lncRNA H19 miR148a3p.docx

lncRNA H19 miR148a3p.docx

《lncRNA H19 miR148a3p.docx》由会员分享，可在线阅读，更多相关《lncRNA H19 miR148a3p.docx（16页珍藏版）》请在冰豆网上搜索。

lncRNAH19miR148a3p

RegulationoflaryngealsquamouscellcancerprogressionbythelncRNAH19/miR-148a-3p/DNMT1axis

ABSTRACT

Laryngealsquamouscellcarcinoma（LSCC）isahighlyaggressivemalignantcancer.TheregulationofLSCCprogressionbylongnon-codingRNA（lncRNA）wasnotwellunderstood.Inthisstudy,wereportedthatthelncRNAH19wasupregulatedinLSCC.TheexpressionlevelsofH19wereinverselycorrelatedwiththesurvivalrateofLSCCpatients.KnockdownofH19expressioninhibitedLSCCcellmigration,invasionandproliferation.WeidentifiedmicroRNAmiR-148a-3pasaninhibitorytargetforH19.OverexpressionofmiR-148a-3preducedLSCCmigration,invasionandproliferationcell,whileinhibitionofmiR-148a-3pdidtheopposite.TheinhibitionofLSCCprogressioninducedbyH19knockdownrequiredtheactivityofmiR-148a-3p.WealsoidentifiedDNAmethyltransferaseenzymeDNMT1asatargetofmiR-148a-3p.CellularDNAmethylationlevelswereinhibitedbybothmiR-148a-3poverexpressionandH19knockdown.Insummary,ourstudydemonstratedthatthelncRNAH19promotedLSCCprogressionviamiR-148a-3pandDNMT1.

INTRODUCTION

Headandnecksquamouscellcarcinomaisthesixthmostcommoncancerintheworld,amongwhichthelaryngealsquamouscellcarcinoma（LSCC）isahighlyaggressivemalignancy[1].AlthoughencouragingprogressinthediagnosisandtreatmentforLSCChasbeenachievedinthepast20years,theoverallsurvivalrateremainsunfavorable.Arecentstudyhasshownthattheoverall1-and2-yearsurvivalratesforLSCCpatientswithouttreatmentareonly56.4%and26.5%,respectively[2].RecurrenceandmetastasisarebelievedtobethemajorfactorsthattolimitthesuccessfultreatmentofLSCC[3].InordertodevelopeffectivetherapyforLSCC,theeffortstowardsunderstandingtheunderlyingpathologicalmechanismsofLSCChavebeenintensifiedrecently.

NoncodingRNAsaresubdividedintosmallncRNAs（<200nt）andlongncRNAs（>200nt）basedontheirsize.SmallncRNAshavebeenshowntoactprimarilyasnegativeregulatorsofgeneexpression.AnumberofmicroRNAs,whichbelongtothesmallncRNAfamily,havebeendemonstratedtofunctionasoncogenesortumorsuppressorgenesforLSCCinourpreviousstudies[4–8].LongncRNAs（lncRNAs）arepoorlyconservedamongspecies[9, 10],butaccumulatingevidencesindicatethatlncRNAscouldplayimportantrolesinavarietyofbiologicalprocessesandmaywellbealsoinvolvedinthedevelopmentofcancerandotherhumandiseases[11, 12].Specifically,ourpreviousstudieshavesuggestedtheinvolvementoflncRNAHOTAIRinLSCC[13, 14].TheoverallpathophysiologicalcontributionoflncRNAstoLSCC,however,islargelyunknown.

ThelncRNAH19istranscribedfromamaternallyexpressedimprintedgenelocusonhumanchromosome11.TheH19geneencodesa2,600ntcapped,spliced,andpolyadenylatednoncodingRNAthatispredominantlycytoplasmic[15, 16].AlthoughH19hasbeenintensivelystudiedinthefieldofgenomicimprinting,thebiologicalfunctionofH19asanon-codingRNAhasonlyrecentlybeguntobeelucidated.AccumulatingevidenceinrecentstudieshasconsistentlydemonstratedthattheexpressionlevelsofH19areupregulatedinavarietyofcancertypes,includinggastriccancer[17],esophagealcancer[18]colorectalcancer[19–21],breastcancer[22, 23],bladdercancer[24, 25],andhepatocellularcarcinoma[26, 27].TheupregulationofH19incancertissuessuggestsitspossibletumorigenicproperties,althoughthedetailedmolecularmechanismremainstobeinvestigated.

Inthepresentstudy,wefoundthatthelncRNAH19wasupregulatedinLSCCandthattheexpressionlevelsofH19wereinverselycorrelatedwiththesurvivalrateofLSCCpatients.Consistently,wefoundthatknockdownofH19expressioninhibitedLSCCcellproliferation,migrationandinvasion.WeidentifiedmicroRNAmiR-148a-3pasatargetforH19.TheexpressionofmiR-148a-3pwasinhibitedbyH19,andtheoverexpressionandtheinhibitionofmiR-148a-3pwererespectivelyassociatedwithreducedandelevatedLSCCproliferation,migrationandinvasion.Importantly,theinhibitionofLSCCprogressioninducedbyH19knockdownrequiredtheactivityofmiR-148a-3p.WealsodeterminedthatDNAmethyltransferaseenzymeDNMT1asaninhibitorytargetofmiR-148a-3p.CellularDNAmethylationwasinhibitedbybothmiR-148a-3poverexpressionandH19knockdown.Takentogether,ourstudydemonstratedthatthelncRNAH19couldpromoteLSCCprogressionviamiR-148a-3pandDNMT1,andthatDNAmethylationwasinvolvedintheregulatorymechanism.

RESULTS

H19wasupregulatedinLSCCandwasinverselycorrelatedwithpatientsurvivalrate

SincelncRNAshavebeenimplicatedinthedevelopmentofcancers,wefirstsoughttoexaminethelandscapeoflncRNAgeneexpressioninLSCCwithahybridizationmicroarraydesignedforlncRNAs.WefoundthattheexpressionlevelsofH19weresignificantlyupregulatedinLSCCtissuesamplescomparedtothoseinnormaltissues（p <0.001, Figure1A）.WithprimersspecifictoH19,wevalidatedourfindingsbyqPCRanalysis,andfoundthatH19levelsweresignificantlyhigher（5.54-fold）inLSCCtumortissuesthanthoseinadjacentnon-neoplastictissues（3.342±1.436versus0.596±0.259）（p <0.01, Figure1B）.Furthermore,wedeterminedthattheexpressionlevelsofH19weresignificantlycorrelatedwiththeprogressionofLSCC,includingtumorgrade,differentiation,necknodalmetastasis,andclinicalstage（Table1）.BasedonthelevelsofH19expression,wecategorized82LSCCpatientsintohigh（n =41）andlow（n =41）H19expressiongroups.WithKaplan-Meieranalysis,wefoundthatpatientswithhighH19expressionhadsignificantlypooreroverallsurvivalratecomparedtothosewithlowH19expression（χ2 =8.704, p =0.003）（Figure1C）.Takentogether,theseresultsindicatedthatH19wasupregulatedinLSCCandwaspositivelycorrelatedwithLSCCprogression.

Figure1:

H19isupregulatedinLSCCandisinverselycorrelatedwithpatientsurvivalrate.（A）BoxplotofH19expressionlevelsinLSCCtissuesandadjacentnon-neoplasticnormaltissuesasdeterminedbylncRNA-specificmicroarrayanalysis（p<0.001）.（B）RealtimePCRanalysisofH19expressionlevelsinLSCCtissuesandadjacentnon-neoplasticnormaltissues（**p<0.01）.（C）TheKaplan-MeieroverallsurvivalratecurveforLSCCpatients（n=82）withhighandlowH19expressionlevels（p=0.003）.

Table1:

RelationshipbetweenH19expressionlevelandclinicopathologicparametersofLSCC

Characteristics（n）

H19level

P

Sex

0.638

Male（55） 

3.295±1.580

Female（27） 

3.440±1.142

Age

0.122

≥58（41） 

3.590±1.426

 <58（41）

3.095±1.438

Tclassification

<0.01

T1–2（49） 

2.890±1.333

T3–4（33） 

4.014±1.357

Differentiation

0.017

G1（58） 

3.0681.332

G2（24） 

3.9931.553

Lymphnodemetastasis

<0.01

Negative（52） 

2.825±1.281

Positive（30） 

4.240±1.278

Primarylocation

0.103

Supraglottic（35） 

3.639±1.357

Glottic（47） 

3.121±1.482

Clinicalstage

<0.01

I–II（45） 

2.867±1.356

III–IV（37） 

3.92±1.351

H19knockdowninhibitedLSCCcellmigration,invasionandproliferation

InordertoinvestigatethefunctionofH19inLSCCdevelopment,weknockeddowntheexpressionofH19bytransfectinglentivirusesencodingcontrolshRNAorH19shRNAintoHep-2cells,awell-establishedLSCCcellline.TheH19expressionlevelsweresignificantlyreducedbythistreatmentafter24h（p <0.01, Figure2A）.Wethenperformedwoundhealingcellmigrationassayonthesecells.WefoundthatthemigrationofHep-2cellswassignificantlyinhibitedbyH19knockdown（p <0.05, Figure2B）.Wealsoperformedtranswellassaytoexaminecellinvasionability,andfoundthatcomparedtoshRNAcontrol,theshRNAtargetingH19ledtosignificantlydecreasednumberoftransmembranecells（p <0.01, Figure2C）.Moreover,byMTSassay,wediscoveredthatH19knockdownalsosignificantlyinhibitedcellproliferation（p <0.05, Figure2D）.Furthermore,wegeneratedLSCCstemcells（LSCC-SCs）fromLSCCpatientandknockeddownH19expression（Figure2E）.TheseLSCC-SCsweresubjectedtosphereformationandMTSassaystoexaminewhetherH19influencesLSCC-SCproliferation.TheresultsrevealedthatdownregulatedofH19significantlysuppressedLSCC-SCgrowth（Figure2F and 2G）.Takentogether,decreasedH19expressionledtoimpairedcellmigration,invasionandproliferationinLSCCcells.

Figure2:

H19knockdowninhibitsLSCCcellmigration,invasionandproliferation. （A）H19expressionlevelsinHep-2cellslentivirusesencodingcontrolshRNAorH19shRNA.（B）Woundhealingcellmigrationassay,（C）Transwellcellinvasionassayand（D）MTScellproliferationassayinHep-2cellstransfectedwithlentivirusesencodingcontrolshRNAorH19shRNA.（E）H19expressionlevelsinLSCC-SCstransientlytransfectedwithcontrolsiRNAorH19siRNA.（F）SphereformationinLSCC-SCstransfectedwithcontrolsiRNAorH19siRNA.（G）MTScellproliferationassayinLSCC-SCstransfectedwithcontrolsiRNAorH19siRNA.（H）H19expressionlevelsinsubcutaneousxenograftLSCCtumorstransfectedwithlentivirusesencodingcontrolshRNAorH19shRNA.（I）SubcutaneousxenograftLSCCtumorsdevelopedinnudemicefromHep-2cellstransfectedwithlentivirusesencodingcontrolshRNAorH19shRNA.（J）Weightquantificationoftumortissuesdepictedin（I）.（K）ImmunohistochemistrystainingofBrdUintumortissuesdepictedin（I）.Scalebar=100μm.（L）QuantificationofBrdUpositivecells.*p <0.05and**p <0.01comparedtothecontrolgroup.

Inadditionto invitro experiments,weusedamousexenograftmodeltostudytheoncogenicroleofH19inLSCCdevelopment invivo.Allmicesubcutane