Circular RNA circNHSL1 promotes gastric cancer progression through the miR13063p/SIX1/vimentin axis | SpringerLink

CircularRNAcircNHSL1promotesgastriccancerprogressionthroughthemiR-1306-3p/SIX1/vimentinaxis|SpringerLink
AbstractBackground

MountingevidencesindicatethatcircularRNAs(circRNAs)playvitalrolesinthedevelopmentandprogressionofvariouscancers.However,thedetailfunctionsandunderlyingmechanismsofcircRNAsingastriccancerremainlargelyunknown.

Methods

Theexpressionprofileofmetastasis-relatedcircRNAswasscreenedbyRNA-seqanalysis.qRT-PCRwasusedtodeterminethelevelandprognosticvaluesofcircNHSL1ingastriccancertissues.Invitrocellwoundhealingandtranswell(migrationandinvasion)andinvivotumOrigenesisandmetastasisassayswereperformedtoevaluatethefunctionsofcircNHSL1.Luciferasereporter,RNAimmunoprecipitation(RIP)andrescuedassayswereemployedtoconfirmtheinteractionsbetweencircNHSL1,miR-1306-3pandSIX1.It’swidelyacceptedthatasamesenchymalMarker,Vimentinpromotesinvasionandmetastasisinvariouscancers.LuciferasereporterassaywasusedtodeterminetheregulationofSIX1onVimentin.Inaddition,Insituhybridization(ISH)wasperformedtodetectthelevelandprognosticvaluesofmiR-1306-3p.

Results

WefoundthatthelevelofcircNHSL1wassignificantlyup-regulatedingastriccancer,andpositivelycorrelatedwithclinicopathologicalfeaturesandpoorprognosisofpatientswithgastriccancer.Functionally,circNHSL1promotedcellmobilityandinvasion,aswellasinvivotumorgenesisandmetastasis.MechaNISTically,circNHSL1actedasamiR-1306-3pspongetorelievetherepressiveeffectofmiR-1306-3ponitstargetSIX1.Moreover,SIX1enhancedVimentinexpressioninthetranscriptionallevelthroughdirectlybindingtothepromoterdomainofVimentin,therebypromotingcellmigrationandinvasion.Inaddition,miR-1306-3pwasdown-regulatedandnegativelycorrelatedwithpathologicalfeaturesandpoorprognosisingastriccancer.

Conclusions

CircNHSL1promotesgastriccancerprogressionthroughmiR-1306-3p/SIX1/Vimentinaxis,andmayserveasanoveldiagnosticmarkerandtargetfortreatmentofgastriccancerpatients.

Background

Gastriccanceristhefifthmostcommoncancerandthethirdmajorcauseofcancer-relateddeathsworldwide[1].Directinfiltration,hematogenousmetastasis,transcoelomicspreadandlymphaticmetastasisarethemainpathwaysofmetastasisforgastriccancer,whicharetheleADIngcausesofpoorprognosisforpatientsintheadvancedstage.Despiteofmanyadvancementsindiagnosesandtreatmentsforgastriccancer,mostpatientsarefoundtobeintheadvancedstageassoonasbeingdiagnosed,andtheoverall5-yearsurvivalrateisstilllessthan30%[2,3].Hence,itisextremelyurgenttoelucidatethemolecularmechanismsunderlyinggastriccancerdevelopmentandprogressionanddiscovernovelmoleculartargetsforearlydiagnosesandtreatments.

CircularRNAs(circRNAs)haveemergedasanewtypeofendogenousnon-codingRNA[4,5]andarecharacterizedbyacontinuouscovalentlyclosedloopwithabacksplicesitebetween5′-and3′-end,whichisdifferentfromtheformationoflinearRNA[6,7,8].ThecircularstructureofcircRNAsisresponsIBLefortheirstableexistenceandhighabundanceindifferentspecies.Previousstudieshaveprovedtheirbroadlyevolutionaryconservation,abundantpresenceinexosomesandplasma,tissueandspace-timespecificity[9,10].Thereby,circRNAshavegreatpotentialasdiagnoses,treatmentsandprognosisbiomarkersforvariousdiseases,especiallycancers.Withthedevelopmentofhigh-throughputsequencingtechniqueandbioinformaticsanalysis,multiplecircRNAswerediscoveredandconfirmedtobeinvolvedindiversifiedBIOLOGicalprocesses,suchascellcycle,proliferation,apoptosis,autophagy,migrationandinvasion,indifferenttypesofcancers[11,12,13,14,15].Inrecentyears,circRNAshavebeenidentifiedtoplayrolesinseveralmolecularmechanisms,suchasspongingmicroRNAs(miRNAs),proteintranslationandbindingtoRNA-bindingproteins[16,17],ofwhichmiRNAspongeisthemostcommonrolesofcircRNAsinthedevelopmentandprogressionoftumors.DespiteseveralcircRNAshavebeenreportedingastriccancer,fewtumormetastasis-relatedcircRNAsandtheirfunctionsandmolecularmechanismshavebeenclearlyelucidated.

Asamemberofthehomeoboxgenefamily,Sineoculishomeoboxhomolog1(SIX1)encodesahomeodomain-containingtranscriptionfactor[18,19].EctopicexpressionofSIX1ubiquitouslyexistsinnumeroustypesofcancerandplaysimportantrolesinbothtumorinitiationandprogression[20,21].TheoverexpressionofSIX1correlateswithadvancedclinicopathologicalcharacteristicsandpoorprognosisinesophagealcancer,colorectalcancerandhepatocellularcarcinoma[22,23,24].IthasbeenreportedthatSIX1enhancesTGFβsignalingandtranscriptionallypromotesVEGF-Cexpression,therebypromotinglymphangiogenesisandmetastasisofcervicalsquamouscarcinoma[25].SIX1couldtranscriptionallysuppresstheexpressionofmiR-200familyandpost-transcriptionallypromoteZEB1expression,consequentlyrepressingE-cadherinexpressionandpromotingepithelial-mesenchymaltransition(EMT)incolorectalcancer[26].Inaddition,SIX1decreasestheexpressionofp53throughacompetitivemechanisminvolvingsimultaneousdownregulationofribosomalproteinL26(RPL26)andupregulationofmiR-27a-3pinbreastcancer[21].However,littleisknownaboutthefunctionsandregulatorymechanismsofSIX1ingastriccancer.VimentiniswidelyacceptedasamesenchymalbiomarkertopromoteEMTofvariouscancercells,therebypromotinginvasionandmetastasisinvitroandinvivo[27].BioinformaticsanalysisindicatedthatSIX1sharesthebindingsitesofthepromoterdomainofVimentin.

Inthisstudy,weidentifiedanovelmetastasis-relatedcircRNAcircNHSL1fromexonsoftheNHSlike-1(NHSL1),withacircBaseIDofhsa_circ_0006835.WefoundthatcircNHSL1wasup-regulatedinbothgastriccancertissuesandcelllines,andcorrelatedwithadvancedclinicalstage,distantmetastasis,lymphnodemetastasisandpoorprognosis.Importantly,circNHSL1promotedinvasionandmetastasisofgastriccancerbyactingasamiR-1306-3pspongetorelieveitsrepressionontargetSIX1.FurThermore,wefirstlydemonstratedthatSIX1enhancedtheexpressionofVimentinintranscriptionallevelbydirectlybindingtothepromoterdomainofVimentin.Collectively,ourdatashowthatcircNHSL1actsasanoncogenicgeneingastriccancerprogressionthroughmiR-1306-3p/SIX1/Vimentinaxis,andmayserveasanoveldiagnosticmarkerandtargetfortreatmentofgastriccancerpatients.

MethodsClinicalspecimensandethicalapproval

Therearetwogroupsofgastriccancerandadjacentnormaltissueswerecollected.Thefirstgroupof57pairedgastriccancerandmatchednormalsamplesfrom2013to2014wasobtainedfrompatientswithprimarygastriccancerduringsurgicalresectioninShanghaiGeneralHospitalandimmediatelyfixedinformalin.Thegroupofsampleswasembeddedinparaffintoconstructtissuemicroarray(TMA),andthefinalTMAcontained54pairedgastriccancersamples.Anothergroupoffreshfrozenspecimens(93pairs)wascollectedfrom2015to2017andstoredinliquidnitrogen.Noneofthepatientsreceivedradiotherapyorchemotherapybeforesurgery.AllclinicopathologicaldiagnoseswereconfirmedbytwopathologistsaccordingtotheguidelinesoftheUnionforInternationalCancerControl(UICC).ThepresentstudywasapprovedbytheEthicsCommitteeofShanghaiGeneralHospital.Writteninformedconsentwasobtainedfromallsubjectsbeforeenrollmentinthisstudy.

Celllinesandcultureconditions

Humangastriccancercelllines(MKN-28,AGS,MKN-45,BGC-823,MGC-803,HGC-27,SGC-7901)andnormalhumangastricepithelialcells-1(GES-1)wereobtainedfromTypeCultureCollectionoftheChineseAcademyofScience(Shanghai,China).AllcelllinesweremaintainedinRPMI1640mediumwith10%fetalbovineserum(FBS)(Gibco,GrandIsland,NY,USA)and1%penicillin-streptomycinat37 °Cinahumidifiedatmospherecontaining5%CO2.

Transfection,oligonucleotidesandplasmids

ToregulatecircNHSL1,miR-1306-3pandSIX1expression,oligonucleotidesandplasmidswereconstructed.ThefollowingsiRNAstargetingcircNHSL1weredesignedbyRiboBio(Guangzhou,China):si-circ-1target,5′-ACACAGCAAAGGATAAA-3′;si-circ-2target,5′-ACAGCAAAGGATAAAG-3′;andsi-circ-3target,5′-CAAAGGATAAAGATGGAAA-3′.FulllengthcircNHSL1wasclonedintothepEX-3(GenePharma,Shanghai,China)overexpressionvector.Themimics,inhibitorandnegativecontrolsforhsa-miR-1306-3pwerepurchasedfromRiBoBio(Guangzhou,China).TheshRNA-SIX1sequenceswereasfollows:5′-CCAGCTCAGAAGAGGAATT-3′(target),5′-CCAGCTCAGAAGAGGAATT-3′(sense),and5′-AATTCCTCTTCTGAGCTGG-3′(antisense).TheSIX1genewasclonedintopLVXplasmids(HarOLife,Shanghai,China).TheoligonucleotidesandplasmidsweretransfectedintocellswithLipofectamine™2000(Invitrogen,Carlsbad,CA,USA)accordingtothemanufacturer’sinstructions.

RNAextraction,nuclear-cytoplasmicfractionation,RNaseRtreatmentandquantitativereal-timePCR(qRT-PCR)

TotalRNAwasextractedfromgastriccancertissuesandcelllinesusingTRIzol(TaKaRa,Shiga,Japan)accordingtothemanufacturer’sinstructions.NuclearandcytoplasmicRNAfractionationwasisolatedwithPARIS™Kit(Invitrogen,USA)followingthemanufacturer’sinstruction.ForRNaseRtreatment,10 μgtotalRNAwasincubatedfor15 minat37 °Cwith40 URNaseR(EpicentreTechnologies,Madison,USA).ForcircRNAandmRNA,RNAwasreverselytranscribedintoCDNAusingaPrimeScript™RTMasterMixreagentkit(TaKaRa,Shiga,Japan).FormiRNA,cDNAwassynthesizedbythePrimeScript™RTreagentkit(TaKaRa,Shiga,Japan).RNAexpressionwasquantifiedbyqRT-PCRwithSYBRPremixExTaq™(TaKaRa,Shiga,Japan).GAPDHorU6wereusedasinternalcontrolsforcircRNA,mRNAormiRNA.The2-ΔΔCtmethodwasusedtoanalyzerelativeexpressionlevels.ThespecificprimerswerelistedinAdditional file 1:TableS1.

Proteinextractionandwesternblotting

Totalproteinwasextractedfromgastriccancercellsusingradioimmunoprecipitationassaybufferwith1%proteaseinhibitorphenylmethanesulfonyfluoride(BeyotimeBiotechnology,Jiangsu,China).BCAproteinassaykit(BeyotimeBiotechnology,Jiangsu,China)wasusedtomeasureproteinconcentrations.SDS-polyacrylamidegelelectrophoresis(SDS-PAGE)wasperformedwith30 μgoftotalproteinforeachsample.Then,theproteinwastransferredontopolyvinylidenefluoride(PVDF)membrane(Millipore,MA,USA).Next,themembranewasblockedin5%non-fatmilkatroomtemperaturefor1.5 h,andthenwasincubatedwithprimaryantibodyat4 °Covernight.Nextday,thesecondaryantibodywasincubatedatroomtemperaturefor1 h.EachProteinbandwasvisualizedbyECLchemiluminescentreagent(Millipore,MA,USA).Theantibodieswerelistedasfollows:SIX1(1:1000,CellSignalingTechnology,MA,USA);Vimentin(1:2000,Abcam,MA,USA);GAPDH(1:5000;CellSignalingTechnology,MA,USA).

Immunohistochemicalanalysis(IHC)

Afterdewaxingandrinsing,theTMAwasboiledin10 mMsodium-citratebufferfor5 mintoretrieveantigen.Then,theTMAwasblockedin3%hydrogenperoxidefor10 minincaseoftheinterventionofendogenousperoxidaseactivity.Thereafter,theTMAwasincubatedwiththeprimaryanti-SIX1antibody(1:200,CellSignalingTechnology,MA,USA)at4 °Covernight.Nextday,theTMAwasincubatedwithsecondaryantibodyatroomtemperaturefor30 min.Next,theTMAwasstainedwithDABandhematoxylin.Lastly,theTMAwascoveredwithcoverslipsformicroscopicobservation.

Insituhybridization(ISH)

TheTMAwasdealtwithProteinaseKfor10 minat37 °Cafterde-waxingandre-hydration,followedbyincubatingwithhybridizationmixfor1 hat57 °C.Then,theTMAwasblockedfor15 mininblockingsolutionandhybridizedwithdigoxigenin(DIG)-labeledmiR-1306-3pprobesat50 °Cfor16 h.Nextday,theTMAwastreatedwith0.5%blockingreagentfor30 minafterwashingtwice.Then,theTMAwasincubatedwithanti-DIGandhorseradishperoxidasefor2 hatroomtemperature,followedbywashingtwicewithTBSTanddehydrationwithxylene.Finally,theTMAwascoveredwithcoverslips.Thestainingscoreswereassessedbytwoindependentpathologistsblindedtotheclinicopathologicaldata.Thestainingscoreswerebasedontwoindicators:thestainingintensityandtheproportionofpositivelystainedcells.Thestainingintensitywasscoredwiththefollowingscoresystem,0(negativestaining),1(weakstaining),2(moderatestaining)and3(strongstaining).Theproportionofpositivelystainedcellswasevaluatedwithfivelevels,0( 10%),1(10–25%),2(25–50%),3(50–75%)and4( 75%).Theproductsoftheabovetwoindicatorswereconsideredthefinalscore.Thefinalscoresweredividedintotwogrades,lowexpression(≤4),highexpression( 4).

Cellwoundhealingandtranswellassays

Forcellwoundhealingassay,thecellswerefirstlyculturedtofullconfluencein6-wellplates.Subsequently,thecellswerescratchedwitha200 μlmicroPipettetipinthecenterofthewell.Then,thecellswereincubatedwithserum-freemedium.Representativeimageswerecapturedat0 hand24 hafterinjury.Thewidthofwoundhealingwasquantifiedandcomparedwithbaselinevalues.Allexperimentswererepeatedindependentlyintriplicate.

Fortranswellassay,2 × 104cellsofeachgroupin200 μlserum-freemediumwereseededintheupperchamber(8.0 μmpore,Corning,USA)without(migration)orwith(invasion)Matrigel(BDBioscience,USA).600 μlRPIMmediumwith10%FBSwasaddedtothelowerchamber.Afterincubatingfor24 h,theupperchamberswerefixedwith4%polymethanolfor30 minandthenstainedwith0.1%crystalvioletfor30 min.Thecellsthatmigratedorinvadedtothereversesideoftheupperchamberswerephotographed.Fiverandomfieldswereselectedtocalculatecellsthatmigratedorinvaded.

Luciferasereporterassay

Theluciferasereporterplasmids(pGL3-Firefly-RenillacontainingcircNHSL1sequenceandMutantsequence,pGL3-Firefly-RenillacontainingSIX13′-UTRsequenceandMutantsequence)weresynthesizedbyGenePharmaCo.(Shanghai,China).Thefireflyluciferasereporterplasmids(pGL4.27-LuccontainingVimentinpromoterandMutantsequence)andrenillaluciferaseplasmids(pRL)weregeneratedbyHarOLifeCo.(Shanghai,China).Theluciferasereporterplasmidswereco-transfectedintocellswithmimics,inhibitor,LV-shSIX1andpRLorpLVX-shSIX1andpRLusingLipofectamine™2000reagent.After36 h,thefireflyluciferaseandrenillaluciferaseactivityweredetected.TheeffectsofmiR-1306-3porSIX1onluciferasereporterplasmidswerecalculatedwiththeratiooffireflyluciferase/Renillaluciferaseactivity.Allexperimentswereindependentlyrepeatedintriplicate.

RNAimmunoprecipitation(RIP)assay

RIPassaywasconductedwithmagnaRIPTMRNA-bindingProteinImmunoprecipitationkit(Millipore,Billerica,MA).MKN-28cellsweretransfectedwithmiR-1306-3pmimicsornegativecontrol.ThecellswerelysedincompleteRNAlysisbufferafter48 h.Then,theRIPimmunoprecipitationbufferincludingmagneticbeadsconjugatedwithnegativecontrolmouseIgGorhumananti-AGO2antibody(Mouse,Millipore,Billerica,USA)wasaddedintocelllysates.Subsequently,thelysateswererotatedovernight.Nextday,afterincubatingwithProteinaseKfor30 min,theimmunoprecipitatedRNAwasextracted.Last,qRT-PCRandagarosegelelectrophoresiswereperformedtoidentifytheexpressionofcircNHSL1andmiR-1306-3p.

Animalexperiments

Toestablishxenograftmousemodels,theshRNAagainstcircNHSL1(thesametargetwithsi-circ-1)andnegativecontrolwereclonedintopLL3.7vector,andthefull-lengthcDNAofcircNHSL1oranegativecontrolwereclonedintoPLCDH-ciRvector,containingafrontandbackcircularframe.Then,thestablecelllineswithknockdownoroverexpressionofcircNHSL1wereconstructedwithMKN-28orSGC-7901cells.Forinvivotumorigenesisassay,1.0 × 107MKN-28orSGC-7901cellsin150 μlPBSweresubcutaneouslyinjectedintoleftinguinalregionofmaleBALB/cathymicnudemice(4 weeksold).Tumorvolumeswerecalculatedbytheformula:tumor = (length×width2)/2andmeasuredeverythreedays.Finally,themiceweresacrificed,andthevolumeandweightoftumorsweredetected.

Forinvivolivermetastasisassay,1.0 × 107cellswereintravenouslyinjectedintoileocolicveinofnudemiceaswedidpreviously[28].Forinvivoperitonealmetastasisassays,1.0 × 107cellswereinjectedintotheabdominalcavityofnudemice.After4 weeks,theliverswereremovedandtheperitonealmetastaticnoduleswereobserved.Theliverswereparaffin-embeddedandfinallyvalidatedbyhematoxylinandeosin(HE)staining.TheanimalexperimentswereapprovedbytheInstitutionalAnimalCareandUseCommitteeofShanghaiGeneralHospital,andwereperformedaccordingtotheguidelinesforthecareanduseoflaboratoryanimals.

Statisticalanalysis

TheSPSS22.0softwarewasconductedforstatisticalanalyses.Datawerecalculatedbytheχ2orFisher’sexacttest.ThecorrelationswereanalyzedbyPearson’stest(r,P).PairedandunpairedcontinuousvariableswerecomparedbyStudent’st-testortheMann-WhitneyUtest.ThesurvivalcurvesweredrawnusingtheKaplan-Meiermethodandwereanalyzedbylog-ranktests.p  0.05wasconsideredstatisticallysignificantinalltests.

ResultsCircNHSL1isup-regulatedingastriccancertissuesandcorrelateswiththeprogressionandpoorprognosis

Tocharacterizemetastasis-relatedcircRNAtranscripts,RNA-seqanalysisbetween3gastriccancertissueswithmetastasisand2gastriccancertissueswithoutmetastasiswasconducted(Fig. 1a).Wefoundatotalof38differentiallyexpressedcircRNAswithacut-offcriteriaoffoldchange 2.0andp  0.05,ofwhich37wereup-regulatedand1wasdown-regulatedingastriccancertissueswithmetastasis.Inthetop10up-regulatedcircRNAs,wefoundthatcircNHSL1,alsonamedhsa_circ_0006835accordingtotheannotationofcircBase(http://www.circbase.org/),wasthehighestup-regulatedcircRNA ingastriccancertissueswithmetastasis,whichwassplicedfromNHSL1genelocatedatchr6:138,743,180–138,893,726andfinallyformedasense-overlappingcirculartranscriptof335 nt(Fig.1b).Sangersequencingconfirmedthehead-to-tailsplicing(Fig.1b).TodetectthelevelofcircNHSL1andNHSL1,wedesignedtwosetsofprimers,divergentprimersthatwereexpectedtoamplifycircNHSL1andconvergentprimerstoamplifylinearNHSL1mRNA.WefoundthatthelevelofcircNHSL1wasobviouslyhigherinmultiplegastriccancercellsthaninGES-1cell(Fig.1c).Amongthesecelllines,MKN-28andAGScellsexhibitedthehighestlevelofcircNHSL1,whileSGC-7901andMGC-803cellsthelowestlevel(Fig.1c).Todetectwhetherthehead-to-tailsplicingofcircNHSL1resultsfromtrans-splicingorgenomicrearrangements,weextractedcDNAandgDNAfromMKN-28andSGC-7901cells.ThegelelectrophoresisresultsshowedthatcircNHSL1wasdetectedonlyincDNA,butnotingDNA,(Fig.1dande)indicatingthattheloopstructureofcircNHSL1comesfromreversesplicing.ToconfirmthestABIlityofcircNHSL1,MKN-28andSGC-7901cellsweretreatedwithRNaseR,aprocessive3′to5′exoribonuclease.AsshowninFig.1f,circNHSL1resistedthedigestionofRNaseR,butthelinearformofNHSL1wasdigestedsharply.Inaddition,theresultsofNuclear-cytoplasmicfractionationillustratedthatcircNHSL1waspredominantlylocalizedinthecytoplasm(Fig.1g).TheseresultsindicatethatcircNHSL1ishighlystableincytoplasmofgastriccancercells,implyingitspotentialtobeanappropriatediagnosticorprognosticmarker.

Fig.1

CircNHSL1isup-regulatedingastriccancertissuesandcorrelatedwiththeprogressionandpoorprognosis.aCircRNAmicroarraybetween3gastriccancertissueswithoutmetastasisand2gastriccancertissueswithmetastasis.bSchematicillustrationoftheformationofcircNHSL1viathecircularizationofexonsinNHSL1gene.Sangersequencingconformedthehead-to-tailsplicingofcircNHSL1.cRelativeexpressionofcircNHSL1incelllinesbyqRT-PCR.dandeThegelelectrophoresisvalidatedtheexistenceofcircNHSL1.DivergentprimersamplifiedcircNHSL1incDNAbutnotgDNA.GAPDHwasusedasalinearcontrol.fRelativeexpressionofcircNHSL1andNHSL1mRNAinbothMKN-28andSGC-7901cellswasdetectedbyqRT-PCRinthepresenceorabsenceofRNaseR.gcircNHSL1wasmainlylocatedinthecytoplasmbynuclear-cytoplasmicfractionationassay.hRelativecircNHSL1expressionin93pairedfreshfrozennormalgastrictissuesandgastriccancertissues.iThelevelofcircNHSL1wassignificantlyhigheringastriccancertissueswithM1stagethanwithM0stage(p  0.01).jKaplan-Meiersurvivalanalysis(log-ranktest)showedthatgastriccancerpatientswithhighcircNHSL1expressionhavealowerOSandDFSthanthesewithlowcircNHSL1expression(p  0.01).Alldataarepresentedasthemean ± SEM.*p  0.05,**p  0.01

Fullsizeimage

TodeterminethelevelofcircNHSL1,wecollected93pairsoffreshfrozengastriccancertissuesandmatchednormaltissues.qRT-PCRresultsshowedthatconsistentwiththeresultsofgastriccancercells,circNHSL1expressionwashigherinmost(80.65%,75/93)gastriccancertissuesthaninnormalgastrictissues(Additional file 2:FigureS1,Fig.1h).TofurtheranalyzethecorrelationbetweenthelevelofcircNHSL1withclinicopathologicalfeaturesandprognosis,thesesamplesweredividedintotwogroups,highcircNHSL1groupandlowcircNHSL1groupbasedonthemedianexpressionofcircNHSL1.AsshowninTable 1,highlevelofcircNHSL1waspositivelycorrelatedwithUICCstages,pathologicalTstages,lymphaticmetastasis,distantmetastasisandgrades.Also,thelevelofcircNHSL1washigherintissueswithM1stagethanthesewithM0stage(Fig.1i),whichconfirmedtheRNA-seqresultsthatcircNHSL1isanoncogenicandmetastasis-relatedcircRNA.Furthermore,theKaplan-MeieranalysisshowedthatgastriccancerpatientswithhighcircNHSL1expressionhadthesignificantlypoorerOSandDFScomparedwithpatientswithlowcircNHSL1expression(Fig.1j).Insummary,circNHSL1wasconfirmedtobeahighlystablecircRNAandanappropriatediagnosticandprognosticmarkerforgastriccancer.

Table1CorrelationbetweencircNHSL1expressionandclinicopathologicalparametersingastriccancer(n = 93)FullsizetableCircNHSL1promotesmigrationandinvasionofgastriccancercellsinvitro

TotestthefunctionsofcircNHSL1ingastriccancercells,threesi-RNAstargetedthejunctionsitesofcircNHSL1andoverexpressionplasmidsofcircNHSL1weredesignedandtransfectedintoMKN-28andAGScellsorSGC-7901andMGC-803cells,respectively.CircNHSL1expressionwassignificantlysilencedbysi-circ-1andsi-circ-2,whileNHSL1mRNAdidnotchange(Fig. 2a,Additional file 3:FigureS2b).Similarly,circNHSL1wasobviouslyoverexpressed,andnosignificantchangeinNHSL1mRNAwasobserved(Fig.2b,Additionalfile3:FigureS2a).Amongthe3si-RNAs,si-circ-1hadthehighestknockdownefficiencyinMKN-28andSGC-7901cells,whilesi-circ-2inAGScells.Themobilityofgastriccancercellswasprominentlydecreasedbydown-regulationofcircNHSL1,andtheeffectwasalsoconfirmedbythetranswellmigrationandinvasionassaysinMKN-28,AGSandSGC-7901cells(Fig.2c,Additionalfile3:FigureS2eandf).Nevertheless,overexpressionofcircNHSL1promotedthemobility,migrationandinvasionofgastriccancercellsinSGC-7901,MGC-803andMKN-28cells(Fig.2d,Additionalfile3:FigureS2candd).Takentogether,thesefindingssuggestthatcircNHSL1promotestheprogressionofgastriccancerinvitro.

Fig.2

CircNHSL1promotesmigrationandinvasionofgastriccancercellsinvitro.aandbRelativeexpressionofcircNHSL1andNHSL1mRNAwasdetectedbyqRT-PCRingastriccancercellsaftertransfectionofsi-circNHSL1,pEX-3-circNHSL1ornegativecontrol.cThecellmobility,migrationandinvasionwereevaluatedbywoundhealingandtranswellmigrationandinvasionassaysafterknockdownofcircNHSL1inMKN-28andAGScells.dThecellmobility,migrationandinvasionwereevaluatedbywoundhealingandtranswellassaysafteroverexpressionofcircNHSL1inSGC-7901andMGC-803cells.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01

FullsizeimageCircNHSL1promotestheprogressionofgastriccancerviaSIX1

AccordingtothehypothesisofcompetingendogenousRNA(ceRNA)[29,30],circRNAsmaypromotetheexpressionoftargetgenesbyspongingmiRNAs.SincecircNHSL1islocatedinthecytoplasmandexhibitshighstability,wespeculatedthatcircNHSL1couldactasamiRNAspongetoincreasetargetgeneexpression.TodiscoverthegenespositivelycorrelatedwithcircNHSL1expression,RNA-seqforcDNAbetweenabove3gastriccancertissueswithoutmetastasisand2gastriccancertissueswithmetastasisinRNA-seqforcircRNAwasperformed(Fig. 3a).ThelevelofNHSL1mRNAwaslowingastriccancertissueswithmetastasis.CombinedwiththeresultsthatNHSL1expressionwasnotchangedbydown-regulationorup-regulationofcircNHSL1ingastriccancercells(Fig.2aandb),circNHSL1didnotregulateNHSL1mRNAexpressionthroughspongingmiRNAs.Thetop10up-regulatedgenesingastriccancertissueswithmetastasiswereselected.CircNHSL1silencingsignificantlydecreasedMAP4K4andSIX1expressioninbothMKN-28andAGScells(Fig.3b,Additional file 4:FigureS3a),whileonlyAMOTandSIX1expressionincreasedinbothSGC-7901andMGC-803cellsafterup-regulationofcircNHSL1(Fig.3c,Additionalfile4:FigureS3b).Thereby,weassumedthatcircNHSL1positivelyregulatedSIX1expression.Toconfirmthehypothesis,aluciferasereporterplasmidwithwildtypeofSIX1mRNA3′-UTRwasconstructed,andluciferasereporterassayswereperformed.CircNHSL1silencingsignificantlydecreasedtheluciferaseactivity,whilecircNHSL1overexpressionobviouslyincreasedtheluciferaseactivity(Fig.3d).Furthermore,down-regulationorup-regulationofcircNHSL1decreasedorincreasedSIX1proteinexpressioningastriccancercells,respectively(Fig.3eandf,Additionalfile4:FigureS3candd).WeanalyzedthelevelofcircNHSL1andSIX1in61pairedgastriccancertissuesamongabove93pairedgastriccancertissues,andfoundthatthelevelofcircNHSL1positivelycorrelatedwiththelevelofSIX1(p  0.01)(Fig.3g).Similarly,thelevelofSIX1inthehighcircNHSL1groupwassignificantlyhigherthaninthelowcircNHSL1group(Fig.3h).

Fig.3

CircNHSL1promotestheprogressionofgastriccancerviaSIX1.aCircRNAmicroarraybetween3gastriccancertissueswithoutmetastasisand2gastriccancertissueswithmetastasis.bandcRelativeexpressionof10mRNAcandidateswasdetectedbyqRT-PCRingastriccancercellsaftertransfectionofsi-circNHSL1,pEX-3-circNHSL1ornegativecontrol.dRelativeluciferaseactivitiesweredetectedingastriccancercellsaftertransfectingluciferasereporterplasmidwithwildtypeofSIX1mRNA3′-UTRwithsi-circNHSL1,pEX-3-circNHSL1ornegativecontrol.eandfTheeffectsofcicrNHSL1andSIX1onthemRNAandproteinexpressionsofSIX1andVimentinweredetectedbyqRT-PCRandwesternblotting.gPearsoncorrelationanalysisdeterminedthesignificantlypositivecorrelationbetweenthelevelsofcircNHSL1andSIX1in61pairedgastriccancertissues(p  0.01).hThelevelofSIX1washigherintissueswithhighcircNHSL1expressionthanwithlowcircNHSL1expression(p  0.01).iandjTheeffectsofcicrNHSL1andSIX1onthemobility,migrationandinvasionwereconfirmedbywoundhealingandtranswellassays.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01

Fullsizeimage

TotesttheeffectsofSIX1onthefunctionsofcircNHSL1,cellwoundhealing,transwellmigrationandinvasionassayswereperformed.TheresultsshowedthatoverexpressionofSIX1reversedtheabilityofcircNHSL1silencingtosuppressthemobility,migrationandinvasionofgastriccancercells(Fig.3i),whiledown-regulationofSIX1decreasedtheenhancedabilityofmobility,migrationandinvasioninducedbyoverexpressionofcircNHSL1(Fig.3j).Inaddition,circNHSL1promotedtheexpressionofmRNAandproteinofVimentin,whileoverexpressionofSIX1relievedthesuppressionofcircNHSL1silencingonVimentinexpression,anddecreaseinSIX1attenuatedthepromotionofcircNHSL1overexpressiononVimentinexpression,indicatingthatcircNHSL1promotedVimentinexpressionviaSIX1(Fig.3eandf).Takentogether,circNHSL1promotesgastriccancerprogressionviaSIX1.

SIX1promotestheprogressionofgastriccancerbytranscriptionallyregulatingvimentinexpression

Asatranscriptionfactorofthehomeoboxgenefamily,SIX1mayregulatetargetgeneexpressionintranscriptionallevel,therebyexertingbiologicalfunctions.WeanalyzedthepotentialbindingDNAsequencelogoofSIX1(Additional file 5:FigureS4a)andfoundtwotheoreticalbindingsitesinthetop2000 ntofthepromoterdomainofVimentingene(http://jaspar.genereg.net)(Fig. 4d).Hence,wespeculatedthatSIX1mayregulateVimentinexpressioninthetranscriptionallevel.PearsoncorrelationanalysisshowedthatthemRNAexpressionlevelofSIX1waspositivelycorrelatedwiththatofVimentininabove61pairedgastriccancertissues(p  0.05)(Fig.4a).SIX1promotedVimentinexpressioninthelevelofmRNAandprotein,asshowninFig.4bandc.Furthermore,aluciferaseplasmidwiththetop2000 ntofthepromoterdomainofVimentingene(pLuc-WT)andaluciferaseplasmidwithmutantsequencesinbothtwobindingsitesofthetop2000 ntofthepromoterdomain(pLuc-Mutant)weregenerated(Fig.4d).LuciferasereporterassaysdemonstratedthatSIX1enhancedtheluciferaseactivityofpLuc-WTinadose-dependentmanner,butnotthatofpLuc-Mutant(Fig.4eandf),suggestingthatSIX1enhancedVimentinexpressionbydirectlybindingtothepromoterdomainofVimentin.Then,wetestedthefunctionsofSIX1ingastriccancercells.TheresultsillustratedthatSIX1promotedcellmobility,migrationandinvasionofgastriccancercells(Fig.4g-j).Tosummary,SIX1promotesgastriccancerprogressionbypositivelyregulatingVimentinexpressioninthetranscriptionallevel.

Fig.4

SIX1promotestheprogressionofgastriccancerbytranscriptionallyregulatingVimentinexpression.aThelevelofSIX1positivelycorrelatedwiththelevelofVimentinin61pairedgastriccancertissuesbyPearsoncorrelationanalysis(p  0.01).bandcRelativeexpressionofSIX1andVimentinwasdetectedbyqRT-PCRandwesternblottingingastriccancercellsaftertransfectionofLV-shSIX1,pLVX-SIX1ornegativecontrol.dSchematicillustrationofthesequencesofwildtypeofVimentinpromoterdomainandmutantsequencesinthebindingsitesofSIX1onVimentinpromoterdomainareshown.eandfRelativeluciferaseactivitiesweredetectedingastriccancercellsaftertransfectingluciferasereporterplasmidswithwildtypeofVimentinpromoterdomainormutantVimentinpromoterdomainwithLV-shSIX1,pLVX-SIX1ornegativecontrol.g-jTheeffectsofSIX1onthemobility,migrationandinvasionweredetectedbywoundhealingandtranswellassays.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01

FullsizeimageCircNHSL1actsasamiRNAspongeofmiR-1306-3p

PreviousstudieshaverevealedthatcircRNAscanserveasmiRNAspongestoabrogatethefunctionsofmiRNAs.Inthefollowingstudies,weexploredwhethercircNHSL1promotedgastriccancerprogressionbyspongingmiRNAs.Firstly,wepredictedthepotentialtargetmiRNAsofcircNHSL1withmiRandadatabase(http://www.microrna.org)andthepossibleupstreammiRNAsofSIX1withTargetScandatabase(http://www.targetscan.org).TheresultsshowedthatcircNHSL1andSIX1sharemicroRNAresponseelements(MREs)ofmiR-1306-3pwithhighscores(Fig. 5d,Additionalfile5:FigureS4b).Next,thelevelofmiR-1306-3pwasexaminedinabove61pairedgastriccancertissuesandmatchednormaltissues,andtheresultsindicatedthatmiR-1306-3pwasremarkablydown-regulatedin80.33%(49/61)gastriccancertissuesthaninnormalgastrictissues(Additional file 6:FigureS5a).PearsoncorrelationanalysisshowedthatthelevelofcircNHSL1negativelycorrelatedwiththelevelofmiR-1306-3pinabovegastriccancertissues(p  0.01)(Fig.5a).Inaddition,knockdownoroverexpressionofcircNHSL1causedup-regulationordown-regulationofmiR-1306-3p,respectively,ingastriccancercells(Fig.5b).Then,luciferasereporterplasmidswithawildtypeofcircNHSL1sequence(WT)andmutantcircNHSL1sequenceinthebindingsitesofmiR-1306-3p(Mutant)(Fig.5d)weregenerated.LuciferasereporterassaysillustratedthatoverexpressionofmiR-1306-3psuppressedtheluciferaseactivityofWTandknockdownofmiR-1306-3pincreasedtheluciferaseactivityofWT,butdidnotchangetheluciferaseactivityofMutant(Fig.5c),demonstratingthedirectinteractionbetweencircNHSL1andmiR-1306-3p.IthasbeenwidelyknownthatmiRNAsbindtoMREsthroughRNA-inducedsilencingcomplex(RISC),ofwhichArgonaute2(AGO2)proteinisthekeycomponent.Thereby,ananti-AGO2RIPassaywasperformedinMKN-28cellstopulldowncircNHSL1andmiR-1306-3pwithananti-AGO2antibody(IgGasnegativecontrol,noneasInput).TheresultsshowedthatbothcircNHSL1andmiR-1306-3pwereefficientlypulleddownbyanti-AGO2antibodycomparedwithIgG,andsignificantlyenrichedbyoverexpressionofmiR-1306-3pcomparedwithnegativecontrol(Fig.5eandf).Collectively,thesedatademonstratedthatcircNHSL1actsasaspongeofmiR-1306-3pingastriccancer.

Fig.5

CircNHSL1promotesgastriccancerprogressionbyservingasamiRNAspongeofmiR-1306-3p.aPearsoncorrelationanalysisdeterminedthesignificantlynegativecorrelationbetweenthelevelsofcircNHSL1andmiR-1306-3pin61pairedgastriccancertissues(p  0.01).bTheeffectsofcircNHSL1ontheexpressionofSIX1wasdetectedbyqRT-PCR.cTheeffectsofmimics,inhibitorandnegativecontrolontheluciferaseactivitiesweredetectedingastriccancercellsaftertransfectingluciferasereporterplasmidswithwildtypeofWT,Mutantornegativecontrol.dSchematicillustrationofthesequenceofwildtypeofSIX13′-UTR(WT)andmutantsequencesonthecomplementarysitesofSIX13′-UTRwithmiR-1306-3p(Mutant).eandfAnti-AGO2RIPassaywasperformedinMKN-28cellsaftertransfectionwithmimicsornegativecontrol,followedbyagarosegelelectrophoresis(e)andqRT-PCR(f)todetecttheexpressionofcircNHSL1andmiR-1306-3p.gTheeffectsofcircNHSL1andmiR-1306-3pontheluciferaseactivitiesofwildtypeofSIX1mRNA3′-UTRingastriccancercellsweredetected.handiTheeffectsofcircNHSL1andmiR-1306-3ponthemRNAandproteinexpressionsofSIX1andVimentinweredetectedbyqRT-PCRandwesternblotting.jandkTheeffectsofcircNHSL1andmiR-1306-3ponthemobility,migrationandinvasionwereconfirmedbywoundhealingandtranswellassays.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01

FullsizeimageMiR-1306-3preversestheabilityofcircNHSL1topromotegastriccancerprogression

ToexplorewhethercircNHSL1exertsitsbiologicalfunctionbyspongingmiR-1306-3p,rescuedexperimentswereperformedwithup-regulationordown-regulationofmiR-1306-3ponthebasisofectopiccircNHSL1expression.LuciferasereporterassaysdemonstratedthatmiR-1306-3pinhibitorincreasedtheluciferaseactivityoftheluciferasereporterplasmidwithwildtypeofSIX1mRNA3′-UTR,whichwassuppressedbyknockdownofcircNHSL1,whilemiR-1306-3pmimicsdecreasedtheluciferaseactivityinducedbyoverexpressionofcircNHSL1(Fig.5g).Furthermore,miR-1306-3preversedtheabilityofcircNHSL1toenhancemRNAandproteinexpressionofSIX1andVimentiningastriccancercells(Fig.5handi).Then,woundhealingandtranswellmigrationandinvasionassayswereconducted.TheresultsshowedthatmiR-1306-3pattenuatedtheabilityofcircNHSL1topromotemobility,migrationandinvasionofgastriccancercells(Fig.5jandk).Insummary,circNHSL1promotesgastriccancerprogressionthroughspongingmiR-1306-3p.

MiR-1306-3pisdown-regulatedingastriccancertissuesandnegativelycorrelateswithclinicopathologicalfeaturesandprognosis

FewstudiesaboutmiR-1306-3phavebeenreported,andtheexpressionlevel,functionsandrolesofmiR-1306-3pincancershavenotbeenexplored.Therefore,miR-1306-3phasyettobeunderstood.Inthisstudy,weusedanothergroupofgastriccancertissues(aTMAincluding54pairedgastriccancertissuesandmatchednormaltissues)todetectthelevelofmiR-1306-3pwithISH.TheresultsindicatedthatconsistentwithqRT-PCRresultsinabove61pairedfreshfrozengastriccancertissues,thelevelofmiR-1306-3pwassignificantlyhigherinnormalgastrictissuesthaningastriccancertissues(Fig. 6aandb).FurtheranalysisshowedthatmiR-1306-3pwasmarkedlyhigheringastriccancertissuesofUICCstageII,distantmetastasisM0stage,welldifferentiation(G1stage)andpathologicalT2andT3stagesthanintheseofUICCstageIII,distantmetastasisM1stage,moderateandpoordifferentiation(G2andG3stages)andpathologicalT4stage,respectively(Fig.6c-f).MiR-1306-3pexpressioningastriccancertissuesnegativelycorrelatedwithdifferentiation,pathologicalTstage,distantmetastasisandUICCstage(Table 2).TheKaplan-MeieranalysisshowedthatgastriccancerpatientswithlowmiR-1306-3pexpressionhadashorterOSandDFS(Fig.6gandh),indicatinglowmiR-1306-3pexpressionpredictedapoorprognosis.Ingeneral,miR-1306-3pactsasatumorsuppressorandhighmiR-1306-3ppredictsapromisingprognosisingastriccancer.

Fig.6

MiR-1306-3pisdown-regulatedingastriccancertissuesandcorrelatedwiththeprogressionandpoorprognosis.TheexpressionofmiR-1306-3pwasdetectedbyISHwithTMAincluding54pairedgastriccancertissuesandpairednormaltissues.aRepresentimagesofmiR-1306-3pexpressioninnormalgastricmucosaandgastriccancertissueswithStageII,StageIII,M0,M1,G1,G2,G3,pT2,pT3andpT4.bThelevelofmiR-1306-3pingastriccancertissueswassignificantlylowerthaninnormalgastrictissues.c-fThelevelofmiR-1306-3pingastriccancertissueswithStageII(c),M0(d),welldifferentiation(e)andpT2andpT3(f)wassignificantlyhigherthanthesewithStageIII,M1,moderateandpoordifferentiationandpT4.g-hKaplan-Meiersurvivalanalysis(log-ranktest)showedthatgastriccancerpatientswithlowmiR-1306-3pexpressionhavealowerOSandDFSthanthesewithhighmiR-1306-3pexpression(p  0.01).Alldataarepresentedasthemean ± SEM.**p  0.01

FullsizeimageTable2CorrelationbetweenmiR-1306-3pexpressionandclinicopathologicalparametersingastriccancer(n = 54)FullsizetableSIX1isadirecttargetofmiR-1306-3p

AccordingtotheTargetScandatabase,SIX1mRNAcontainstheMREofmiR-1306-3p,implyingthatmiR-1306-3pmaydirecttargetSIX1.WeanalyzedtheexpressionofmiR-1306-3pandSIX1inabove61pairedgastriccancertissues,andfoundSIX1wasup-regulatedin77.05%(47/61)gastriccancertissues(Fig. 7a)andthelevelofmiR-1306-3pnegativelycorrelatedwiththelevelofSIX1(Fig.7b).Then,thelevelofSIX1wasdeterminedinabovementionedTMAofgastriccancerusingimmunohistochemistry.PearsoncorrelationanalysisindicatedthatmiR-1306-3pISHscorenegativelycorrelatedwithSIX1IHCscoreinbothgastriccancertissuesandnormalgastrictissues(Fig.7candd).Inaddition,overexpressionofmiR-1306-3psignificantlydecreasedtheexpressionofSIX1mRNAandprotein,whiledown-regulationofmiR-1306-3premarkablyincreasedit,inMKN-28andSGC-7901cells(Fig.7eandf,Additional file 7:FigureS6aandb).Furthermore,luciferasereporterplasmidswiththewildtypeofSIX1mRNA3′-UTR(WT)andmutantSIX1mRNA3′-UTRinthebindingsitesofmiR-1306-3p(Mutant)wereconstructed(Additionalfile5:FigureS4c).LuciferasereporterassaysshowedmiR-1306-3pmimicssignificantlydecreasedtheluciferaseactivityofWT,whilemiR-1306-3pinhibitorremarkablyincreasedit,butnotthatofMutant,inMKN-28andSGC-7901cells(Fig.7gandh,Additionalfile7:FigureS6candd).ThesedatasuggestedthatmiR-1306-3psuppressesSIX1expressionbydirectlybindingto3′-UTRofSIX1mRNA.

Fig.7

MiR-1306-3psuppressesgastriccancerprogressionthroughdirectlytargetingSIX1.aSIX1expressionisup-regulatedin77.05%(47/61)gastriccancertissues.bPearsoncorrelationanalysisdeterminedthesignificantlynegativecorrelationbetweenthelevelsofmiR-1306-3pandSIX1in61pairedgastriccancertissues(p  0.01).canddTheISHscoresofmiR-1306-3pnegativelycorrelatedwiththeIHCscoresofSIX1inbothnormalgastricmucosatissues(c)andgastriccancertissues(d)ofTMA.eandfTheeffectsofmiR-1306-3pandSIX1onthemRNAandproteinexpressionsofSIX1andVimentinweredetectedbyqRT-PCR(e)andwesternblotting(f).gandhTheeffectsofmimics(g)andinhibitor(h)ofmiR-1306-3pontheluciferaseactivitiesofwildtypeofSIX1mRNA3′-UTR(WT)andmutantSIX1mRNA3′-UTR(Mutant)weredetected.iandjTheeffectsofmiR-1306-3pandSIX1onthemobility,migrationandinvasionweredetectedbywoundhealingandtranswellassays.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01

FullsizeimageMiR-1306-3psuppressesgastriccancerprogressionthroughinhibitingSIX1expression

GiventherolesofcircNHSL1andSIX1onpromotinggastriccancerprogression,wedetectedtheroleofmiR-1306-3ponthemigrationandinvasionofgastriccancercells.TheresultsshowedthatmiR-1306-3pmimicssuppressedthemobility,migrationandinvasionofMKN-28andSGC-7901cells(Fig.7i,Additionalfile7:FigureS6f),whilemiR-1306-3pinhibitorenhancedthemobility,migrationandinvasionofgastriccancercells(Fig.7j,Additionalfile7:FigureS6e).

TofurtherexploretheeffectsofSIX1ontherolesofmiR-1306-3pingastriccancerprogression,rescuedexperimentswereconducted.AsshowninFig.7eandf,Additionalfile7:FigureS6aandb,SIX1reversedtheabilityofmiR-1306-3ptosuppressthemRNAandproteinlevelofSIX1andVimentin.Functionally,SIX1reversedtheabilityofmiR-1306-3ptoinhibitthemobility,migrationandinvasionofgastriccancercells(Fig.7iandj,Additionalfile7:FigureS6eandf).Tosummary,miR-1306-3psuppressesgastriccancerprogressionthroughinhibitingSIX1expression.

CircNHSL1enhancesthegrowthandmetastasisofxenografttumorsofgastriccancercellsinvivo

ToinvestigatethefunctionsofcircNHSL1invivo,thestableMKN-28cellswithsh-circNHSL1orsh-NCandSGC-7901cellswithcircNHSL1orNCwereconstructed,accordingtocircNHSL1expressionin7gastriccancercells.Thexenograftmousemodelwasestablishedbysubcutaneouslyinjectingofgastriccancercells.After28 days,allmiceweresacrificedandtumorsampleswereharvested(Fig. 8aandd).Theweight(Fig.8b)andvolume(Fig.8c)oftumorswithknockdownofcircNHSL1weremarkedlylowerthanthosewithcontrolinMKN-28cells,whiletheweight(Fig.8e)andvolume(Fig.8f)oftumorswithoverexpressionofcircNHSL1weresignificantlyhigherthanthecontroltumorsofSGC-7901cells.

Fig.8

CircNHSL1promotesgrowthandmetastasisofgastriccancercellsinvivo.aImagesofsubcutaneousxenografttumorsofMKN-28cells.bTumorweightofMKN-28cellswasshown.cTumorvolumesofMKN-28cellsmeasuredevery3 daysfor6timeswereanalyzed.dImageofsubcutaneousxenografttumorsofSGC-7901cells.eTumorweightofSGC-7901cellswasshown.fTumorvolumesofSGC-7901cellsmeasuredevery3 daysfor6timeswereanalyzed.gRepresentativeimagesoflivermetastasisassayandHEstainingoftheliverofMKN-28cells.KnockdownofcircNHSL1decreasedthenumberofliversurfacemetastasiscomparedwithnegativecontrol.hRepresentativeimagesoflivermetastasisassayandHEstainingoftheliverofSGC-7901cells.OverexpressionofcircNHSL1increasedthenumberofliversurfacemetastasiscomparedwithnegativecontrol.i-jRepresentativeimagesofperitonealmetastasisassays.KnockdownofcircNHSL1decreasedthenumberofperitonealmetastasisnodulescomparedwithnegativecontrol(i),whereasoverexpressionofcircNHSL1increasedthenumberofperitonealmetastasisnodules(j).**p  0.01

Fullsizeimage

TheroleofcircNHSL1onmetastasiswasconfirmedbyinvivolivermetastasisandperitonealmetastasisassays.WeobservedthatthenumberofliversurfacemetastaticnodulesinmicebearingMKN-28cellswithsh-circNHSL1waslowerthaninmicebearingMKN-28cellswithsh-NC(Fig.8g),whereasthenumberofliversurfacemetastaticnodulesinmicebearingSGC-7901cellswithcircNHSL1wasmuchthaninmicebearingSGC-7901cellswithNC(Fig.8h).NecropsyrevealedthatthenumberofperitonealmetastaticnoduleswasreducedinmicebearingMKN-28cellswithsh-circNHSL1comparedwithsh-NC(Fig.8i),whileSGC-7901cellswithcircNHSL1colonizedthevisceralorgansandformedmultiplemetastaticnodules(Fig.8j),revealingthatcircNHSL1enhancedtumorcolonizationandmetastasis.Overall,thesedatademonstratethatcircNHSL1promotescellgrowthandmetastasisofgastriccancerinvivo.

Discussion

CircRNAsareanewtypeofhighlystableandabundantendogenousnoncodingRNAs.Withthedevelopmentofhigh-throughputsequencingandbioinformaticsanalysis,anincreasingnumberofcircRNAshavebeenidentifiedandconfirmedtoregulatethedevelopmentandprogressionofvarioushumancancersinrecentyears[31,32,33].However,fewcircRNAshavebeenwellfunctionallyandmechanisticallycharacterizedingastriccancer,andthebiologicalfunctionsofmostcircRNAshaveyettobeexplored.Inthisstudy,weidentifiedanovelmetastasis-relatedcircRNAcircNHSL1,whichisdramaticallyup-regulatedingastriccancertissuesandcells.

Invasionandmetastasisarethebiggestobstaclestosuccessfulsurgicalresectionoftumors,aswellastheleadingcauseofhighmortalityofgastriccancerpatients[34].Hence,itisurgenttodiscovermetastasis-relatedgenesandillustratethemolecularmechanismsofinvasionandmetastasisofgastriccancer.Inthepresentstudy,weappliedRNA-seqanalysistoaccesstheexpressionprofileofmetastasis-relatedcircRNAbetween3gastriccancertissueswithoutmetastasisand2gastriccancertissueswithmetastasis.Themosthighlyup-regulatedcircRNAin2gastriccancertissueswithmetastasis,circNHSL1,wasfurtherconfirmedtobeobviouslyhighin61pairedgastriccancertissuesandpositivelycorrelatewithpathologicalTstages,lymphaticmetastasis,distantmetastasisandpoorprognosis.Functionally,circNHSL1promotesinvasionandmetastasisofgastriccancercellsinvitroandinvivo,implyingthatcircNHSL1isatumorpromoteringastriccancer.Duetothestableloopstructure,greatresistancetoexoribonucleaseandhighabundanceinthecytoplasm,circNHSL1maybeanefficientdiagnosticandtherapeutictargetandapromisingbiomarkerforprognosisingastriccancer.

CircRNAshavebeenreportedtohavemultipleanddiversemolecularmechanismsinthedevelopmentandprogressionofvariouscancers,amongwhichceRNAisthemostimportantandfrequentlyreported.CeRNAhypothesissuggeststhatcircRNAsserveasceRNAstopositivelyregulatetheexpressionofmiRNAtargetgenes[35,36].Forexample,circAKT3promotesPIK3R1expressionviamiR-198suppressioningastriccancer[37].CircPSMC3actsasamiR-296-5pspongetopromotetheexpressionofPhosphataseandTensinHomolog(PTEN)[38].Inthepresentstudy,RNA-seqbetweenabove3gastriccancertissueswithoutmetastasisand2gastriccancertissueswithmetastasisusedinRNA-seqforcircRNAswasperformedtoanalyzemetastasis-relatedgenes.Thetop10genesofhighexpressionin2gastriccancertissueswithmetastasiswereselected.AseriesofmolecularexperimentsdemonstratedthatcircNHSL1promotesSIX1expressioningastriccancer.SubsequentrescuedexperimentsconfirmedthatoverexpressionofSIX1reversetheabilityofdecreasedcircNHSL1tosuppressthemobility,migrationandinvasion,whiledown-regulationofSIX1attenuatedtheabilityofenhancedcircNHSL1topromotethemobility,migrationandinvasion.Insummary,circNHSL1promotesmalignanceofgastriccancercellsthroughenhancingSIX1expression.

TheceRNAhypothesisconstructsacomplicatedregulatorynetworkandmechanism.CircRNAscontainoneormoreMREsthatactasmiRNAspongestonegativelymodulatemiRNAactivity,attenuatingtheinhibitoryeffectontheirtargetgenes.Indeed,anincreasingamountofevidencedemonstratedtheposttranscriptionalfunctionofcircRNAs.Themostwell-knownexoniccricRNACDR1as/ciRS-7hasbeendemonstratedtocontain63or70bindingsitesformiR-7andconsideredasoneofthemostpowerfulmiRNAsponge[29,30].CircNRIP1activatesAKT1/mTORpathwaybyspongingmiR-149-5pingastriccancer[39].Inaddition,circRNA-MYLKpromotescellproliferation,migrationandinvasionbydirectlybindingtomiR-29aandsubsequentlyrelievessuppressionfortargetVEGFA,whichactivatesVEGFA/VEGFR2signalingpathwayinbladdercancer[40].Here,bioinformaticsanalysisshowedthatcircNHSL1andSIX1shareMREofmiR-1306-3p,implyingtheformationofcircNHSL1/miR-1306-3p/SIX1axis.LuciferasereporterandRIPassaysconfirmedthedirectinteractionbetweencircNHSL1andmiR-1306-3p.Likewise,lossandgainandluciferasereporterassaysdemonstratedthatmiR-1306-3pdirectlysuppressesSIX1expressionthroughbindingto3′-UTRofSIX1mRNA.DuetotheunknownlevelsandfunctionsofmiR-1306-3pintumors,wedetectedtheexpressionlevelsandfunctionsofmiR-1306-3pingastriccancer.WefirstdemonstratedthatmiR-1306-3pwasdown-regulatedingastriccancertissues,negativelycorrelatedwithclinicalpathologicalfeaturesandpoorprognosisandsuppressedtheprogressionofgastriccancer,implyingthatmiR-1306-3pmayactasatumorsuppressor.Furthermore,miR-1306-3preversestheabilityofcircNHSL1topromoteSIX1expressionandthemobility,migrationandinvasion,whileSIX1reversestheabilityofmiR-1306-3ptosuppressthemobility,migrationandinvasion.Combiningpreviousresults,wedemonstratethatcircNHSL1promotesgastriccancerprogressionthroughservingasamiR-1306-3pspongeandrelievingitssuppressionontargetgeneSIX1expression.

SIX1isatranscriptionfactorofthehomeoboxgenefamily.PreviousstudieshavereportedthatSIX1playsimportantrolesinthedevelopmentandprogressionofmultiplecancers[41,42].SIX1isup-regulatedinpancreaticcancertissuesandpromotescellmigrationandinvasioninvitroandgrowthinvivo[43].SIX1isalsoup-regulatedincolorectalcancer,correlateswithpooroverallsurvivalandpromotescancercellgrowthandmetastasisinvitroandinvivo[44].Additionally,asatranscriptionfactor,SIX1enhancesVEGF-CandZEB1expressiontopromoteEMT,invasionandmetastasisofcancercells[26].Nevertheless,thedetailfunctionsandmechanismsofSIX1ingastriccancerremainunknown.WedetectedthatSIX1isup-regulatedingastriccancertissues,andpromotesthemobility,migrationandinvasionofgastriccancercells.BioinformaticsanalysisindicatedthatthepromoterdomainofVimentin,akeymesenchymalmarkerthatpromotescellEMT,invasionandmetastasis[27],containstwobindingsitesofSIX1.FurthermolecularexperimentsdemonstratedthatSIX1transcriptionallypromotesVimentinexpressionthroughdirectlybindingtothepromoterdomainofVimentin.Takentogether,circNHSL1promotesgastriccancerprogressionbymiR-1306-3p/SIX1/Vimentinaxis.

ItiswidelyacceptedthatcircRNAsexistindifferentspecies,possessingthefeaturesofbroadlyevolutionaryconservation,highabundance,tissuespecificityandspace-timespecificity.ThediscoveryofthenovelcircRNAcircNHSL1wouldbeenlightening.IthasbeenreportedthatsomecircRNAsexiststablyinplasmaandexosomes[9,45,46],whichmakesitpossibletobebiomarkersandtargetsfordiagnosis,prognosisandtherapyincertaindiseases.Therefore,whethercircNHSL1canbedetectedinplasmaandexosomesneedsfurtherinvestigation.Inaddition,onecircRNAmaycontainmultipledifferentmiRNAbindingsites,andonemiRNAcanalsobindtoseveralcircRNAs.Hence,circRNAsandmiRNAsmaycrosstalkwitheachotherinbiologicalprocesses.Moreover,lotsofunknowncircRNAsandtheirfunctionsinthedevelopmentandprogressionofcancersremaintobediscovered.Accordingly,moreendeavorsandfurtherstudiesareneededtorevealthefunctionsandmechanismsofcircRNAsincancers.

Conclusion

Insummary,weidentifiedthatanovelmetastasis-relatedcircRNAcircNHSL1isup-regulatedingastriccancertissuesandpositivelycorrelateswithclinicopathologicalfeaturesandpoorprognosis.Furthermore,wedemonstratedthatcircNHSL1promotesinvasionandmetastasisofgastriccancercellsinvitroandinvivobytargetingthemiR-1306-3p/SIX1/Vimentinaxis.OurfindingsfirstlyidentifytheroleofcircNHSL1,whichmayofferaneffectivebiomarkerfordiagnosisandprognosisandapromisingtargetfortherapyingastriccancer.

Thedatasetsusedforthecurrentstudyareavailablefromthecorrespondingauthoronreasonablerequest.

Changehistory27March2020

Afterpublicationofthearticle[1],theauthorsreportederrorsofinter-duplicationinFigure3.

Abbreviations3’UTR:

3′-untranslatedregion

ceRNA:

CompetingendogenousRNA

CircRNA:

CircularRNA

DFS:

Disease-freesurvival

EMT:

Epithelial–mesenchymaltransition

HE:

Hematoxylinandeosin

IHC:

Immunohistochemicalanalysis

ISH:

Insituhybridization

miRNA:

MicroRNA

MREs:

microRNAresponseelements

NHSL1:

theNHSlike-1

Overallsurvival

PTEN:

PhosphataseandTensinHomolog

PVDF:

Polyvinylidenefluoride

qRT-PCR:

Real-timequantitativepolymerasechainreaction

RIP:

RNAimmunoprecipitation

RPL26:

RibosomalproteinL26

SIX1:

Sineoculishomeoboxhomolog1

TMA:

Tissuemicroarray

UICC:

theUnionforInternationalCancerControl

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Acknowledgements

Notapplicable.

Funding

ThisworkwassupportedbygrantsfromtheNationalNaturalScienceFoundationofChina(817725276)receivedbyC.H.,ShanghaiMunicipalEducationCommission-GaofengClinicalMedicineGrantSupport(20161425)receivedbyC.H.,ShanghaiJiaotongUniversityMedicalCrossFund(YG2017MS28)receivedbyC.H.,ShanghaiMunicipalScienceandTechnologyCommittee(14411966800)receivedbyC.H.,andtheTechpoolFund(UF201419)receivedbyC.H..Nofundingbodieshadanyroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

AuthorinformationAuthornotes

ZhonglinZhu,ZeyinRongandZaiLuocontributedequallytothiswork.

AffiliationsDepartmentofGeneralSurgery,ShanghaiGeneralHospital,ShanghaiJiaotongUniversitySchoolofMedicine,650XinsongjiangRoad,SongjiangDistrict,Shanghai,201600,ChinaZhonglinZhu, ZeyinRong, ZaiLuo, ZhilongYu, JingZhang, ZhengjunQiu  ChenHuangAuthorsZhonglinZhuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarZeyinRongViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarZaiLuoViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarZhilongYuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarJingZhangViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarZhengjunQiuViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarChenHuangViewauthorpublicationsYoucanalsosearchforthisauthorinPubMedGoogleScholarContributions

CHandZZdesignedtheexperiments,analyzedthedataandrevisedthemanuscript.ZZwrotethemanuscript.ZRandZLperformedmostoftheexperiments.ZY,JZandZQperformedtheexperiments.Alloftheauthorsdiscussedtheresultsandreviewedthemanuscript.

Correspondingauthor

CorrespondencetoChenHuang.

EthicsdeclarationsEthicsapprovalandconsenttoparticipate

EthicsapprovalwasgrantedbytheEthicsCommitteeofShanghaiGeneralHospital.AnimalexperimentswerecarriedoutaccordingtotheShanghaiGeneralHospitalAnimalCareandUseGuidelines,andtheexperimentalprotocolswereapprovedbytheShanghaiResourceCenterofLaboratoryAnimalsoftheChineseAcademyofScience.Writteninformedconsentswereobtainedfromallpatients.

Consentforpublication

AllauthorsgiveconsentforpublicationofthemanuscriptinMolecularCancer.

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SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.

Additionalfiles
Additionalfile1:

TableS1.ThesequencesofprimersforqRT-PCR.(DOCX17kb)

Additionalfile2:

FigureS1.TherelativeexpressionofcircNHSL1in93pairedfreshfrozennormalgastrictissuesandgastriccancertissues.CircNHSL1expressionwassignificantlyhigherinmost(80.65%,75/93)gastriccancertissuesthaninnormalgastrictissues.(TIF180kb)

Additionalfile3:

FigureS2.CircNHSL1promotesmigrationandinvasionofgastriccancercellsinvitro.aandbRelativeexpressionofcircNHSL1andNHSL1mRNAwasdetectedbyqRT-PCRingastriccancercellsaftertransfectionofsi-circNHSL1,pEX-3-circNHSL1ornegativecontrol.canddThecellmobility,migrationandinvasionwereevaluatedbywoundhealingandtranswellmigrationandinvasionassaysafteroverexpressionofcircNHSL1inMKN-28cells.eandfThecellmobility,migrationandinvasionwereevaluatedbywoundhealingandtranswellassaysafterknockdownofcircNHSL1inSGC-7901cells.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01.(TIF5301kb)

Additionalfile4:

FigureS3.TheeffectsofcircNHSL1ontheexpressionofthetop10genecandidates.aTheeffectsofknockdownofcircNHSL1ontheexpressionofthetop10genecandidatesinAGScells.bTheeffectsofoverexpressionofcircNHSL1ontheexpressionofthetop10genecandidatesinMGC-803cells.canddTheeffectsofknockdownandoverexpressionofcircNHSL1ontheexpressionofSIX1mRNA(c)andprotein(d)inAGSandMGC-803cells.(TIF381kb)

Additionalfile5:

FigureS4.SchematicillustrationofthecomplementarysitesofSIX1mRNA3′-UTRwithmiR-1306-3p.aThepotentialbindingDNAsequencelogoofSIX1protein.bThepredictionofbindingsitesofSIX1mRNA3′-UTRwithmiR-1306-3pbasedonTargetScandatabase.cTheluciferasereporterplasmidscontainingthewildtypeofSIX1mRNA3′-UTR(WT)andmutantsequenceinthebindingsitesofSIX1mRNA3′-UTRwithmiR-1306-3p(Mutant)wereconstructed.(TIF252kb)

Additionalfile6:

FigureS5.TheexpressionofmiR-1306-3p.aThelevelofmiR-1306-3pwasdown-regulatedin80.33%(49/61)gastriccancertissues.bTheefficienciesoftransfectionwithmimicsandinhibitorofmiR-1306-3pweredeterminedinMKN-28andSGC-7901cells.(TIF272kb)

Additionalfile7:

FigureS6.MiR-1306-3psuppressesgastriccancerprogressionthroughdirectlytargetingSIX1.aandbTheeffectsofmiR-1306-3pandSIX1onthemRNAandproteinexpressionsofSIX1andVimentinweredetectedbyqRT-PCR(a)andwesternblotting(b).canddTheeffectsofinhibitor(c)andmimics(d)ofmiR-1306-3pontheluciferaseactivitiesofwildtypeofSIX1mRNA3′-UTR(WT)andmutantSIX1mRNA3′-UTR(Mutant)weredetectedinMKN-28andSGC-7901cells.eandfTheeffectsofmiR-1306-3pandSIX1onthemobility,migrationandinvasionweredetectedbywoundhealingandtranswellassaysinMKN-28andSGC-7901cells.Alldataarepresentedasthemean ± SEMofthreeexperiments.**p  0.01.(TIF5078kb)

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Zhu,Z.,Rong,Z.,Luo,Z.etal.CircularRNAcircNHSL1promotesgastriccancerprogressionthroughthemiR-1306-3p/SIX1/vimentinaxis.MolCancer18,126(2019).https://doi.org/10.1186/s12943-019-1054-7

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Received:25May2019

Accepted:14August2019

Published:22August2019

DOI:https://doi.org/10.1186/s12943-019-1054-7

KeywordsCircNHSL1miR-1306-3pSIX1VimentinMetastasisGastriccancer