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EffectsofSOCS1/3genesilencingontheexpressionofC/EBPαandPPARγduringdifferentiationandmaturationofrodentpreadipocytesRunningtitle:PreadipocyteCategoryofstudy:basicscienceWordcountof Wordcountof:Introduction:SOCS-1andSOCS-3aretwoimportantnegativeregulatorsintheinsulinsignalingpathwayandtheiroverexpressionmayaggravateinsulin.Additionally,subjectswithinsulinareoftenobeseandhaveincreasedexpressionsofSOCS-1andSOCS-3.Thus,wespeculatedthatSOCS-1andSOCS-3maybeinvolvedinabnormaldepositionofadiposetissuesduringinsulin.Results:OurresultsshowedthattheexpressionsofSOCS-1,SOCS-3,C/EBPαandPPARγweremarkedlyincreasedintheadiposetissuesofrats,buttheexpressionsofC/EBPαandPPARγweresignificantlyreducedfollowinggenesilencingofSOCS-1andSOCS-3.Discussion:OurfindingssuggestthatoverexpressionofSOCS-1andSOCS-3mayenhancetheexpressionofC/EBPαandPPARγ,resultinginabnormaldepositionofadiposetissuesduringinsulin.Methods:Inthepresentstudy,IUGRratswithinsulinwerepreparedandtheproteinexpressionsofSOCS-1,SOCS-3,C/EBPαandPPARγweremeasured.Inaddition,smidscarryingshRNAtargetingtheSOCS-1andSOCS-3genewerepreparedindependentlyandwereusedtotransfectpreadipocytestoinducethematurationanddifferentiationofpreadipocytes.At72haftertransfection,theexpressionofC/EBPαandPPARγ,twoimportantmoleculespromotingthedifferentiationofpreadipocytes,wasdetected.Numerousepidemiologicalstudieshaveshownthatadultswithintrauterinegrowthretardation(IUGR)haveasignificantlyincreasedriskforobesityandinsulin(IR)(1-3).OurpreviousstudiesshowedthatIUGRratsaged12weeksdevelopedIR,overexpressedSOCS-1andSOCS-3,andthatsilencingoftheSOCS-1andSOCS-3genesimprovedglucosetransportinmusclecells(4).Thus,wespeculatedthatSOCS-1/3maybeinvolvedinthedevelopmentofIRinmusclecellsofIUGRrats.AvailableevidencesuggeststhatSOCS-1andSOCS-3bindtotheinsulinreceptor,inhibitingitsphosphorylationandsubsequentinsulinsignaltransduction(5,6).Inaddition,SOCS-1andSOCS-3canbindtosubstratesofinsulinreceptors(IRS1andIRS2),resultinginubiquitinationofinsulinreceptorsubstrates,whichalsoinhibitinsulinsignaltransduction(7).IRreferstoreducedsensitivityofperipheraltissuestoinsulin,includingmusculartissuesandadiposetissues,whichhavethehighestsensitivitytoinsulin.Adipocytes,themaincomponentofwhiteadiposetissues,notonlystoreenergybutarealsoimportantendocrineorgansinvolvedintheregulationofglucoseandfatmetabolism(8).Matureadipocytesaredifferentiatedfrompreadipocytes,whichareregulatedbytranscriptionalfactorssuchasC/EBPα,PPARγandADD1/SREBP1c3.PPARγcaninducemRNAtranscriptionofC/EBPα,whichmaypromotePPARγexpressionviapositivefeedback.Furthermore,PPARγandC/EBPαareessentialforthemaintenanceofdifferentiationandmaturationofadipocytes(9,10).Thenormaldifferentiationofadipocytesmaintainsthebalanceofenergymetabolismand,conversely,theabnormaldifferentiationofadipocytesmayinterrupttheeffectsofinsulin,resultinginIR.WehavepreviouslyestablishedIUGRmodelinratsandconstructedsmidscontainingshRNAexpressingSOCS-1/3.Inthepresentstudy,wesoughttoinvestigatetheexpressionofSOCS-1,SOCS-3,C/EBPαandPPARγinadiposetissuesofIUGRrats.At72haftersilencingoftheSOCS-1/3genesandinductionofpreadipocytedifferentiationandmaturation,wedeterminedtheexpressionsofC/EBPαandPPARγ,twokeymoleculesinthedifferentiationofpreadipocytes.WespeculatedthatSOCS-1andSOCS-3mayregulatethedifferentiationofwhichisthemaincauseofIRofadiposetissuesandeventhewholebody.（此段翻译不佳。或有理解不对，主要观点如下：1建立IUGRSOCS1/3是我们之前已完成的工作;2本研究首先检测未干扰时IUGR大鼠中SOCS1/3，C/EBPαandPPARγ水平；3沉默前脂肪细胞中SOCS1/3后，诱导细胞分化成熟，诱导72h时再检测C/EBPαandPPARγ）处具体信息不止是shRNA的序列，还有上面提到的糖脂代谢状况、胰岛素 。PreparationofIUGRratswithIRandconstructionofsmidscarryingshRNAstargetingSOCS1/3genesToprepareIUGRratsnutritionaldeprivationwasperformedatthepregnancystageandsufficientnutritionwasadministeredforneonateratsafterbirth.Levelsoffastingglucoseandinsulinweremeasuredat12weekstoconfirmthepresenceofobesity处具体信息不止是shRNA的序列，还有上面提到的糖脂代谢状况、胰岛素 。NutritionaldeprivationwasperformedatthepregnancystageandsufficientnutritionwasadministeredforneonateratsafterbirthaimingtoprepareIUGR(intrauterinegrowthrestriction)rats.Thelevelsoffastingglucoseandinsulinweremeasuredintheseratsaged12weekstoconfirmthepresenceofobesityandIR.FourshRNAstargetingratSOCS-1andSOCS-3genesweredesigned,whichincludedtheU6promoterandFGP.Then,theshRNApairwiththebestsilencingefficiencywasselected.DetailsareshowninourpreviousstudyImmunoblottingSubcutaneousandperirenaladiposetissuesaswellascellsat72haftertransfectionwerecollectedfollowedbyproteinextractionwithaproteinextractionkit(ProMab,USA)accordingtothemanufacturer’sinstructions.TheBCAmethod(PierceCo,USA)wasemployedtomeasureproteinconcentrations.Proteins(50μg)weresubjectedtoelectrophoresisandtransferredontoaPVDFmembrane,whichwasthenblockedin5%non-fatmilkfor2h.Membraneswereincubatedwithprimaryantibody(1:1000)at4℃overnightandthenwithsecondaryantibody(1:2000)atroomtemperaturefor2h.VisualizationwasperformedwiththeECLmethod.ProteinbandswerecollectedbyscanningwiththeAMBISRadioyticandVisualImagingsystem(AmbisInc)andopticaldensitywasdeterminedastheexpressionC/EBP-αandPPAR-γ.Eachexperimentwasperformedintriplicate.（此段标题和最后一句Cultureofpreadipocytes.Primarycultureofpreadipocyteswasperformedaspreviouslydescribed(11,12).（）Briefly,whiteadiposetissuessurroundingtheratwerecollectedandbloodwasremovedbywashingAdiposetissueswerethenmixedwithfivevolumesof0.1%typeIcollagenaseGibco,USA)followedbyincubationat37℃for45minundercontinuousshakingfordigestionThemixturecontainingthecellswasfilteredthrougha200micronmeshfilterandthefiltratewascollectedintoacentrifugetubefollowedbycentrifugationat2000rpm/minfor10minat4℃.Theseprocedureswereperformedthreetimesandthecellswerecounted.Thecelldensitywasadjustedto1×105cells/cm2andthencellswereseededinto6-welltesandmaintainedinDMEMlowglucose(Gibco,USA)containing10%fetalcalfserum(FBS;Gibco,USA)at37℃inanatmospherewith5%CO2.TransfectionofadipocyteswithsmidscarryingshRNAs.Pre-adipocyteswerepassagedthreetimesandthenseededinto6-welltes(4-5×104cells/well)andmaintainedinantibiotic-DEME.Whenthecellconfluencereached70%,transfectionwasperformed.siRNA-Lipofectamine™2000compoundwaspreparedinthefollowingways:1).250μlofOpti-MEM®Iwasmixedwith5μlofLipofectamine™2000followedbyincubationatroomtemperaturefor5min;2)250μlofOpti-MEM®Iwasmixedwith7.5μlofshRNAfollowedbyincubationatroomtemperaturefor5min;and3)bothmixturesaboveweremixedgentlyfollowedbyincubationatroomtemperaturefor20min.TheshRNA-LipofectamineTM2000compoundwasaddedtoawellcontainingcellsandmediumfollowedbygentletap.Incubationwasperformedat37℃inanatmospherewith5%CO2for12hfollowedbyinductionofdifferentiationandmaturation.Differentiationinductionofpre-adipocytes.At24haftertransfection,acocktailofsteroidswasusedfordifferentiationinductionofpre-adipocytes(13).CellsweremaintainedinDMEMfordifferentiation(0.5mmol/LIBMX,1.0μmol/LDEX,and10μg/LINS)containing10%fetalbovineserum(FBS)for48handthemediumwasrefreshedwithDMEMcontaining10%FBSforanother24hfollowedbydetection.DetectionofC/EBP-αandPPAR-γmRNAexpressionbyrealtimePCR.At72hafterdifferentiationinduction,cellsintwogroupswereharvestedandtotalRNAwasextractedusingTrizolreagents(Invitrogen,USA)followedbymeasurementofabsorbanceat260nmand280nmfordetectionofRNApurity.TheratioofA260/A280of>2wasacceptable.RNAwasthenKit(Fermentas,USA)onanABI7500HTPCRinstrument(ABI,USA)followedbyreal-timePCR.ThesequencesofprimersforPPARγwereasfollows:forward:5’-ACCACAGTTGATTTCTCCAG-3’,reverse:5’-TGTTGTAGAGCTGGGTCTTT-3’,andtheanticipatedsizewas178bp.TheprimersforC/EBPαwereasfollows:forward:5’-CGACTTCTACGAGGTGGAG-3’,reverse:5’-ATGTAGGCGCTGATGTCTAT-3’,andtheanticipatedsizewas192bp.Theprimersforβ-actinwereasfollows:forward:5’-GGCATCCATGAAACTATT-3’,reverse:5’-GATCTTCATGGTGCTAGGAG-3’,andanticipatedsizewas165bp.PrimersweresynthesizedinShanghaiSangonBio-engineeringCo.,.Themixture(25μL)forPCRincluded1μLoftemte,10μmol/Lprimers(1μLforeach),12.5μLofSYBRPremixExTaqandddH2O.ConditionsforPCRwereasfollows:pre-denaturationat95℃for5min,40cyclesofdenaturationat94℃for20s,annealingat58℃for20sandextensionat72℃for20sandafinalextensionat72℃for5min.The△△CTmethodwasemployedforthedeterminationofexpressionsoftargetStatisticalysis.Datawereexpressedasmean±standarddeviation.Comparisonsbetweentwogroupswereconductedusingttests.SPSSversion15.0statisticssoftwarewasemployedforstatisticalysis.Avalueofp<0.05wasconsideredstatisticallysignificant.ExpressionofSOCS-1andSOCS-3proteininIUGRandhealthyrats.Westernblottingassayswereperformedtodetectproteinexpressionslevelsoftargetgenes.TheresultsshowedthattheexpressionofSOCS-1andSOCS-3inthesubcutaneousfatofIUGRratswasdramaticallyincreasedwhencomparedtolevelsinhealthyrats(p<0.05;Fig.1).ExpressionofC/EBPαandPPARγproteinsinIUGRandhealthyrats.RT-PCRwasemployedtomeasurethebaselinemRNAexpressionofC/EBPαandPPARγinIUGRandhealthyrats.ResultsshowedthatmRNAexpressionofC/EBPαandPPARγinIUGRratswasmarkedlyincreasedcomparedtolevelsinhealthyrats(p<0.05;Fig.2).ExpressionofC/EBPαandPPARγfollowingsilencingoftheSOCS-1gene.WesternblottingassaysrevealedthatC/EBPαandPPARγproteinexpressionintheIUGRAgroupat3dafterdifferentiationinductionwasmarkedlyreducedaftersilencingoftheSOCS-1gene<0.05;Fig.3).Real-timePCRrevealedthatthemRNAexpressionofC/EBPαandPPARγwasalsodramaticallydecreasedaftersilencingoftheSOCS-1gene(p<0.05;Fig.4).ExpressionofC/EBPαandPPARγfollowingsilencingoftheSOCS-3gene.AsshowninFigure5,theexpressionofC/EBPαandPPARγproteinsat3dafterdifferentiationinductionwassignificantlyreducedaftersilencingoftheSOCS-1gene.AsshowninFigure6,themRNAexpressionofC/EBPαandPPARγwasalsomarkedlyreduced.Inclinicalpractice,therapidaccumulationofadiposetissueshasbeennotedinIUGRsubjects,resultinginobesityandIR.ThethirdUSNationalHealthandNutritionSurveyrevealedthatthebodyweightofSGAsubjectswasstilllowerthanthatofnormalsubjectsalthoughsufficientnutritionhadbeenadministered.Inaddition,SGAsubjectshadhighproportionsofadiposetissueandtheamountofsubcutaneousadiposetissueandhipcircumferencewerelargerthanthoseinnormalsubjects(14).OurpreviousstudyshowedthattheIUGRratshadahigherBMIrelativetohealthyrats(4).Collectively,thesefindingssuggestexcessivepre-adipocytedifferentiateintomatureadipocytes.Themolecularmechanismsunderlyingtheregulationofadipocytedifferentiationhavebeenextensivelystudiedandsomemoleculeshavebeenfoundtobeinvolvedinthedifferentiationofpre-adipocytesintomatureadipocytes.Ofthesemolecules,PPARγandC/EBPαyimportantroles.PPARγcaninducethematurationofadipocytesandregulatetheexpressionofadipogenesis-relatedgenesandC/EBPαcanregulatetheterminaldifferentiationofadipocytes(15,16).Thus,theover-differentiationofadipocytesandabnormalaccumulationofadiposetissuesmightberelatedtotheincreasedexpressionofPPARγandC/EBPα.Basedontheresultsofthecurrentstudy,theexpressionofPPARγandC/EBPαasmeasuredinananimalmodeldemonstratedtheabovehypothesis.AsPPARγandC/EBPαcanpromotethematurationofadipocytesleadingtoobesity,obesitywasinvestigatedinthepresentstudy.Asitiswellknown,insulincanpromoteadipogenesisandinhibitthedegradationofadiposetissues.AtthelatestageofIUGR,IRandhyperinsulinemiamaydevelop.AlthoughthedevelopmentofIRinvolvesmultiplemoleculesandmultiplesignalingpathways,thespecificmechanismsforIRdevelopmentarestillpoorlyunderstood.IthasbeendemonstratedthatSOCS1and3canpromotethedevelopmentofIR(5-7),andobesesubjectswithIRhavebeenfoundtohaveincreasedexpressionofSOCS1and3(17,18).Thus,weexaminedtheexpressionofSOCS1and3inthesubcutaneousadiposetissuesofratsandtheresultsdemonstratedincreasedexpressionofSOCS1and3comparedtonormalrats.ItispossiblethatincreasedexpressionofSOCS1and3caninduceIR,leadingtohyperinsulinemia,whichfacilitatestheformationandaccumulationofadiposetissues.Alternatively,SOCS1and3maydirectlypromotetheexpressionofPPARγandC/EBPα,resultinginadipogenesisandobesity.Toinvestigatethishypothesis,wedesignedshRNAstargetingSOCS1and3,whichwereusedtosilencetheexpressionofSOCS1and3duringthedifferentiationofpreadipocytesandthenmeasuredtheexpressionofPPARγandC/EBPα.Inductionofpreadipocytedifferentiationwithacocktailofsteroidsisaclassicmethod,hashighefficiencyandcanbecompletedwithin1week.AsshowninthestudyofNtambietal.(16),theexpressionofPPARγandC/EBPαreachedahighlevelthreedaysafterdifferentiationinduction.Therefore,wemeasuredexpressionofPPARγandC/EBPαatthissametimepoint.OurresultsshowedthattherewaspositivecorrelationbetweensilencingoftheSOCS1/3geneandtheexpressionofPPARγandC/EBPαandthatsilencingoftheSOCS1/3