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2 01 년6

2 월

박 사학 위논 문

Promotion of odontoblastic cell differentiation via Wnt1 signaling by microRNA-277

조 지

2016년 2월 박사학위논문

Promotion of odontoblastic cell differentiation via Wnt1 signaling by microRNA-27

조선대학교 대학원

치 의 학 과

조 지 호

Promotion of odontoblastic cell differentiation via Wnt1 signaling by microRNA-27

MicroRNA-27의 Wnt1 신호전달을 통한 상아질모세포 분화 촉진

2016년 2월 25일

조선대학교 대학원

치 의 학 과

조 지 호

Promotion of odontoblastic cell differentiation via Wnt1 signaling by microRNA-27

지도교수 김 수 관

이 논문을 치의학 박사학위신청 논문으로 제출함.

2015년 10월

조선대학교 대학원

치 의 학 과

조 지 호

조지호의 박사학위 논문을 인준함

위원장 한림대학교 교 수 박 준 우 인

위 원 조선대학교 교 수 김 동 기 인

위 원 조선대학교 교 수 전 홍 성 인

위 원 조선대학교 교 수 김 도 경 인

위 원 조선대학교 교 수 김 수 관 인

2015년 12월

조선대학교 대학원

TABLE OF CONTENTS

TABLE OF CONTENTS

LI ST OF FI GURES

ABSTRACT

I .I NTRODUCTI ON

I I .MATERI ALS AND METHODS

1.Cellcultures 3

2.miRNA isolationandAffymetrixmiRNA arrayanalysis 3 3.Quantitativerealtime-PCR (qRT-PCR)andquantitativePCR (qPCR) 4 4.miRNAsandtransfection 5 5.AlizarinredS staining 5 6.Cellproliferationassay 6

7.Immunoblotting 6

8.Vectorconstructionandluciferaseactivityassay 7 9.Statisticalanalysis 8

Ⅲ.RESULTS

1.miR-27expressionissignificantlyup-regulatedduringMDPC-23

odontoblasticcelldifferentiation 9 2.miR-27acceleratesmineralizationinMDPC-23odontoblasticcells 9 3.miR-27up-regulationinMDPC-23cellspromotesdifferentiationby

increasingtheWnt1expression 10

Ⅳ.DI SCUSSI ON

Ⅴ.REFERENCES

Ⅵ.FI GURE LEGENDS

Ⅶ.FI GURES

ABSTRACT i nKOREAN

LI ST OF FI GURES

Fig.1.The relative expression ofmiR-27 during MDPC-23 odontoblastic cell

differentiation 21

Fig.2.The effectofmiR-27 on themineralization in MDPC-23 odontoblastic

cells 22

Fig.3.TheeffectofmiR-27onALP,ColI,DMP-1andDSPP mRNA

expressions 23

Fig.4.ThemeasurementofMDPC cellproliferationbyup-regulatedmiR-27 24

Fig.5.TheeffectofmiR-27ontheexpressionofWnt1duringMDPC-23 odontoblasticcelldifferentiation 25

Fig.6.TheactivationofWnt1signalingbymiR-27 26

ABSTRACT

Promotion ofodontoblasticcelldifferentiation viaWnt1signaling by microRNA-27

Cho,Ji-Ho

Advisor:Prof.Kim,Su-Gwan,D.D.S.,Ph.D.

DepartmentofDentistry,

GraduateSchoolofChosun University

MicroRNA (miRNA,miR)is essentialin regulating celldifferentiation either by inhibiting mRNA translation orby inducing its degradation.However,the roleofmiRNA in odontoblasticcelldifferentiation isstillunclear.In thisstudy, therefore, the molecular mechanism of miR-27 on regulating odontoblast differentiation was examined in MDPC-23 mouse odontoblastic cells derived from mousedentalpapillacells.

In thepresentstudy,theresultsdemonstratethattheexpression ofmiR-27 increased significantly during MDPC-23 odontoblastic cell differentiation.

Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 cells and accelerates mineralization without cellproliferation.The over-expression ofmiR-27 significantly increased the expression levels ofWnt1 mRNA and protein.In addition,thetargetgeneprediction resultsrevealed that the mRNA of Wnt1 has an miR-27 binding site in its 3’UTR and is increasedbymiR-27.

These results suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting Wnt1 signaling.Therefore,miR-27 is a critical candidate as an odontoblastic differentiation molecular target for the developmentofmiRNA basedtherapeuticagentsindentalmedicine.

---

Keywords: miR-27, odontoblasts, differentiation, Wnt1

I.INTRODUCTION

Dentin forms the bulk of the tooth,and is a mineralized tissue formed by odontoblasts [1].Odontoblasts are differentiated from ectomesenchymalcells,and are involved in the secretion of the organic matrix during odontoblast differentiation [1,2]. This matrix contains a mixture of collagenous and non-collagenous proteins,and itbecomes mineralized and forms dentin,the main hard tissueofatooth [1,2].Many researcherssuggestthatsignaling moleculesin the bone morphogenetic protein, fibroblast growth factor and wingless (Wnt) families as well as transcription factors such as Runx2 are involved in the odontoblast differentiation [2-4]. However, the exact molecular mechanisms underlyingodontoblastdifferentiationarestillunclear.

CanonicalWntsignaling isvery importantforthedifferentiation ofseveralcells including odontoblast[4,5].Wntligands bind a heterodimeric complex formed by the LRP5/6 co-receptor and a member of the frizzled receptor families [4]. Activation ofthe receptorinhibits the destruction complex such as adenomatous polyposis coli(APC),anaphase-promoting complex,and glycogen synthase kinase 3b (GSK3b)[4].Wntsignaling mustbetightly regulated forproperdifferentiation [4,5]. However, the exact molecular mechanisms of Wnt signaling underlying odontoblastdifferentiationarestillunclear.

MicroRNA (miRNA,miR)isanendogenous,smallnon-codingsinglestrandRNA thathavebeenshowntoplayaroleinnumerouscellularprocess,includingcellular differentiation [6],organism development [7],proliferation [8] and apoptosis [9].

Therefore,currentresearch focuses on the utility ofmiRNAs as diagnostic and prognostic tools as wellas potentialtherapeutic targets.Despite the numerous studiesonmiRNAs,theirbiologicalfunctionsormechanismsofactionarenotwell understood.

There is a few study thatpost-transcriptionalregulation of gene expression, mediated by miRNAs, plays an important role in the control of odontoblast differentiation [2,10,11].Liu etal.showed thatthe miR-143 and miR-145 control odontoblastdifferentiation and dentin formation through Krüppel-like factor4 and Osterix transcriptionalfactor signaling pathways [11].Sun et al.reported that miR-338-3p promote odontoblast differentiation through targeting Runx2 [2]. miR-27 could regulate adipogenesis, myeloblasts differentiation, osteoblast differentiation,and skeletalmuscle development[4,12,13].Nevertheless,atpresent, theroleofmiR-27inregulatingodontoblastdifferentiationremainsunknown.

In this study,therefore, the molecular mechanism of miR-27 on regulating odontoblastdifferentiation wasinvestigated in MDPC-23mouseodontoblastic cells derivedfrom mousedentalpapillacells.

II.MATERIALS AND METHODS

1.Cellcultures

MDPC-23 odontoblastic cells provided by Dr.J.E.Nör(University ofMichigan, Ann Arbor, MI, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% heatinactivated fetalbovine serum (FBS)and antibiotics(Invitrogen,Carlsbad,CA,USA)in a5% CO2atmosphereat37^oC.To inducecelldifferentiation andmineralization,confluentMDPC-23cellsweretreated with 50 ㎍/mlascorbic acid and 10 mM β-glycerophosphate forup to 10 days [14].

2.miRNA isolation andAffymetrix miRNA array analysis

TotalRNA including miRNAs from MDPC-23 cells from days 0,4 and 7 of differentiation wereisolated with miRNeasy minikit(Qiagen,Valencia,CA,USA) following themanufacturer’sinstructions.Theconcentration,purity and amountof totalRNA werequantifiedusingtheNano-DropND-1000ultravioletSpectrophotometer (ThermoScientific,Wilmington,DE,USA).ThemiRNA array wasscanned using an Affymetrix GeneChipPlatform (DNA link,Seoul,Korea).Foreachsample,total RNA wassubjectedtoatailingreaction(2.5mM MnCl2,ATP,PolyA Polymerase, incubation for 15 min at37^oC) followed by ligation ofthe biotinylated signal molecule to the targetRNA sample (16 Flash Tag ligation mix biotin,T4 DNA ligase,incubationfor30minatroom temperature)andthereaction

was terminated with addition ofstop solution.Each sample was hybridized to a GeneChipH miRNA Array at48^oC and60rpm for16h,then washedandstained on Fluidics Station 450 and scanned on a GeneChipScanner3000 7G (Affymetrix, Fremont,CA,USA).The image data were analyzed with the miRNA QC Tool softwareforqualitycontrol.

3.Quantitativerealtime-PCR (qRT-PCR)andquantitativePCR (qPCR)

Reverse transcription ofthe miRNA was performed using a miScriptReverse Transcription kit(Qiagen,Valencia,CA,USA)starting from 1 ㎍ oftotalRNA.

TaqMan miRNA assayskits(ABI,Carlsbad,CA,USA)wereused toexaminethe specificmiRNA expression by qRT-PCR according to themanufacturer’sprotocol. The qRT-PCR results,which were recorded as threshold cycle numbers,were normalized againstan internalcontrol(U6 RNA),and the comparative threshold cyclemethodwasusedtodeterminethelevelsofmiRNA expression.Thelevelof miR-27 (5’-AGGGCTTAGCTGCTTGTGAGCA-3’) was measured by qRT-PCR.

Thedeviationsinthesamplesrepresentfourindependentexperiments.

Toperform qPCR,thetotalRNA wasisolatedusing TRIzolreagent(GibcoBRL, Grand Island, NY, USA) according to the manufacturer’s instructions. Reverse transcription was carried out with 1 ㎍ of totalRNA using the TermoScript RT-PCR system (Invitrogen, Carlsbad, CA, USA). The levels of alkaline phosphatase (ALP) (ALP-F, 5’-CTCTCCGAGATGGTGGAGGT-3’; ALP-R, 5’-GTC TTCTCCACCGTGGGTCT-3’),type Icollagen (ColI)(ColI-F,5’-TAAGTTGCC AAGAACGTGCC-3’; Col I-R, 5’-AATTGAAAGCCAGGAGGCAT-3’), dentin matrix

protein 1 (DMP-1) (DMP-1-F,5’-CGGCTGGTGGTCTCTCTAAG-3’; DMP-1-R, 5’-ATCTTCCTGGGACTGGGTCT-3’),dentin sialophosphoprotein (DSPP) (DSPP-F, 5’-ATAGCACCAACCATGAGGCT-3’; DSPP-R, 5’-CTTTTGTTGCCTTTGTTGGG –3’), Wnt1 (Wnt1-F, 5’-CAGCGTTCATCTTCGCAATCACCT-3’; Wnt1-R, 5’-AAGGTTCATGAGGAAGCGTAGGTC–3’) and GAPDH (GAPDH-F, 5’-TGC ATCCTGCACCACCAACT-3’; GAPDH-R, 5’-CGCCTGCTTCACCACCTTC-3’) induction were measured by qPCR, and visualized by DNA agarose gel electrophoresis.Thedifferencesin expression werepresented asahistogram after densitometryusingaVersaDoc™ imagingsystem (Bio-Rad,Hercules,CA,USA).

4.miRNAsandtransfection

miR-27andscrambledmiR-27werepurchasedfrom Ambion(Austin,TX,USA). The miR-27,mimic miR-27 and pGL3-promoter-Wnt1-3’UTR were transfected into cultured MDPC-23cellsusing Lipofectamine^™ 2000(Invitrogen,Carlsbad,CA, USA)according tothemanufacturer’sinstruction.Theexperimentalgroup ofcells wastreatedwith10ng/mlmiRNAsfor2days.

5.Alizarin redS staining

Thecellswerefixedwith 70% ethanolfor20min andstainedwith1% Alizarin redS (Sigma–AldrichCorp.,St.Louis,MO,USA)in0.1% NH4OH atpH 4.2–4.4.

ThemineralizationassayswereperformedbystainingMDPC-23cellswithAlizarin red S solution.Thecellswereevaluated at0,4,7and 10days.To quantify the

intensity ofmineralization,wemeasured density ofstained nodulesby colorimetric spectrophotometry.Thestainedcellswerecollectedby centrifugation at13,000rpm for10min at4^oC.Celllysatewassolubilized with 0.5 mlof5% SDS in 0.5 N HClfor30min atroom temperature.Solubilized stain (0.1ml)wastransferred to wellsofa96-wellplate,andabsorbancewasmeasuredat405nm.

6.Cellproliferation assay

Thecellswereseeded ata density of4X 10⁴cells/wellin 24-wellplatesand allowed to attach to the wellovernight.Afterincubation,the cultured cells were transfectedwithmiR-27using Lipofectamine^™ 2000.Thecellswereincubatedwith miR-27atvariousdefined concentrations(2,20and 200ng/ml)for1and 2days at37^oC.Afterincubationunderthedefinedconditions,thecellswereincubatedfor another 4 h in 20 ㎕ of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT).Todissolvetheformazan transformedfrom MTT,the cellswereresuspendedin 150㎕ dimethylsulfoxideand theopticaldensity ofthe solutionwasdeterminedusing aspectrometeratanincidentwavelengthof495nm [15].Theexperimentswererepeatedfourtimes,independently.

7.Immunoblotting

TodeterminethelevelofWnt1in MDPC-23cellstransfected with miR-27,the proteinswereextracted.Thecellswerewashedtwicewithice-coldPBS andlysed using a RIPA buffer for protein extraction according to the manufacturer’s

instructions.Thetotalproteinconcentrationsweredeterminedusing abicinchoninic acid protein assay (Pierce,Rockford,IL,USA).An equalamountofprotein was resolved by 10% SDS–PAGE and transferred to a nitrocellulose membrane for immunoblotting.The anti-Wnt1 (1:1000 dilution;Santa Cruz Biotechnology,Santa Cruz,CA,USA)antibodywasusedastheprimaryantibody.Theimmunoreactivity was visualized using an ECL system (Amersham Biosciences,Piscataway,NJ, USA)andaSignalVisualEnhancersystem (Pierce,Rockford,IL,USA)wasused tomagnifythesignal.

8.Vectorconstruction andluciferaseactivity assay

The3’UTR ofWnt1wasamplified from genomicDNA isolated from MDPC-23 cells using the QIAamp DNA minikit(Qiagen)according to the manufacturer’s instruction,using Wnt1-3’UTR forward primer (5’-CCGCTCGAGGACCCAGCCTAC CTCCCT-3’) and Wnt1-3’UTR reverse primer (5’-ATAGTTTAGCGGCCGCTCGTT GATAAAAATAGATATT-3’). pGL3-promoter-Wnt1-3’UTR was constructed by cloning the 3’UTR ofWnt1 (736 bp)into the downstream ofthe luciferase gene in the pGL3-promotervectorforluciferase activity assay.Forluciferase activity assay,MDPC-23 cells were cultured in 24-wellplates and co-transfected with 5 ng pGL3-promoter-Wnt1-3’UTR,5 ng Renilla for normalization,and 100 ng of miR-27-wild type or same amount of synthesized mimic miR-27. Luciferase activity was measure 2 days aftertransfection using the Dual-luciferase reporter assay system (Promega,Madisom,WI,USA) according to the manufacturer’s instruction.

9.Statisticalanalysis

Allexperimentswereperformedatleastintriplicate.Theresultswerepresented asthemean ± SEM.Statisticalsignificancewasanalyzedusing aStudent'st-test fortwogroupsandoneway analysisofvarianceformulti-groupcomparisons.All statisticalanalyses wereperformed using SPSS version 12.0(SPSS Inc.,Chicago, IL,USA).A pvalue<0.05wasconsideredstatisticallysignificant.

Ⅲ.RESULTS

1. miR-27 expression is significantly up-regulated during MDPC-23 odontoblasticcelldifferentiation.

The expression pattern of miRNA during MDPC-23 cell differentiation was determined by miRNA microarray with cellsamples from days 0,4 and 7 of differentiation (n = 4)using Affymetrix Genechip.This experimentshowed that miR-27 in MDPC-23 cells from days 4 and 7 ofdifferentiation was significantly up-regulatedover3foldcomparedtocontrolcellsofday 0(Fig.1A).ThemiRNA arraydatawasverifiedby examining theexpressionpatternofmiR-27bymiRNA qRT-PCR. In miRNA qRT-PCR analysis, miR-27 levels were increased in MDPC-23 cells maintained in odontoblastic celldifferentiation medium (Fig.1B). TheseresultsshowedthatmiR-27wassignificantlyup-regulatedduring MDPC-23 celldifferentiation and over-expression ofmiR-27 enhanced thecelldifferentiation processinMDPC-23cells.

2.miR-27acceleratesmineralization in MDPC-23odontoblasticcells.

The effect of miR-27 on odontoblastic cell mineralization was measured.

MDPC-23cellswerecultured for10daysin differentiation mediatransfectedwith miR-27and themineralized noduleswereevaluated by Alizarin red S staining.In controlMDPC-23cells,mineralized nodulesappeared after7 daysofculture.The

miR-27 transfected MDPC-23 cells showed mineralized nodules after4 days,and themineralizednodulesincreasedwithtimeduringthecultureperiod(Fig.2).

To study the potentialrole of miR-27 in MDPC-23 differentiation,the cells transfected with miR-27 were analyzed.The qPCR results showed thatmiR-27 enhanced the differentiation ofMDPC-23 odontoblastic cells,indicated by higher expression oftheodontoblastmarkergenesALP,ColI,DMP-1and DSPP,along withanincreaseintheiractivitiescomparedtocontrolcells(Fig.3).

Asshown in Fig.4,theup-regulated miR-27did notalterthecellproliferation intheMDPC-23cells.

Taken together,these data were suggesting that miR-27 was promoting the odontogenic specific differentiation without alteration the cell proliferation in MDPC-23.

3.miR-27 up-regulation in MDPC-23 cells promotes differentiation by increasing theWnt1expression.

The molecular mechanisms by which miR-27 promotes differentiation of MDPC-23cellswereinvestigated.PutativemiR-27targetswerepredicted using a targetprediction program,TargetScan,which revealed thatthe 3’UTR ofWnt1 mRNA contains a complementary site for the miR-27.Accordingly,qPCR and immunoblottingwereperformedtoobservethealterationofWnt1expressioninthe MDPC-23 cells after the transfection of miR-27. As shown in Fig. 5, the over-expression ofmiR-27 significantly increased the expression levels ofWnt1 mRNA (Fig.5A)andprotein(Fig.5B)comparedtocontrol.

The 3’UTR of Wnt1, containing the miR-27 binding sites, was subcloned downstream of a luciferase reporter gene in pGL3 promoter vector and co-transfected with miR-27 or mimic miR-27 into MDPC-23 cells followed by luciferaseactivity assay in orderto determinewhetherWnt1isadirecttargetof miR-27(Fig.6A).Theluciferaseactivity assay showedthattherelativeluciferase activity increased significantly by miR-27 compared to pGL vector alone, pGL3-Wnt1 3’UTR and mimic miR-27 (Fig.6B).These results hypothesized that theup-regulated Wnt1 expression by miR-27 inducesthepromotion ofMDPC-23 odontoblasticcelldifferentiation.

Ⅳ.DISCUSSION

In recent years,the role of miRNA has been studied in biological events, including celldifferentiation [6],development[7],proliferation [8],adipogenesis[16]

and osteoblastogenesis [17].However,the physiologicalrole of miR-27 in the regulationofodontoblastdifferentiationisnotentirelyclear.Inthisstudy,therefore, the molecularmechanism ofmiR-27 on regulating odontoblastdifferentiation was examined in MDPC-23 odontoblastic cells.This study reports here thatmiR-27 promotedodontoblastdifferentiationbymodulatingWnt1signalpathway.

In the present study,the results were shown that miR-27 was significantly up-regulated during MDPC-23 celldifferentiation and over-expression ofmiR-27 enhanced the celldifferentiation process in MDPC-23 cells (Fig.1).In addition, over-expressed miR-27 accelerated mineralization in MDPC-23 cells (Fig.2)with increase of ALP mRNA and ColImRNA (Fig.3).Furthermore,to determine whether miR-27 induces the odontogenic specific differentiation in MDPC-23,it wasmeasured theexpressionallevelofDMP-1and DSPP,which arewellknown arepresentativemarkertoidentify theodontogenicdifferentiation [2,10].Asshown in PCR results,theexpressionsofDMP-1andDSPP weregradually up-regulated in MDPC-23 cells transfected with miR-27 (Fig. 3). On the other hand, over-expressed miR-27 did notalterthe cellproliferation in the MDPC-23 cells (Fig.4).Takentogether,thesedatasuggestthatmiR-27mightbeaccelerating the differentiationofMDPC-23cellswithoutincreasingcellproliferating.

Although the roles ofmiRNAs associated celldifferentiation were demonstrated

inseveralcelltypes,theroleofmiR-27inducedodontoblasticcelldifferentiation is notfully understood [10-12].However,ithasbeen reported thatWnt/β-catenin is an upstream regulatory molecules for DSPP for dentin mineralization in mouse dentalpaollarcells [18].Moreover,Koizumin etal.,have been reported thatthe expressionofDSPP andDMP-1inodontogenicdifferentiatingbybonemorphogenic protein 2wasmediatedby Wnt/β-cateninsignaling pathway [19].Consequently,in the present study, to investigate the cellular mechanism of miR-27-induced odontoblasticcelldifferentiation,themiR-27targetgeneprediction associated with differentiation using TargetScan program was performed.The results ofmiR-27 target gene prediction revealed that Wnt1 was a prime molecule to promote ondontoblastdifferentiation.In addition,theover-expression ofmiR-27significantly increased theexpression levelsofWnt1mRNA and protein (Fig.5and 6),which is suggesting that up-regulated Wnt1 by miR-27.Recently,itis reported that miR-27 promotes osteoblastdifferentiation by modulating Wntsignaling through theexpressionaldown-regulation ofAPC in thehuman fetalosteoblastic1.19 cell line [4].These data suggested that up-regulated miR-27 inMDPC-23 mouse odontoblasticcellsderived from mousedentalpapilla cellspromotesodontoblast differentiation by targeting Wnt1 signaling. However, the mechanisms of odontoblastdifferentiation induced by miR-27 are notyetcompletely understood.

Further studies will reveal the precise cellular and molecular mechanisms of odontoblastdifferentiationinducedby miR-27.Overall,miR-27mightbeconsidered a criticalcandidateasan odontoblasticcelldifferentiation moleculartargetforthe developmentofmiRNA basedtherapeuticagentsinthedentalmedicine.

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9.Lynam-Lennon N,MaherSG,ReynoldsJV.TherolesofmicroRNA in cancer and apoptosis. Biol Rev Camb Philos Soc. 2009;84:55-71. doi: 10.1111/j.1469-185X.2008.00061.x.

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19.KoizumiY,Kawashima N,Yamamoto M,Takimoto K,Zhou M,SuzukiN, Saito M, Harada H, Suda H. Wnt11 expression in rat dental pulp and promotionaleffects ofWnt signaling on odontoblastdifferentiation.Congenit Anom.2013;53:101-108.doi:10.1111/cga.12011.

Ⅵ.FIGURE LEGENDS

Fig.1.Therelativeexpression ofmiR-27during MDPC-23odontoblasticcell differentiation.Confluentcultures ofMDPC-23 cells were maintained in complete medium with the addition of differentiation cocktail(50 ㎍/ml ascorbic acid and 10 mM β-glycerophosphate). Cells were harvested following 0,4 and 7 days incubation and endogenous expression levelof miR-27wasmeasured.TherelativeexpressionofmiR-27wasaccessedby miRNA array using affymetrix Genechip (A)and by qRT-PCR (B).Each data pointrepresents the mean ± SEM offourexperiments.^**p<0.01 or

***p<0.001vs.day0.

Fig.2.TheeffectofmiR-27onthemineralizationinMDPC-23odontoblasticcells.

MDPC-23 odontoblastic cellmineralization was altered by over-expressed miR-27.(A) Mineralized nodule formation in MDPC-23 cells.MDPC-23 cellsweretransfectedwithmiR-27for10days,andthemineralizationwas evaluated by Alizarin red S staining.(B)Quantification ofmineralization wasaccessedbycolorimetricspectrophotometry.Eachdatapointrepresents themean± SEM ofthreeexperiments.^*p<0.05or^**p<0.01vs.control(the controlcellsweremeasuredintheabsenceofmiR-27transfection).

Fig.3.TheeffectofmiR-27onALP,ColI,DMP-1andDSPPmRNA expressions.

TotalRNA wasisolatedusingTRIzolreagentandreversetranscriptionwas carriedoutwith1㎍ oftotalRNA usingtheTermoScriptRT-PCR system.

The PCR products were electrophoresed on a 1.5% agarose gel and visualizedwithethidium bromide.

Fig.4.ThemeasurementofMDPC cellproliferationbyup-regulatedmiR-27.The cells wereincubated with miR-27 atvarious defined concentrations (2,20 and 200 ng/ml) for 1 and 2 days at37^oC.The cellproliferation was determined by MTT assays after miR-27 transfection.The experiments wererepeatedfourtimes,independently.

Fig.5.The effectofmiR-27 on the expression ofWnt1 during MDPC-23 odontoblastic cell differentiation. The over-expression of miR-27 significantly increased the expression levels of Wnt1 mRNA (A) and protein (B) compared to control.MDPC-23 cells were transfected with miR-27 according to the defined conditions.(Lower panel) Quantitative datafor(Upperpanel)wereanalyzed by using Imagegauge3.12software afterGAPDH orβ-actinnormalization.

Fig.6.TheactivationofWnt1signalingbymiR-27.(A)Theschematicdiagram to generatethereportervector.The3’UTR ofWnt1wascloned by PCR and subclond into the downstream of luciferase gene in the pGL3 promoter vector.(B)The luciferase activity ofgenerated reportervectorcontaining

3’UTR ofWnt1 was significantly up-regulated by miR-27.MDPC-23cells were co-transfected with generated reporter vector containing 3’UTR of Wnt1,miR-27 and mimic miR-27 according to the defined condition.And then luciferase activity was accessed by manufacturer’s instruction.Each data pointrepresents the mean ± SEM ofthree experiments.^*p<0.05 vs. otherexperimentalgroups.

Ⅶ.FIGURES

Fig.1.Therelativeexpression ofmiR-27during MDPC-23odontoblasticcell differentiation.

Fig.2.TheeffectofmiR-27onthemineralizationinMDPC-23odontoblasticcells.

Fig.3.TheeffectofmiR-27onALP,ColI,DMP-1andDSPP mRNA expressions.

Fig.4.ThemeasurementofMDPC cellproliferation by up-regulatedmiR-27.

Fig.5.The effectofmiR-27 on the expression ofWnt1 during MDPC-23 odontoblasticcelldifferentiation.

Fig.6.TheactivationofWnt1signaling by miR-27.

ABSTRACT i n KOREAN

MicroRNA-27의 Wnt1신호전달을 통한 상아질모세포 분화 촉진

조 지 호

조선대학교 대학원 치의학과 ( 지도교수:김 수 관)

상아질은 치아의 가장 중요한 구성 성분으로 치관과 치근의 대부분을 구성하며,상 아질모세포는 제Ⅰ형과 Ⅱ형 교원질과 같은 유기기질과 당단백 및 dentin sialophosphoprotein을 합성하고 분비한다.상아질의 기질이 침착됨에 따라 상아질모세 포가 세포돌기를 원심 쪽으로 내어 상아세관에 묻히게 되고,결과적으로 상아질모세포 는 치수 가장자리 상아질의 내면에 위치하여 상아질을 유지하게 된다.그러나 상아질 모세포의 분화기전과 상아질의 형성과정을 조절하는 인자 그리고 그와 관련된 분자생 물학적 기전은 아직까지 명확히 알려져 있지 않다.생체에서 내인성으로 존재하는 microRNA(miRNA,miR)는 21-25개의 염기서열로 구성된 매우 작은 noncoding RNA 분자로서,RNA 간섭을 통하여 그 기능을 나타내며 주로 표적 유전자의 3’-UTR 결

합부위에 상보적인 염기서열을 가지면서 그 표적유전자의 단백합성을 억제하거나 촉진 시킨다.또한 miRNA는 세포 내 다양한 유전자를 조절함으로써 세포분화,성장과 증식 등 대부분의 생명현상에 관여하는 것으로 보고되고 있다.따라서 본 연구에서는 상아 질모세포로서 MDPC-23세포주의 분화과정에서 miR-27에 의한 상아질모세포 분화의 촉진과 그 분자적 기전을 밝히고자 하였다.

상아질모세포의 분화과정에서 miR-27에 의한 상아질모세포의 분화기전을 분석하기 위해,생쥐 치유두세포 유래의 상아질세포 전구세포주인 MDPC-23세포주에서 분화유 도와 miR-27을 처리한 후,miRNA array분석,정량 PCR 분석,AlizarinredS 염색,세 포증식 분석,immunoblotting및 luciferaseactivity분석 등을 시행하였다.

MDPC-23 세포주의 분화과정에서 miR-27의 발현은 증가하였다. miR-27은 MDPC-23세포주의 석회화를 촉진시켰으나,MDPC-23세포주의 증식에는 영향을 주 지 않았다.또한 miR-27은 MDPC-23세포주의 분화과정에서 Wnt1mRNA와 단백의 발현을 증가시켰으며,MDPC-23세포주의 분화과정에서 luciferase활성을 증가시켰다.

본 연구의 결과로 상아질모세포 MDPC-23세포주의 분화과정에서 그 발현이 증가 된 miR-27은 Wnt1의 활성을 증가시켜 상아질모세포의 분화를 촉진시키는 것으로 사 료된다.

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중심어: miR-27, 상아질모세포, 세포분화, Wnt1