaswellasALPactivity.

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathwaysAllauthorsChunshengWang,HuanweiSunYimingZhonghttps://doi.org/10.1080/21691401.2019.1573189Publishedonline:04March2019

Figure2.NGR1promotedMC3T3-E1cellsdifferentiationinadose-dependentmanner.(A)TheexpressionofRunx-2waselevatedbyNGR1atproteinlevels.(B)TheexpressionofOsxwasincreasedinMC3T3-E1cellspre-treatedwithNGR1.(C)ALPactivitywasenhancedbyNGR1.MC3T3-E1cellswerepre-treatedwithNGR1(0–50 μmol/L)for48 h.NGR1:NotoginsenosideR1;Osx:Osterix;ALP:alkalinephosphatase.*p  .05,**p  .01,or***p  .001comparedtocontrol.

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Figure2.NGR1promotedMC3T3-E1cellsdifferentiationinadose-dependentmanner.(A)TheexpressionofRunx-2waselevatedbyNGR1atproteinlevels.(B)TheexpressionofOsxwasincreasedinMC3T3-E1cellspre-treatedwithNGR1.(C)ALPactivitywasenhancedbyNGR1.MC3T3-E1cellswerepre-treatedwithNGR1(0–50 μmol/L)for48 h.NGR1:NotoginsenosideR1;Osx:Osterix;ALP:alkalinephosphatase.*p  .05,**p  .01,or***p  .001comparedtocontrol.

NGR1promotedRunx-2andOsxexpressionandALPactivitybyactivatingMAPKandJAK1/STAT3signallingpathways

InordertoaddressthatNGR1stimulatedpreosteoblastcellsdifferentiationthroughmodulatingMAPKandJAK1/STAT3signallingpathways,weinitiallyexaminedthephosphorylatedexpressionofregulatoryfactorsofMAPKandJAK1/STAT3signallingpathwaysatproteinlevels.OurresultsvalidatedthatNGR1directlyactivatedMAPKandJAK1/STAT3signallingpathways(p  .01orp  .001)(Figure3(A)).WenextblockedMAPKandJAK1/STAT3pathwaysbystimulatingMC3T3-E1cellswithSBandRU,separately.TheresultsthatthephosphorylatedexpressionofMAPK,JAK1,andSTAT3wasreducedrevealedthatMAPKandJAK1/STAT3signallingpathwayswereinactivated(Figure3(B)).AsweobservedinFigure3(C,D),theexpressionofRunx-2andOsxwasaccordinglysuppressedbySBandRUinMC3T3-E1cellspre-treatedwithNGR1.Notably,SBandRUsimilarlyreversedthemodulatoryeffectsofNGR1onALPactivityinMC3T3-E1cells(p  .05)(Figure3(E)).Asaconsequence,NGR1promotedRunx-2andOsxexpressionandALPactivitybyactivatingMAPKandJAK1/STAT3signallingpathways.

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathwaysAllauthorsChunshengWang,HuanweiSunYimingZhonghttps://doi.org/10.1080/21691401.2019.1573189Publishedonline:04March2019

Figure3.Runx-2,Osx,andALPactivitywereenhancedbyNGR1byimprovingphosphorylationofMAPK,JAK,andSTAT3.(A)ThephosphorylatedexpressionofMAPK,JAK1,andSTAT3wasincreasedinpreosteoblastMC3T3-E1cellstreatedwithNGR1.(B)SBandRUrepressedthephosphorylationofMAPK,JAK1andSTAT3,respectively.(C)SBandRUdecreasedtheproteinexpressionofRunx-2inducedbyNGR1.(D)SBandRUinhibitedtheproteinexpressionofOsxinducedbyNGR1.(E)SBandRUinactivatedALPactivityelevatedbyNGR1.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup;MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)andSB(10 μmol/L)orRU(300 nmol/L)intheNGR1+SBorNGR1+RUgroup,respectively.NGR1:NotoginsenosideR1;Osx:Osterix;ALP:alkalinephosphatase;SB:SB203580;RU:Ruxolitinib;CTRL:control;p-:phosphorylated-;t-:total-;MAPK:mitogen-activatedproteinkinase;JAK1:januskinase1;STAT3:signaltransducerandactivatoroftranscription3.*p  .05,**p  .01,or***p  .001comparedtoCTRL;#p  .05comparedtoNGR1.

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Figure3.Runx-2,Osx,andALPactivitywereenhancedbyNGR1byimprovingphosphorylationofMAPK,JAK,andSTAT3.(A)ThephosphorylatedexpressionofMAPK,JAK1,andSTAT3wasincreasedinpreosteoblastMC3T3-E1cellstreatedwithNGR1.(B)SBandRUrepressedthephosphorylationofMAPK,JAK1andSTAT3,respectively.(C)SBandRUdecreasedtheproteinexpressionofRunx-2inducedbyNGR1.(D)SBandRUinhibitedtheproteinexpressionofOsxinducedbyNGR1.(E)SBandRUinactivatedALPactivityelevatedbyNGR1.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup;MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)andSB(10 μmol/L)orRU(300 nmol/L)intheNGR1+SBorNGR1+RUgroup,respectively.NGR1:NotoginsenosideR1;Osx:Osterix;ALP:alkalinephosphatase;SB:SB203580;RU:Ruxolitinib;CTRL:control;p-:phosphorylated-;t-:total-;MAPK:mitogen-activatedproteinkinase;JAK1:januskinase1;STAT3:signaltransducerandactivatoroftranscription3.*p  .05,**p  .01,or***p  .001comparedtoCTRL;#p  .05comparedtoNGR1.

miR-23aelevatedtheexpressionofRunx-2andOsxandALPactivitybyup-regulatingmiR-23a

GiventhatmiR-23aacceleratesdifferentiationofosteoblasts[20,22]andNGR1couldmodulatebiologicalprocessesbyregulatingtheexpressionofmiRs[23],weconsideredthattheremightbeamodulatoryrelationshipbetweenNGR1andmiR-23a.Onthebaseofourfindings,thatNGR1promotedRunx-2andOsxexpressionandenhancedALPactivity,wefurtherconfirmedthattheexpressionofmiR-23awasmarkedlyenhancedbyNGR1(p  .01;Figure4).

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathwaysAllauthorsChunshengWang,HuanweiSunYimingZhonghttps://doi.org/10.1080/21691401.2019.1573189Publishedonline:04March2019

Figure4.NGR1up-regulatedtheexpressionofmiR-23a.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup.NGR1:NotoginsenosideR1;CTRL:control;miR-23a:microRNA-23a.**p  .01comparedtoCTRL.

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Figure4.NGR1up-regulatedtheexpressionofmiR-23a.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup.NGR1:NotoginsenosideR1;CTRL:control;miR-23a:microRNA-23a.**p  .01comparedtoCTRL.

SincemiR-23awasincreasedinMC3T3-E1cellspre-treatedwithNGR1,wehypothesizedthatNGR1mightpromotetheexpressionofRunx-2andOsxaswellasALPactivitythroughup-regulatingtheexpressionofmiR-23a.Forsubstantiatingourassumption,wesilencedtheexpressionofmiR-23abytransfectionofmiR-23ainhibitorandstimulatedtransfectedMC3T3-E1cellswithNGR1.OurresultsshowedthatmiR-23ainhibitorsignificantlyreducedtheexpressionofmiR-23ainMC3T3-E1cells(p  .01;Figure5(A)).Moreover,down-regulatedmiR-23aobviouslyrestrainedRunx-2andOsxexpression(p  .01)(Figure5(B,C)).Similarly,ALPactivityincreasedbyNGR1wasreducedbymiR-23ainhibitor(p  .05)(Figure5(D)).Summarily,NGR1enhancedRunx-2andOsxexpressionandALPactivitybyelevatingmiR-23aexpression.

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathwaysAllauthorsChunshengWang,HuanweiSunYimingZhonghttps://doi.org/10.1080/21691401.2019.1573189Publishedonline:04March2019

Figure5.NGR1acceleratedthedifferentiationofMC3T3-E1cellsbyincreasingtheexpressionofmiR-23a.(A)TheexpressionofmiR-23awasdown-regulatedbymiR-23ainhibitor.(B)miR-23ainhibitorreducedtheproteinexpressionofRunx-2inducedbyNGR1.(C)miR-23ainhibitordecreasedtheproteinexpressionofOsxinducedbyNGR1.(D)miR-23ainhibitorinactivatedtheactivityofALPstimulatedbyNGR1.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup;MC3T3-E1cellsweretreatedwithNGR1andtransfectedwithmiR-23ainhibitorintheNGR1+miR-23ainhibitorgroup;MC3T3-E1cellsweretreatedwithNGR1andthecorrespondingnegativecontrolofmiR-23aintheNGR1+NCgroup.miR-23a:microRNA-23a;NGR1:NotoginsenosideR1;CTRL:control;NC:negativecontrol;Osx:Osterix;ALP:alkalinephosphatase.*p  .05or**p  .01comparedtoCTRL;#p  .05or##p  .01comparedtoNGR1+NC.

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Figure5.NGR1acceleratedthedifferentiationofMC3T3-E1cellsbyincreasingtheexpressionofmiR-23a.(A)TheexpressionofmiR-23awasdown-regulatedbymiR-23ainhibitor.(B)miR-23ainhibitorreducedtheproteinexpressionofRunx-2inducedbyNGR1.(C)miR-23ainhibitordecreasedtheproteinexpressionofOsxinducedbyNGR1.(D)miR-23ainhibitorinactivatedtheactivityofALPstimulatedbyNGR1.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup;MC3T3-E1cellsweretreatedwithNGR1andtransfectedwithmiR-23ainhibitorintheNGR1+miR-23ainhibitorgroup;MC3T3-E1cellsweretreatedwithNGR1andthecorrespondingnegativecontrolofmiR-23aintheNGR1+NCgroup.miR-23a:microRNA-23a;NGR1:NotoginsenosideR1;CTRL:control;NC:negativecontrol;Osx:Osterix;ALP:alkalinephosphatase.*p  .05or**p  .01comparedtoCTRL;#p  .05or##p  .01comparedtoNGR1+NC.

Down-regulatedmiR-23aexpressionblockedMAPKandJAK1/STAT3signallingpathwaystriggeredbyNGR1

WehaveelucidatedthatNGR1elevatedthephosphorylationofMAPK,JAK1,andSTAT3.TheobservationthatRunx-2andOsxexpressionandALPactivitystraightlyenhancedbyNGR1bytriggeringMAPKandJAK1/STAT3signallingpathwaysandpromotingmiR-23aexpressionraisedourhypothesisthatmiR-23amightparticipateinactivatingMAPKandJAK1/STAT3signallingpathways,whichweretriggeredbyNGR1.Consequently,weexaminedthephosphorylatedexpressionofMAPK,JAK1,andSTAT3inMC3T3-E1cellsaftersuppressingtheexpressionofmiR-23abytransfectingMC3T3-E1cellswithmiR-23ainhibitor.OurresultsrevealedthatreducedmiR-23aexpressionsignificantlyinhibitedphosphorylationofMAPK,JAK1andSTAT3inMC3T3-E1cellstreatedwithNGR1(p  .05;Figure6(A,B)).Analyzingtheaboveevidence,wemightdistinctlyconcludethatNGR1triggeredMAPKandJAK1/STAT3signallingpathwaysbyelevatingmiR-23aexpression.

NotoginsenosideR1promotesMC3T3-E1differentiationbyup-regulatingmiR-23aviaMAPKandJAK1/STAT3pathwaysAllauthorsChunshengWang,HuanweiSunYimingZhonghttps://doi.org/10.1080/21691401.2019.1573189Publishedonline:04March2019

Figure6.NGR1enhancedthephosphorylatedexpressionofMAPK,JAK1andSTAT3bypromotingtheexpressionofmiR-23a.(A)miR-23ainhibitorrepressedthephosphorylationofMAPKpromotedbyNGR1.(B)miR-23ainhibitordecreasedthephosphorylatedexpressionofJAK1andSTAT3intheMC3T3-E1cellspre-treatedwithNGR1.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup;MC3T3-E1cellsweretreatedwithNGR1andtransfectedwithmiR-23ainhibitorintheNGR1+miR-23ainhibitorgroup;MC3T3-E1cellsweretreatedwithNGR1andthecorrespondingnegativecontrolofmiR-23aintheNGR1+NCgroup.NGR1:NotoginsenosideR1;NC:negativecontrol;p-:phosphorylated-;t-:total-;MAPK:mitogen-activatedproteinkinase;JAK1:januskinase1;STAT3:signaltransducerandactivatoroftranscription3.*p  .05or**p  .01comparedtoCTRL;#p  .05or##p  .01comparedtoNGR1+NC.

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Figure6.NGR1enhancedthephosphorylatedexpressionofMAPK,JAK1andSTAT3bypromotingtheexpressionofmiR-23a.(A)miR-23ainhibitorrepressedthephosphorylationofMAPKpromotedbyNGR1.(B)miR-23ainhibitordecreasedthephosphorylatedexpressionofJAK1andSTAT3intheMC3T3-E1cellspre-treatedwithNGR1.MC3T3-E1cellsweretreatedwithNGR1(50 μmol/L)for48 hintheNGR1group;MC3T3-E1cellswerenottreatedwithNGR1intheCTRLgroup;MC3T3-E1cellsweretreatedwithNGR1andtransfectedwithmiR-23ainhibitorintheNGR1+miR-23ainhibitorgroup;MC3T3-E1cellsweretreatedwithNGR1andthecorrespondingnegativecontrolofmiR-23aintheNGR1+NCgroup.NGR1:NotoginsenosideR1;NC:negativecontrol;p-:phosphorylated-;t-:total-;MAPK:mitogen-activatedproteinkinase;JAK1:januskinase1;STAT3:signaltransducerandactivatoroftranscription3.*p  .05or**p  .01comparedtoCTRL;#p  .05or##p  .01comparedtoNGR1+NC.

Discussion

Inourpresentwork,wehaveelucidatedthatNGR1promotedpreosteoblastdifferentiation.Onthebasisofitspositiveeffects,ourresearchprovidedacompellingconclusionforapotentialmechanismbywhichNGR1accelerateddifferentiationbyactivatingMAPKandJAK1/STAT3pathways.Furthermore,ourobservationrevealedthatmiR-23aexertedacrucialfunctionindifferentiation.Itisappreciatedthatdown-regulatedmiR-23ablockedMAPKandJAK1/STAT3pathwayswhichweretriggeredbyNGR1.

Althoughthepro-osteogenicfunctionofNGR1hasbeenevaluatedinosteoblastinvitroinapreviousstudy[13],itsroleinpreosteoblastisacompellingquestion.SinceALPisalatedifferentiationMarkerofosteoblastsfrompreosteoblasts,playingpivotalrolesinbonemineralizationandsuggestingosteoblastactivity[24,25],wedissectedALTactivityinMC3T3-E1cellstreatedwithNGR1.TheincreasedALPactivitysuggestedthatNGR1promotedpreosteoblastdifferentiationwhichwasalsoprovedbyenhancementofRunx-2andOsxexpression.Runx-2hasbeenreportedtoinduceosteoblastdifferentiation[26].Osxisidentifiedasanessentialmultifunctionalregulatorinbonegrowth[27].Hence,NGR1possessedapro-osteogenicfunction.Inaddition,NGR1promotedpreosteoblastsdifferentiationinadose-dependentmanner.MAPKandJAK1/STAT3signallingpathwaysareinvolvedinmultipletypecellsdifferentiation[28–30].Ourobservation,thatreducedphosphorylatedexpressionofMAPK,JAK1andSTAT3bypharmacologicaltreatmentrepressedRunx-2andOsxexpressionaswellasALPactivity,confirmingourhypothesisthatNGR1mightacceleratedifferentiationbytriggeringMAPKandJAK1/STAT3signallingpathways.Thatis,pre-treatmentwithNGR1couldenhancedifferentiationofpreosteoblastwhichplayscrucialrolesinboneformationandfractureshealing,indicatingapotentialtherapeuticcureforboneinjury.

Noticeably,wedetectedthehigherexpressionofmiR-23ainpreosteoblastcellspre-treatedwithNGR1.Consistentwithourfindings,previousstudiessuggestedthataberrantexpressionofmiR-23aclusterprovokesdysfunctionofosteocytedifferentiation[20].AlthoughNGR1hasbeenprovedtoexertapro-osteogenicfunctioninosteoblastdifferentiation,themechanismisundefined,especiallywhetherthereisacorrelationbetweenmiR-23aandNGR1.InordertoexplorewhetherNGR1regulatedmiR-23aexpressionforinfluencingtheproductionofRunx-2andOsxandALPactivity,wesuppressedmiR-23aexpressionbytransfectingpreosteoblastswithmiR-23ainhibitor.Ourresultsfurtherelucidatedthatup-regulatingmiR-23aexpression,NGR1promotedpreosteoblastdifferentiation.RecentfindingsdemonstratedthatmiR-23aclusterisnecessaryforsustainingstage-specificHoxAfactorexpressionwhichcouldphysicallyinteractwithRunx-2duringpreosteoblastdifferentiation[31].Hasasanet al.reportedthatRunx-2negativelymodulatesmiR-23aclusterexpressionandtheyprovideddirectevidenceforapotentialmechanismthatmiR-23aclustertargetsSATB2whichhasbeenprovedtosynergizewithRunx-2tofacilitateboneformation[22].Similarly,ourfindingsdemonstratedtheremightexhibitanegativefeedbackeffectofmiR-23aontheexpressionofRunx-2.AsforOsx,therearescarcereportsaboutdirectregulationofmiR-23aonOsx.Therefore,wesupposedthatNGR1mightup-regulatemiR-23aexpressionwhichcircuitouslypromotesOsxexpression.

TheregulatoryeffectsofmiRsonsignallingpathways,especiallystudieshavevalidatedthatmiR-23atargetsMAPKandJAK/STATsignallingpathwaysinprostatecancer[32],provokedourattention.ConsideringMAPKandJAK1/STAT3signallingpathwaysareinvolvedinosteoblastcellsdifferentiationprocess[30,33,34],wefocusedonthealternationofphosphorylatedexpressionofMAPK,JAK1,andSTAT3whichweremodulatedbyaconcomitantsynergismbetweenNGR1andmiR-23ainhibitor,subsequentlyverifiedinourstudies.TheresultsshowedthatmiR-23ainhibitorreducedthephosphorylationofMAPK,JAK1,andSTAT3.Mechanistically,weconfirmedthatNGR1maypotentiateMAPKandJAK1/STAT3signallingpathwaysbyup-regulatingmiR-23aexpression.

Takentogether,wehavevalidatedthatNGR1promotedpreosteoblastcellsdifferentiation.Basedonitseffectiveaccelerationonpreosteoblastcellsdifferentiation,weproposedapotentialmechanismthatNGR1exertedup-regulatoryeffectsonRunx-2,OsxandALPactivitybyincreasingmiR-23aexpression.Further,NGR1activatedMAPKandJAK1/STAT3signallingpathwaysbyup-regulatingtheexpressionofmiR-23a.

Disclosurestatement

Nopotentialconflictofinterestwasreportedbytheauthors.

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