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RESEARCHARTICLEOpenAccessLongnoncodingRNAexpressionprofileinfibroblast-likesynoviocytesfrompatientswithrheumatoidarthritisYuZhang1,Yu-ZhongXu1,NingSun1,Jian-HongLiu1,Fang-FangChen1,Xiao-LongGuan1,AngLi1,FeiWang1,Qin-FeiZhao1,Hai-YongWang1,Shu-ShengSong1,WeiYu1,Jian-NingZhao2andXiao-JunLi1,3*AbstractBackground:LongnoncodingRNAs(lncRNAs)haverecentlyreceivedwideattentionaskeymoleculesthatmediateavarietyofphysiologicalandpathologicalprocessesbyregulatinggeneexpression;however,knowledgeoflncRNAsinrheumatoidarthritis(RA)islimited.
Thus,weinvestigatedthelncRNAexpressionprofileinfibroblast-likesynoviocytes(FLSs)frompatientswithRAandexploredthefunctionofabundantlyexpressedlncRNAs.
Methods:LncRNAandmRNAmicroarrayswereperformedtoidentifydifferentiallyexpressedlncRNAsinRAFLSscomparedwithnormalFLSs.
Quantitativepolymerasechainreaction(qPCR)wasusedtovalidatetheresults,andcorrelationanalysiswasusedtoanalyzetherelationshipbetweentheseaberrantlyexpressedlncRNAsandclinicalcharacteristics.
Areceiveroperatingcharacteristic(ROC)curvewasconstructedtoevaluatethediagnosticvalueofthelncRNAsidentified.
Results:Accordingtothegeneexpressionprofiles,135lncRNAsweredifferentiallyexpressedbetweenRAandnormalFLSs.
Furthermore,qPCRdatashowedthatlncRNAENST00000483588wasup-regulatedandthatthreelncRNAs(ENST00000438399,uc004afb.
1,andENST00000452247)weredown-regulatedinRAFLSs.
TheexpressionlevelofENST00000483588waspositivelycorrelatedwiththelevelofC-reactiveproteinandtheSimplifiedDiseaseActivityIndexscore.
Moreover,theareasundertheROCcurvewere0.
85,0.
92,0.
97,and0.
92forENST00000483588,ENST00000438399,uc004afb.
1,andENST00000452247,respectively.
Conclusions:TheresultsindicatethatthedysregulationofENST00000483588,ENST00000438399,uc004afb.
1,andENST00000452247maybeinvolvedinthepathologicalprocessesofRAandthattheselncRNAsmayhavepotentialvalueforthediagnosisandassessmentofthediseaseactivityofRA.
Keywords:Rheumatoidarthritis,Fibroblast-likesynoviocytes,LongnoncodingRNA,ExpressionprofileBackgroundRheumatoidarthritis(RA)isasystemicautoimmunediseasecharacterizedbychronicjointinflammationandvariabledegreesofboneandcartilagedestruction.
ThethickenedsynoviuminRAmainlyincludestwotypesofcells:macrophage-likesynovialcellsandfibroblast-likesynovialcells(FLSs).
AskeyeffectorcellsinRA,FLSshaveattractedincreasingattention[1].
ThemammaliangenomeencodesthousandsofnoncodingRNAs(ncRNAs).
Overthepastfewdecades,mostoftheresearchatten-tioninthisfieldhasbeendevotedtoexploringtheroleofsmallncRNAssuchasmicroRNAs(~21–25nucleo-tides),whichregulategeneexpressionatthetranscrip-tionalandpost-transcriptionallevels.
MicroRNA-124awasfoundtobeakeyregulatoroftheproliferationandsecretionofmonocytechemoattractantprotein1inRAFLSs[2].
Inaddition,microRNA-18awasreportedtoactivateFLSsthroughafeedbackloopinnuclearfactor-kappaBsignaling[3].
Moreover,anovelp53/micro-RNA-22/cyr61axisinsynovialcellswasreportedtoplay*Correspondence:xiaojunli62@126.
com1DepartmentofClinicalLaboratoryScience,JinlingHospital,SchoolofMedicine,NanjingUniversity,305EastZhongshanRoad,Nanjing210002,China3StateKeyLaboratoryofAnalyticalChemistryforLifeScience,DepartmentofChemistry,NanjingUniversity,Nanjing210093,ChinaFulllistofauthorinformationisavailableattheendofthearticle2016TheAuthor(s).
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Zhangetal.
ArthritisResearch&Therapy(2016)18:227DOI10.
1186/s13075-016-1129-4aroleinregulatinginflammationinRA[4].
Recently,longnoncodingRNAs(lncRNAs),whichhadpreviouslybeenthoughttobenonfunctionalRNAs,wereshowntoplayimportantrolesinchromatinremodeling,transcrip-tioncontrol,post-transcriptionalprocessing,andproteinmetabolism.
LncRNAshavealsobeenfoundtoplayimportantrolesinmanydiseasessuchascancer[5,6],Alzheimer'sdisease[7],cardiovasculardisease[8],dia-betesmellitus[9],andsystemiclupuserythematosus[10].
However,knowledgeoflncRNAsinRAFLSsremainslimited.
Inthisstudy,anlncRNAexpressionprofileforRAwasestablished,andtherelationshipsbetweentheexpressionlevelsofaberrantlyexpressedlncRNAsandclinicalindiceswereanalyzed.
Moreover,weexploredthevalueoftheselncRNAsindiagnosingorassessingRAdiseaseactivity.
MethodsPatientsandspecimensRAsynovialtissuespecimenswereobtainedfromthekneejointsof10patientswithRA,whowereundergo-ingtotalkneearthroplasty(twomenandeightwomen,agerange38–65years).
Inaddition,normalsynovialtis-suespecimenswereobtainedfromthekneejointsof10patientswithtrauma,whowereundergoingarthroscopicsurgeryforkneeligamentinjuryormeniscusinjury(threemenandsevenwomen,agerange35–61years).
TheclinicalcharacteristicsofthepatientsareshowninAdditionalfile1:TableS1.
AllpatientswithRAincludedinthestudyfulfilledthe2010AmericanCollegeofRheumatology/EuropeanleagueAgainstRheumatism(ACR/EULAR)classificationcriteriaforRA[11].
Writ-teninformedconsentwasobtainedfromeachpartici-pantpriortosamplecollection.
ThestudywasapprovedbytheHumanEthicsCommitteeofJinlingHospital.
IsolationandcultureofFLSsTissuespecimensweremincedintosmallpiecesanddigestedfor2hourswith2mg/mloftypeIIcollagenase(Invitrogen,Carlsbad,CA,USA)inhigh-glucoseDulbecco'smodifiedEagle'smedium(DMEM)at37°C[12].
Aftercen-trifugationat210*gfor5minutes,theprecipitatewasresuspendedwith1mlofhigh-glucoseDMEMcontaining10%fetalbovineserum(FBS),100units/mlpenicillin,and100units/mlstreptomycin,andthenculturedin25-cm2cellcultureflasks(Corning)inahumidified5%CO2incu-bator.
After10hours,4mlofhigh-glucoseDMEMcon-taining10%FBSwasaddedtothecellcultureflask.
Allexperimentswereconductedusingcellsatpassage3.
FlowcytometryFLSsatpassage3wereidentifiedbyflowcytometrybasedontheexpressionofCD68(amacrophagemarker)andCD90(afibroblastmarker)[13].
Cellswerewashedthreetimeswithphosphate-bufferedsaline(PBS)andwerethenincubatedwithfluoresceinisothiocyanate(FITC)-conjugatedanti-CD68antibody,phycoerythrin(PE)-conjugatedanti-CD90antibody,FITC-conjugatedmouseIgG2b,orPE-conjugatedmouseIgG1(MiltenyiBiotec,Germany)for20minutesinthedark.
CellswerewashedwithPBSandthenanalyzedonaFACSCaliburflowcytometer(BDBiosciences,SanDiego,CA,USA).
MicroarrayanalysisSamplelabelingandarrayhybridizationwereperformedaccordingtotheAgilentOne-ColorMicroarray-BasedGeneExpressionAnalysisprotocol(AgilentTechnology).
Briefly,RNAwaspurifiedusingtheRNeasyMiniKit(Qiagen,Germany).
Eachsamplewasthenamplifiedandlabeledwithcyanine-3-CTP.
ThelabeledcRNAswerepurifiedagainwiththeRNeasyMiniKit.
TheproductionofcRNAsneededtoreach1.
65μgtomeettherequire-mentsofthemicroarray.
Thespecificactivityofthela-beledcRNAsneededtoreach9.
0pmolCy3/μgcRNA.
RNAquantityandqualityweremeasuredaccordingtotheA260nm/A280nmratiousingaNanoDropND-1000spectrometer.
RNAintegritywasdetectedbystandarddenaturingagarosegelelectrophoresis.
Foreachmicro-array,0.
6μgcRNA,5μlof10*blockingagent,1μlof25*fragmentationbuffer,andnuclease-freewaterwereaddedtoreachatotalvolumeof25μl:25μlof2*GEHybridizationBufferwasthenaddedtostopthefragmen-tationreaction.
ThehybridizationsolutionandArraystarHumanLncRNAMicroarrayV3.
0wereincubatedat65°Cfor17hoursinanAgilentHybridizationOven.
Ap-proximately30,586lncRNAsand26,109codingtran-scriptscanbedetectedusingthethird-generationlncRNAmicroarray.
Afterwashingthechip,amicroarrayscanner(AgilentDNAMicroarrayScanner)wasusedtomeasurethefluorescenceintensity.
AgilentFeatureExtractionSoftwarewasusedtoanalyzetherawdata.
VolcanoplotsandhierarchicalclusteranalysesThemicroarraydatawerelog-transformedandnormalizedusingquantilenormalization.
Afterfilteringtoremoveunreliabletranscripts,theremainingdatawerestatisticallyanalyzedtoidentifylncRNAsandmRNAswithsignifi-cantlydifferentialnormalization.
Volcanoplotsareusefultoolsforvisualizinggenesexpresseddifferentiallybetweentwogroups.
Transcriptsweredistributedaccordingtostat-isticalsignificance(y-axis)andthemagnitudeofchange(log2ratioofRAFLSs/normalFLSs)(x-axis).
HierarchicalclusteranalysiswasusedtoidentifydistinguishableRNAexpressionprofilesbetweendifferentsamples.
LncRNAclassificationAnalyzingthegenomiccontextoflncRNAscanhelptopredicttheirfunctionalroles.
AccordingtothesequenceZhangetal.
ArthritisResearch&Therapy(2016)18:227Page2of10andrelativepositionbetweenlncRNAsandtheirassoci-atedprotein-codinggenes,thelncRNAsdetectedbymicroarraywerecharacterizedasnaturalantisense,in-tronicantisense,exonsenseoverlapping,intronsenseoverlapping,andbidirectional,andintergenic,amongothers[14].
NaturalantisenselncRNAsareRNAmole-culesthataretranscribedfromtheantisensestrandandoverlapwithcodingtranscripts.
IntronicantisenselncRNAsareRNAmoleculesthataretranscribedfromtheantisensestrand,butdonotshareoverlappingexons.
ExonsenseoverlappinglncRNAscanbeconsideredastranscriptvariantsofprotein-codingmRNAs,astheyoverlapwithcodingtranscriptsfromthesamegenomicstrand.
IntronsenseoverlappinglncRNAsoverlapwiththeintronsofannotatedcodinggenesonthesamegen-omicstrand.
AbidirectionallncRNAisorientedhead-to-headwithaprotein-codinggenewithin1000bp.
IntergeniclncRNAsinvolvenooverlappingorbidirec-tionalcodingtranscriptsnearthelncRNAs.
The"others"categorymainlyincludedlncRNAsforwhichtheassoci-atedgeneswerepseudogenes.
BioinformaticsanalysisPathwayanalysisandGeneOntology(GO)analysiswereappliedtoexplorethepotentialrolesthatthedifferentiallyexpressedmRNAsplayinabiologicalpathwayorGOfunction,includingthreecategories:biologicalprocess,cellularcomponent,andmolecularfunction.
Co-expressionnetworkAgeneco-expressionnetworkwasbuiltaccordingtothenormalizedsignalintensityofspecificallyexpressedlncRNAsandmRNAsusingtheCytoscapeprogram.
Pearsoncorrelationanalysiswasusedtoevaluatethesig-nificanceofthecorrelationbetweentheexpressionlevelsbetweeneachpairofgenes.
Pearsoncorrelationcoeffi-cientswereselectedforinclusioninthenetworkwhentheywereabove0.
95.
ByanalyzingthefunctionofthesemRNAs,wecouldlinklncRNAswithparticularfunc-tionsandsignalingpathways,whichcouldfacilitatethepredictionoftheirbiologicalfunctionsandmechanismsofaction.
Iftheareaofthenodeincreasedwithincreas-ingconnectionsbetweenmRNAsandlncRNAs,thisindicatedthatthelncRNAmayplayanimportantrole.
Quantitativepolymerasechainreaction(qPCR)analysisTheprimersusedforqPCRweresynthesizedbyKangcheng(Shanghai,China).
EachPCRcontained2μltemplatecDNA,10μl2*SYBRGreenmix(TaKaRa,Japan),1μlforwardprimer,1μlreverseprimer,andnuclease-freewatertoatotalvolumeof20μl.
ThePCRconditionswereasfollows:95°Cfor10minutes,followedby40cyclesof95°Cfor10secondsand60°Cfor60sec-onds.
GAPDHmRNAwasusedasanendogenouscontroltonormalizelncRNAexpressionlevelsusingthe2-Ctmethod.
Theprimersequencesusedinthisstudywereasfollows:forGAPDH,5′-GGGAAACTGTGGCGTGAT-3′(forward)and5′-GAGTGGGTGTCGCTGTTGA-3′(re-verse);forENST00000483588,5′-CACGTGAAAGGGGGAGAAA-3′(forward)and5′-CCAACAGCACAGAAGGCGT-3′(reverse);forNR_073012,5′-ATTCCCACGTATGCGGAGTG3′(forward)and5′-ACGGGTGTAGTAGGCGTTTC-3′(reverse);forENST00000567753,5′-CGTGGCAGGACTTTGCTTTC-3′(forward)and5′-GGGGTCTTACTGTGTGGGGT-3′(reverse);foruc003xhp.
3,5′-AGGTATCAGTCTGGGGGACC-3′(forward)and5′-ACGCACAGTGGAGGAATGAG-3′(reverse);forENST00000557804,5′-CAGAACGATCAAGCGACCTC-3′(forward)and5′-CGTGGAAGGAAGATGACCC-3′(reverse);forENST00000438399,5′-AGAAAGTCAAGGGAAGATAAGG-3′(forward)and5′-TAGTCAACCAGGGAAGCAGT-3′(reverse);foruc004afb.
1,5′-AAACTATGCCTGATTTGTGGTC-3′(forward)and5′-CTGGTGTAAGAGCATGGGGT-3′(reverse);forENST00000412143,5′-ATTACTCTTTCCCAGCCCAGC-3′(forward)and5′-GCCCTCCTTGCAGCATCAT-3′(re-verse);forENST00000452247,5′-GACTCCTCCTCCTGCTTTCAC-3′(forward)and5′-GGCAATAGAGCTGGATCTTGT-3′(reverse).
StatisticalanalysisSPSSsoftware19.
0andGraphPadPrism6.
0wereusedtoanalyzethedata.
Thetwo-tailedStudentttestandrank-sumtestwereusedasappropriatetoanalyzetheexpressionlevelsbetweentwogroups.
Therelation-shipsbetweentheexpressionlevelsoflncRNAsandclinicalcharacteristicswereanalyzedbyPearson'scor-relationcoefficient.
Pvaluesbelow0.
05wereregardedasstatisticallysignificant.
Thediagnosticvaluewasevaluatedwithareceiveroperatingcharacteristic(ROC)curve.
ResultsIdentificationofFLSsThepurityofFLSsatpassage3wasdeterminedbyflowcytometry.
ThepurityofthethirdgenerationofFLSsoftheprimaryculturereached99.
0%(Fig.
1).
LncRNAandmRNAexpressionprofileinRAFLSsWeperformedgenome-wideanalysisoftheexpressionprofilesoflncRNAsandmRNAsinthreepairsofFLSssamplesfrompatientswithRAandpatientswithtraumausingArraystarHumanLncRNAMicroarrayV3.
0.
Basedonthecriteriaofafoldchange>2.
0andaPvalue<0.
05,135lncRNAsand103mRNAsweredifferentiallyexpressedbetweentheRAFLSsandnormalFLSs(Additionalfiles2and3:TablesS2andS3).
Ofthe135lncRNAsidentified,62lncRNAswereup-regulatedZhangetal.
ArthritisResearch&Therapy(2016)18:227Page3of10and73lncRNAsweredown-regulatedintheRAFLSs.
Ofthe103mRNAsdetected,36mRNAswereup-regulatedand67mRNAsweredown-regulatedintheRAFLSs.
VolcanoplotsandhierarchicalclusterTovisualizethedifferentiallyexpressedlncRNAsandmRNAs,volcanoplotanalysiswasconductedtofurtherexplorethedifferencebetweentheRAFLSsandnormalFLSs(Fig.
2aandb).
Thedatashowedthat135(0.
44%)ofthelncRNAsand103(0.
39%)ofthemRNAsweresignificantlydifferentiallyexpressed.
Theresultsofhier-archicalclusteranalysesshoweddistinguishablelncRNAandmRNAexpressionprofilesbetweentheRAFLSsandnormalFLSs(Fig.
2candd).
PathwayandGOanalysesPathwayandGOanalyseswereappliedtoexplorethepotentialrolesthatthedifferentiallyexpressedmRNAsmightplayinRA.
Pathwayanalysisshowedthatthedif-ferentiallyexpressedmRNAsinRAFLSswererelatedwithmTOR,HIF-1,Ras,proteoglycans,andapoptosissignalingpathways,amongothers(Additionalfile4:FigureS1).
ThesemRNAswerefurthercharacterizedasbeingmainlyinvolvedinregulatingbiologicalprocessesrelatedtocellgrowth,vascularpermeability,andcellactivation(Additionalfiles5and6:FiguresS2andS3).
ClassificationoflncRNAsOfthe62up-regulatedlncRNAsidentified,weobserved4(6.
5%)naturalantisense,10(16.
1%)intronicanti-sense,4(6.
5%)exonsenseoverlapping,1(1.
6%)intronsenseoverlapping,5(8.
1%)bidirectional,35(56.
5%)intergenic,and3(4.
8%)otherlncRNAs(Fig.
3a).
Ofthe73down-regulatedlncRNAsidentified,weobserved8(11.
0%)naturalantisense,8(11.
0%)intronicantisense,8(11.
0%)exonsenseoverlapping,3(4.
1%)intronsenseoverlapping,1(1.
4%)bidirectional,43(58.
9%)intergenic,and2(2.
7%)otherlncRNAs(Fig.
3b).
qPCRvalidationConsideringtheobservedfoldchanges,thecalculatedPvalues,andtheprimerspecificities,weselectedthefollowingninelncRNAsforfurthervalidationofexpres-sionlevelsbyqPCR:ENST00000483588,NR_073012,ENST00000567753,uc003xhp.
3,ENST00000557804,ENST00000438399,uc004afb.
1,ENST00000412143,andENST00000452247(Table1).
TheqPCRresultsconfirmedthatENST00000483588wasoverexpressedintheRAFLSs,whereastheexpressionlevelsofENST00000438399,uc004afb.
1,andENST00000452247weredecreasedintheRAFLSs(Fig.
4).
ThesefourlncRNAsshowedsimilartrendsinqPCR,asdeterminedbymicroarray.
Neverthe-less,somelncRNAsshowednosignificantdifferencebetweenRAFLSsandnormalFLSsinqPCRincontrasttotheresultsdeterminedinthemicroarray.
CorrelationbetweentheaberrantlyexpressedlncRNAsandclinicalcharacteristicsofpatientswithRAWefurtheranalyzedthecorrelationbetweentheex-pressionlevelsofthelncRNAsENST00000483588,ENST00000438399,uc004afb.
1,andENST00000452247andclinicalcharacteristics(erythrocytesedimentationrate(ESR),C-reactiveprotein(CRP)expression,SimplifiedDiseaseActivityIndex(SDAI)score,andage).
Theexpres-sionlevelofENST00000483588waspositivelycorrelatedwiththeCRPlevel(r=0.
74,P<0.
05)andtheSDAIscore(r=0.
79,P<0.
01)(Fig.
5).
However,noneoftheselncRNAsweresignificantlycorrelatedwithESRorage.
DiagnosticvalueoffourselectedlncRNAsThepotentialdiagnosticvalueoffourlncRNAsforRAwasevaluatedusingpatientswithtraumapatientsascontrolsubjects.
TheareaundertheROCcurvefortheENST00000483588,ENST00000438399,uc004afb.
1,andENST00000452247lncRNAswas0.
85,0.
92,0.
97,and0.
92,respectively,indicatingpotentialdiagnosticvalueforRA(Fig.
6).
LncRNAandmRNAco-expressionnetworkAnlncRNAandmRNAco-expressionnetworkwascon-structedbasedonthecorrelationanalysisbetweenthedifferentiallyexpressedlncRNAsandmRNAs.
Wefur-therfocusedonco-expressionnetworkscenteringonENST00000483588,ENST00000438399,uc004afb.
1,andENST00000452247.
Theco-expressionnetworksshowedFig.
1Identificationoffibroblast-likesynoviocytes(FLSs)byflowcytometryZhangetal.
ArthritisResearch&Therapy(2016)18:227Page4of10thatENST00000483588expressionwaspositivelycorre-latedwithATAD3AandNDUFA4L2mRNAexpressionlevels(Additionalfile7:FigureS4),ENST00000438399expressionwaspositivelycorrelatedwithPTPRQmRNAexpressionandnegativelycorrelatedwithPTHLHmRNAexpression(Additionalfile8:FigureS5),uc004afb.
1expressionwaspositivelycorrelatedwithTNFRSF11BmRNAexpression(Additionalfile9:FigureS6),andENST00000452247expressionwaspositivelycorre-latedwithZNF154mRNAexpressionandnegativelycorrelatedwithWISP3mRNAexpression(Additionalfile10:FigureS7).
DiscussionTodate,expressionprofilestudiesofcellsandtissueshavemainlyfocusedonmRNAsandmicroRNAs.
RecentadvancesinthedepthandqualityoftranscriptomesequencinghaverevealedanincreasingnumberofdistinguishablyexpressedlncRNAsinvariousdiseases.
AlthoughseveralfindingshaveimplicatedlncRNAsinthedevelopmentandprogressionofvariousdiseases,researchonlncRNAsrelatedtorheumaticdiseasesislimited.
Liuetal.
[15]demonstratedthatlncRNA-CIRexpressioninchondrocytespromotedextracellularmatrixdegradationbyaffectingtheexpressionofcolla-gen,aggrecan,andmatrix-degradingenzymes,andplaysanimportantroleinthepathogenesisofosteoarthritis.
Furthermore,studiesoflncRNAsinTcells[16]andmonocytes[17,18]frompatientswithRAhavebeenconducted.
TheaimofourstudywastoexplorelncRNAexpressioninRAFLSstoprovidenewinsightintothepathogenesisofRA.
AschematicdiagramofouroverallstudydesignandmainfindingsisprovidedinAdditionalfile11:FigureS8.
WeanalyzedthreeRAFLSsamplesandthreenormalFLSsamplesusinglncRNAandmRNAmicroarrays.
Fig.
2Volcanoplotandheatmapanalysesofrheumatoidarthritis(RA)fibroblast-likesynoviocyte(FLS)samplesandnormalFLSsamples.
a,blncRNA(a)andmRNA(b)volcanoplotsofRAFLSsversusnormalFLSs(CONT).
Eachsquarerepresentsadifferenttranscript.
Redsquaresrepresentgenesthatpassedthestatisticalandfold-changecutoffs.
c,dlncRNA(c)andmRNA(d)heatmapsshowingdistinguishablelncRNAexpressionprofilesbetweenpatientswithRAandpatientswithtrauma.
Eachcolumnindicatesadifferentsample.
EachrowindicatesonemRNAorlncRNA.
RelativelyhighexpressionisindicatedbyredshadingandrelativelylowexpressionisindicatedbygreenshadingZhangetal.
ArthritisResearch&Therapy(2016)18:227Page5of10Basedonthemicroarraydata,wefound135lncRNAsand103mRNAsthatweredifferentiallyexpressed.
MostoftheselncRNAshavenotbeenfunctionallycharacterized,whereasmostoftheidentifiedmRNAsarewell-known.
Therefore,bioinformaticsanalysisoftheaberrantlyexpressedmRNAswasconductedtohelpbetterunder-standthepotentialroleofFLSsinthepathologicalprocessofRAandspeculateontheputativefunctionofthedifferentiallyexpressedlncRNAs,sincepreviousreportshaveshowedthatlncRNAsparticipateinawidevarietyofpathologicalprocessesbyregulatinggeneexpressionatthelevelsofchromatinremodeling,tran-scriptionalcontrol,andpost-transcriptionalprocessing.
GOandpathwayanalysesshowedthatthedifferentiallyexpressedmRNAsmainlyrelatedtoregulationofgrowth,vascularpermeability,andcellactivationprocessesthatareclearlyassociatedwithRApathogenesis[19–21].
WeusedqPCRtovalidatethelncRNAmicroarrayre-sults.
BasedontheqPCRresults,ENST00000483588,ENST00000438399,uc004afb.
1andENST00000452247weredifferentiallyexpressed,whichwasinagreementwiththemicroarrayresults.
Nevertheless,otherlncRNAsdidnotdiffersignificantlyinRAFLSsandnormalFLSsonqPCR,incontrasttotheresultsfromthemicroarray.
Thedifferenttrendsarelikelyduetothefactthattheex-pandedtestsamplesizefortheqPCRmighthaveexcludedFig.
3Classificationanalysesofup-regulatedlncRNAsa(Up-regulation)anddown-regulatedlncRNAsb(Down-regulation)inrheumatoidarthritisfibroblast-likesynoviocytes,asdetectedbymicroarrayanalysisTable1NinedifferentiallyexpressedlncRNAsinrheumatoidarthritisfibroblast-likesynoviocytes(FLSs)versusnormalFLSsdeterminedbymicroarraySequencenameGenesymbolPvalueFoldchangeRegulationENST00000483588C17orf76-AS1<0.
018.
91upNR_073012PRSS21<0.
013.
48upENST00000567753RP11-524C21.
2<0.
013.
38upuc003xhp.
3BC015784<0.
013.
21upENST00000557804AC068831.
10<0.
012.
97upENST00000438399RP11-534G20.
3<0.
0116.
41downuc004afb.
1AK096159<0.
017.
30downENST00000412143PSORS1C3<0.
015.
01downENST00000452247RP11-573I11.
2<0.
014.
22downFig.
4RelativeexpressionoflncRNAsusingqPCR.
aExpressionofENST00000483588,NR_073012,ENST00000567753,uc003xhp.
3,andENST00000557804inrheumatoidarthritis(RA)fibroblast-likesynoviocyte(FLS)samples(n=10)andnormalFLSsamples(n=10).
bExpressionofENST00000438399,uc004afb.
1,ENST00000412143,andENST00000452247inRAFLSsamples(n=10)andnormalFLSsamples(n=10).
Valuesarethemean±SEM;*P<0.
01forRAversusnormalFLSsamplesZhangetal.
ArthritisResearch&Therapy(2016)18:227Page6of10someofthefalsepositiveresultsobtainedinthemicroarray.
ENST00000483588isa689-bpintronicantisenselncRNAtranscriptfromtheC17orf76-AS1gene.
AsshowninTable1andFig.
4,ENST00000483588wassignificantlyup-regulatedinRAFLSscomparedwithnormalFLSs.
Nakayaetal.
[22]showedthatintronicantisenselncRNAsareenrichedintheintronsofgenesrelatedtoregulationoftranscription,andpotentiallyfunctionasregulatorsofalternativesplicing.
However,inthepresentstudy,theexpressionofFAM211A,thecodinggenecorrespondingtothelncRNAENST00000483588,wasnotsignificantlydifferentbetweentheRAFLSsandnormalFLSs.
ENST00000483588hasanantisenseorientationrelativetothecodinggene,andthetranscribedregionspartiallyoverlapwithoutexons.
Theco-expressionnetworkofENST00000483588showedthatENST00000483588ex-pressionwaspositivelycorrelatedwithATAD3AandNDUFA4L2mRNAexpression.
ATAD3Aencodesamito-chondrialmembraneproteinthathelpsstabilizelargemitochondrialDNA-proteincomplexes,knownasnucle-oids[23].
NDUFA4L2playsaroleinthebiologicaloxida-tionofmitochondria.
Thus,theroleofENST00000483588inmitochondrialfunctionmeritsfurtherstudy.
Interestingly,strongassociationsbetweenENST00000483588expressionandtheCRPlevelandSDAIscoreinpatientswithRAwerefound.
CRPisanimportantclinicalparameterthatiscommonlyusedasamarkerofdiseaseactivityinRA[11,24,25].
RecentstudieshaveshownthatCRPisnotonlyaproductoftheinflamma-toryresponsebutalsoplaysaproinflammatoryroleinRA.
CRPcanactivatecomplementsandinduceosteo-clastdifferentiation[26,27].
TheCRPlevelcanalsopro-videanindicationoftreatmentefficacy,withdecreasedCRPreflectingeffectivetreatmentagainstarthritis,andsustainedhighCRPreflectingapoortreatmentresponse[28,29].
TheSDAIisavalidandsensitivecompositeindextoassessdiseaseactivityanddefinecutoffvaluesrepresentingremissioninRA,asrecommendedbytheACRandEULAR[30].
ConsideringthehighdiagnosticvalueofENST00000483588,wespeculatethatthislncRNAmaybeinvolvedinthepathologicalprocessofRA.
Nevertheless,furtherstudiesareneededtoclarifytheunderlyingmechanisms.
IncontrasttoENST00000483588,theexpressionlevelofENST00000438399inRAFLSswasmarkedlylowerthanthatinnormalFLSs.
ENST00000438399isa3307-bpintergeniclncRNAtranscriptfromtheRP11-534G20.
3genelocatedonchromosome10:29698475–29713107.
Theco-expressionnetworkofENST00000438399showedthatENST00000438399expressionwaspositivelycorre-latedwithPTPRQmRNAandnegativelycorrelatedwithPTHLHmRNA.
PTPRQcaninhibitcellproliferation,andinduceapoptosis[31].
PTHLHcanpromotecellprolifera-tionandexertaprotectiveeffectagainstapoptosis;more-over,itsexpressioncorrelateswiththeseverityofcarcinoma[32].
Inthisstudy,themicroarrayanalysisshowedthattheexpressionlevelofPTPRQwasdown-regulated,whereastheexpressionlevelofPTHLHwasup-regulatedinRAFLSs.
Previousreportshavedemonstratedthatthehumangenomeencodesatleast3289longinter-genicnoncodingRNA,whichareevolutionarilyconservedandmaybeinvolvedindiversebiologicalprocesses,in-cludingcell-cycleregulation,immunesurveillance,andembryonicstemcellpluripotency[33,34].
WhetherENST00000438399interactswithPTPRQorPTHLHneedstobeverified,asdoesitsroleinregulatingthegrowthofRAFLSs.
Moreover,theexpressionlevelsofuc004afb.
1andENST00000452247alsodecreasedinRAFLSs.
uc004afb.
1isa2045-bpintergeniclncRNAtranscriptexpressedfromtheAK096159genelocatedonchromosome9:68,743,530-68,769,869.
Theco-expressionnetworkofuc004afb.
1showedthatuc004afb.
1waspositivelycorrelatedwithFig.
5CorrelationbetweenlncRNAsandtheclinicalcharacteristicsofpatientswithrheumatoidarthritis.
aCorrelationbetweenENST00000483588expressionandC-reactiveprotein(CRP).
bCorrelationbetweenENST00000483588expressionandtheSimplifiedDiseaseActivityIndex(SDAI)scoreZhangetal.
ArthritisResearch&Therapy(2016)18:227Page7of10TNFRSF11BmRNAexpression.
TNFRSF11B,whichen-codesosteoprotegerin,functionsasanegativeregulatorofboneresorption[35].
Itwasdown-regulatedintheRAFLSsinthemicroarrayresults.
ENST00000452247isan869-bpnaturalantisenselncRNAtranscriptexpressedfromtheRP11-573I11.
2gene.
Theco-expressionnetworkofENST00000452247showedthatENST00000452247ex-pressionwaspositivelycorrelatedwithZNF154mRNAexpressionandnegativelycorrelatedwithWISP3mRNAexpression.
ZNF154encodesaproteinthatbelongstotheKrüppelfamilyofzincfingertranscriptionalregulators,themembersofwhicharethoughttofunctioninnormalandabnormalcellgrowthanddifferentiation.
Hyperme-thylationandlowexpressionlevelsofZNF154havebeenconsideredpromisingbiologicalmarkersfortumoridenti-ficationandcancerrecurrencesurveillance[36].
AbnormalexpressionofWISP3hasbeendetectedinavarietyoftumors.
SilencingWISP3expressionsuppressescellproliferationandinducesapoptosisinbladdercancercells[37],andalsosuppressescellproliferationandmigra-tionandWntsignaling,andtheexpressionofadhesionmoleculesingastriccancercells[38].
Moreover,muta-tionsofthisgeneareassociatedwithprogressivepseudo-rheumatoiddysplasia[39,40].
HypomethylationandrelativelyhighexpressionofWISP3havebeendemon-stratedinapreviousstudy[41].
Inthisstudy,themicro-arrayanalysisshowedthattheexpressionlevelofWISP3wasup-regulatedinRAFLSs.
Thepathologicalphenom-enaofbonedestruction,angiogenesis,andabnormalpro-liferationofFLSsarecharacteristicsofRA.
Therefore,theresultsoftheco-expressionnetworksprovidecluesforfurtherresearchintothemolecularpathogenicmecha-nismsunderlyingRA.
ThelncRNAsuc004afb.
1andENST00000452247alsohadhighdiagnosticvalue,withareasundertheROCcurveof0.
965and0.
92,respectively.
Nevertheless,themolecularmechanismsofuc004afb.
1andENST00000452247inRArequirefurtherstudy.
ConclusionsInsummary,ourstudyprovidescomprehensivelncRNAandmRNAprofilesforRAFLSs.
Thedifferentialexpres-sionofENST00000483588,ENST00000438399,uc004afb.
1,andENST00000452247inRAFLSssuggeststhattheselncRNAsmayparticipateinthepathogenesisofRA.
TheROCcurveanalysisindicatedthattheselncRNAsmayalsohavediagnosticvalueforRA.
ExpandingtheRAFLSsam-plesandaddingplasmasampledetectionwouldhelptoclarifytheirsuitabilityinclinicaldiagnosis.
AlthoughtheseFig.
6Receiveroperatingcharacteristic(ROC)curves.
a–dROCcurvesforpatientsrheumatoidarthritis(RA)basedontheexpressionofENST00000483588(a),ENST00000438399(b),uc004afb.
1(c),andENST00000452247(d)inRAfibroblast-likesynoviocytes(FLSs)andnormalFLSsZhangetal.
ArthritisResearch&Therapy(2016)18:227Page8of10resultsarepreliminaryatthisstage,theyarevaluableinexpandingknowledgeontheroleoflncRNAsinrheumaticdiseases,andprovidingtargetsforfurtherresearch,specific-allyontheirlinktothepathogenesisofRA.
AdditionalfilesAdditionalfile1:TableS1.
Clinicalcharacteristicofpatientswithrheumatoidarthritis(RA)andtrauma.
(DOC45kb)Additionalfile2:TableS2.
DifferentiallyexpressedlncRNAsinRAFLSsversusnormalFLSs.
(DOC219kb)Additionalfile3:TableS3.
DifferentiallyexpressedmRNAsinRAFLSsversusnormalFLSs.
(DOC174kb)Additionalfile4:FigureS1.
Pathwayanalysisfordifferentiallyexpressed(DE)mRNAs.
aPathwayanalysisforup-regulatedmRNAs.
bPathwayanalysisfordown-regulatedmRNAs.
(TIF259kb)Additionalfile5:FigureS2.
Geneontology(GO)analysisforup-regulatedmRNAs.
aBiologicalprocess(BP)analysisforup-regulatedmRNAs.
bCellularcomponent(CC)analysisforup-regulatedmRNAs.
cMolecularfunction(MF)analysisforup-regulatedmRNAs.
(TIF432kb)Additionalfile6:FigureS3.
Geneontology(GO)analysisfordown-regulatedmRNAs.
aBiologicalprocess(BP)analysisfordown-regulatedmRNAs.
bCellularcomponent(CC)analysisfordown-regulatedmRNAs.
cMolecularfunction(MF)analysisfordown-regulatedmRNAs.
(TIF468kb)Additionalfile7:FigureS4.
Co-expressionnetworkofthedifferentiallyexpressedlncRNAsandmRNAs.
ENST00000483588wasconnectedto12lncRNAsand9mRNAs.
BluenodesrepresentlncRNAsandyellownodesrepresentprotein-codinggenes.
Aredlinerepresentsapositivecorrelation,andagreenlinerepresentsnegativecorrelation.
(TIF863kb)Additionalfile8:FigureS5.
Co-expressionnetworkofthedifferentiallyexpressedlncRNAsandmRNAs.
ENST00000438399wasconnectedto11lncRNAsand9mRNAs.
BluenodesrepresentlncRNAsandyellownodesrepresentprotein-codinggenes.
Aredlinerepresentspositivecorrelation,andagreenlinerepresentsnegativecorrelation.
(TIF756kb)Additionalfile9:FigureS6.
Co-expressionnetworkofthedifferentiallyexpressedlncRNAsandmRNAs.
uc004afb.
1wasconnectedto17lncRNAsand8mRNAs.
BluenodesrepresentlncRNAsandyellownodesrepresentprotein-codinggenes.
Aredlinerepresentspositivecorrelation,andagreenlinerepresentsnegativecorrelation.
(TIF889kb)Additionalfile10:FigureS7.
Co-expressionnetworkofthedifferentiallyexpressedlncRNAsandmRNAs.
ENST00000452247wasconnectedto18lncRNAsand12mRNAs.
BluenodesrepresentlncRNAsandyellownodesrepresentprotein-codinggenes.
Aredlinerepresentspositivecorrelation,andagreenlinerepresentsnegativecorrelation.
(TIF1529kb)Additionalfile11:FigureS8.
Schematicdiagramofthestudydesignandsummaryofmainfindings.
(TIF814kb)AbbreviationsACR/EULAR:AmericanCollegeofRheumatology/EuropeanleagueAgainstRheumatism;CRP:C-reactiveprotein;DMEM:Dulbecco'smodifiedEagle'smedium;bp:Basepair(s);ESR:Erythrocytesedimentationrate;FBS:Fetalbovineserum;FITC:Fluoresceinisothiocyanate;FLSs:Fibroblast-likesynoviocytes;GO:GeneOntology;lncRNA:LongnoncodingRNA;ncRNA:Non-codingRNA;PBS:Phosphate-bufferedsaline;qPCR:Quantitativepolymerasechainreaction;RA:Rheumatoidarthritis;ROC:Receiveroperatingcharacteristic;SDAI:SimplifiedDiseaseActivityIndexAcknowledgementsThisworkwassupportedbytheNationalScienceFoundationofChina(grants81470071and81171652),andtheClinicalScienceandTechnologyProjectsofJiangsuProvince(grantBL2014072).
AvailabilityofsupportingdataAllofthedatafromthemicroarraytrialsarestoredintheGeneExpressionOmnibus(GEO)database[GEO:GSE83147].
Authors'contributionsYZconceivedanddesignedthestudy,performedthestatisticalanalyses,anddraftedthemanuscript.
YZXandNSparticipatedinthedesignofthestudy,contributedtotheanalysesoftheresults,anddraftedthemanuscript.
JHLparticipatedinthedesignofthestudy,assistedindatainterpretation,andrevisedthemanuscript.
FFCassistedwiththeflowcytometryassaysanddraftedthemanuscript.
XLGandALcollectedthetissuesamples,registeredthepatientdata,andhelpeddraftthemanuscript.
FWandQFZmaintainedtheFLSs,verifiedtheirpurity,andhelpeddraftthemanuscript.
HYWparticipatedintheRNAextractionandqPCRs,andhelpeddraftthemanuscript.
SSSparticipatedinthestatisticalanalysisandhelpedrevisethemanuscript.
WYandJNZhelpedindesigningthestudyandrevisingthemanuscript.
XJLconceivedanddesignedthestudy,contributedtodataanalyses,andrevisedthemanuscript.
Allauthorsreadandapprovedthemanuscript.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
ConsentforpublicationAllpatientsprovidedwrittenconsentforpublication.
EthicsapprovalandconsenttoparticipateThestudywasapprovedbytheEthicsCommitteeofJinglingHospitalwiththefollowingreferencenumber:2014GJJ-023.
Writteninformedconsentwasobtainedfromeachparticipantpriortosamplecollection.
Authordetails1DepartmentofClinicalLaboratoryScience,JinlingHospital,SchoolofMedicine,NanjingUniversity,305EastZhongshanRoad,Nanjing210002,China.
2DepartmentofOsteology,JinlingHospital,SchoolofMedicine,NanjingUniversity,305EastZhongshanRoad,Nanjing210002,China.
3StateKeyLaboratoryofAnalyticalChemistryforLifeScience,DepartmentofChemistry,NanjingUniversity,Nanjing210093,China.
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