Methylation of a Panel of MicroRNA Genes Is a Novel Biomarker for Detection of Bladder Cancer

Transcription

1 EUROPEAN UROLOGY 6 (1) vilble t journl homepge: Urothelil Cncer Methyltion of Pnel of MicroRNA Genes Is Novel Biomrker for Detection of Bldder Cncer Tkshi Shimizu,b, Hiromu Suzuki b,c, *, Msnori Nojim c,d, Hiroshi Kitmur, Eiichiro Ymmoto b,c, Reo Mruym b,c, Msmi Ashid b, Tomo Hthir b, Mshiro Ki b, Noy Msumori, Tkshi Tokino e, Kohzoh Imi f, Tiji Tsukmoto, **, Minoru Toyot b Deprtment of Urology, Spporo Medicl University, Spporo, Jpn; b Deprtment of Moleculr Biology, Spporo Medicl University, Spporo, Jpn; c First Deprtment of Internl Medicine, Spporo Medicl University, Spporo, Jpn; d Deprtment of Public Helth, Spporo Medicl University, Spporo, Jpn; e Medicl Genome Science, Reserch Institute for Frontier Medicine, Spporo Medicl University, Spporo, Jpn; f Division of Novel Therpy for Cncer, The Advnced Clinicl Reserch Center, The Institute of Medicl Science, The University of Tokyo, Tokyo, Jpn Article info Article history: Accepted November 1, 1 Published online hed of print on November, 1 Keywords: Biomrker Bldder cncer DNA methyltion MicroRNA Urinry test Abstrct Bckground: Dysregultion of micrornas (mirnas) hs been implicted in bldder cncer (BC), lthough the mechnism is not fully understood. Objective: We imed to explore the involvement of epigenetic ltertion of mirna expression in BC. Design, setting, nd prticipnts: Two BC cell lines (T4 nd UM-UC-) were treted with 5-z- -deoxycytidine (5-z-dC) nd 4-phenylbutyric cid (PBA), fter which their mirna expression profiles were nlyzed using TqMn rry (Life Technologies, Crlsbd, CA, USA). Bisulfite pyrosequencing ws used to ssess mirna gene methyltion in 5 cncer cell lines, 8 primry tumors, nd 1 preopertive nd 47 postopertive urine smples. Outcome mesurements nd sttisticl nlysis: Receiver operting chrcteristic (ROC) curve nlysis ws used to ssess the dignostic performnce of the mirna gene pnel. Results nd limittions: Of 664 mirnas exmined, 146 were upregulted by 5-z-dC plus PBA. CpG islnds were identified in the proximl upstrem of mirna genes, nd 1 of those were hypermethylted in cell lines. Among them, mir-17, mir-14-, mir-14-, nd mir-9- were frequently nd tumor-specificlly methylted in primry cncers (mir-17: 68.7%; mir-14-: 5.6%; mir-14-: 65.1%; mir-9-: 45.8%). Methyltion of the sme four mirnas in urine specimens enbled BC detection with 81% sensitivity nd 89% specificity; the re under the ROC curve ws.916. Ectopic expression of silenced mirnas in BC cells suppressed growth nd cell invsion. Conclusions: Our results indicte tht epigenetic silencing of mirna genes my be involved in the development of BC nd tht methyltion of mirna genes could be useful biomrker for cncer detection. # 1 Europen Assocition of Urology. Published by Elsevier B.V. All rights reserved. * Corresponding uthor. Deprtment of Moleculr Biology, Spporo Medicl University, S1, W17, Chuo-Ku, Spporo , Jpn. Tel ; Fx: ** Corresponding uthor. Deprtment of Urology, Spporo Medicl University, S1, W16, Chuo-Ku, Spporo 6-854, Jpn. Tel ; Fx: E-mil ddresses: hsuzuki@spmed.c.jp (H. Suzuki), tijit@spmed.c.jp (T. Tsukmoto). -88/$ see bck mtter # 1 Europen Assocition of Urology. Published by Elsevier B.V. All rights reserved.

2 19 EUROPEAN UROLOGY 6 (1) Introduction MicroRNAs (mirnas) re group of smll noncoding RNAs tht negtively regulte the trnsltion nd stbility of prtilly complementry trget mrnas. In tht wy, they ply importnt roles in wide rry of biologic processes, including cell prolifertion, differentition, nd poptosis [1]. Incresing evidence suggests tht dysregultion of mirna expression contributes to the initition nd progression of humn cncer [,]. Altered mirna expression is thought to ply n importnt role in the pthogenesis of bldder cncer (BC) nd in certin tumor phenotypes. For instnce, high-grde BC exhibits upregultion of severl mirnas, including mir-1, which suppresses p5 function [4]. In ddition, mir-1-to-mir-5 expression rtios re elevted in invsive BC cells [5], while mir- fmily members regulte epithelil-to-mesenchyml trnsition by trgeting trnscription repressors ZEB1 nd ZEB in BC cells [6]. Although the mechnisms underlying mirna dysregultion in cncer re not yet fully understood, recent studies hve shown tht the silencing of severl mirnas is tightly linked to epigenetic mechnisms, including histone modifiction nd DNA methyltion [7,8]. For exmple, tretment with histone decetylse (HDAC) inhibitor nd DNA methyltrnsferse (DNMT) inhibitor restored expression of vrious mirnas in cncer cells [7,9], nd the list of mirna genes methylted in cncer is rpidly growing [1]. Studies hve lso shown tht restortion of epigeneticlly silenced mirnas my be n effective strtegy for treting cncer nd tht berrnt methyltion of mirna genes could be useful biomrker for cncer detection [1,11]. In ddition, it ws recently shown tht the silencing of mirna expression in BC is ssocited with DNA methyltion, often involving the CpG islnd (CGI) or CpG shore [1,1]. In n effort to identify novel biomrkers nd tretment trgets in BC, we imed to identify mirnas epigeneticlly silenced in BC cells by screening for mirnas whose expression is upregulted by DNA demethyltion nd HDAC inhibition. We lso investigted the methyltion of mirna genes in urine specimens nd ssessed its clinicl usefulness s biomrker for detection of BC.. Mterils nd methods.1. Cell lines nd tissue smples BC cell lines (T4, UM-UC-, HT-1197, HT-176, SW78, nd 567) nd norml urothelil cell line (SV-HUC-1) were obtined from the Americn Type Culture Collection (ATCC, Mnsss, VA, USA; Supplementry Tble 1). A colorectl cncer cell line HCT116 hrboring genetic disruptions within the DNMT1 nd DNMTB loci (DNMTs KO) hve been described previously [8]. T4 nd UM-UC- cells were treted first with 1 mm or.1 mm 5-z- -deoxycytidine (5-z-dC; Sigm-Aldrich, St Louis, MO, USA) for 7 h, nd then with mm 4-phenylbutyric cid (PBA; n HDAC inhibitor, Sigm-Aldrich) for 7 h, replcing the drug nd medium every 4 h. A totl of 8 primry BC specimens were collected from ptients who underwent rdicl cystectomy (RC) or trnsurethrl resection of bldder tumor (TURBT; 66 mles nd 17 femles; medin ge: 7 yr; rnge: 4 9 yr). Of the 8 ptients, 7 underwent surgicl resection fter initil dignosis, 7 received chemotherpy before surgery, nd re recurrentcses. Smples of nontumorous bldder tissue djcent (< cm) to nd distnt (> cm) from the tumors were lso collected. Six smples of norml urothelil tissue from renl cell crcinom (RCC) ptients who underwent nephrectomy were lso collected. Informed consent ws obtined from ll ptients before collection of the specimens, nd this study ws pproved by the institutionl review bord. Totl RNA ws extrcted using mirvn mirna isoltion kit (Life technologies, Crlsbd, CA, USA). Genomic DNA ws extrcted using the stndrd phenol-chloroform procedure... Urine smples Voided urine specimens were collected from cncer-free individuls (Supplementry Tble ) nd 86 BC ptients. In ddition, postopertive voided urine smples were collected from 6 of the 86 ptients 1 d fter TURBT tretment. As n independent test set, preopertive urine smples were collected from 4 BC ptients, nd postopertive smples were collected from 11 ptients. The postopertive urine smples were collected from ptients in whom tumors were successfully resected without leving residul tumors. The urine (1 ml) ws mixed with 5 ml of ThinPrep PreservCyt solution (Hologic, Bedford, MA, USA) nd stored t 4 8C. Ech smple ws centrifuged t rpm for 1 min, nd genomic DNA ws extrcted from the pelleted sediment using the stndrd phenol-chloroform procedure... MicroRNA expression profiling Expression of 664 mirnas ws nlyzed using TqMn MicroRNA rry v. (Life Technologies). Briefly, 1 mg of totl RNA ws reversetrnscribed using Megplex Pools kit (Applied Biosystems, Foster City, CA, USA), fter which mirnas were mplified nd detected using polymerse chin rection (PCR) with specific primers nd TqMn probes. U48 snrna (RNU48, Life Technologies) served s n endogenous control..4. Quntittive rel-time polymerse chin rection of mirna Expression of selected mirnas ws nlyzed using TqMn microrna ssys. Briefly, 5 ng of totl RNA were reverse-trnscribed using specific stem-loop rel-time primers, fter which they were mplified nd detected using PCR with specific primers nd TqMn probes. U6 snrna (RNU6B, Life Technologies) served s n endogenous control..5. Methyltion nlysis Bisulfite conversion of genomic DNA, methyltion-specific PCR (MSP), bisulfite sequencing, nd bisulfite pyrosequencing were crried out s described previously [8]. Primer sequences nd PCR product sizes re listed in Supplementry Tble. Primer loctions for methyltion nlysis re shown in Supplementry Figure Trnsfection of microrna precursor molecules BC cells (1 1 6 cells) were trnsfected with 1 pmol of Pre-miR mirna Precursor Molecules (Life Technologies) or Pre-miR mirna Molecules Negtive Control #1 using Cell Line Nucleofector kit R (Lonz, Bsel, Switzerlnd) with Nucleofector I electroportion device (Lonz) ccording to the mnufcturer s instructions. The vibility of the mirna precursor trnsfectnts ws nlyzed using wter-soluble tetrzolium slt (WST) ssys [8]. Cell invsion ws ssessed using Mtrigel invsion ssys [8].

3 EUROPEAN UROLOGY 6 (1) Gene expression microrry nlysis One-color microrry-bsed gene expression nlysis ws crried out ccording to the mnufcturer s instructions (Agilent Technologies, Snt Clr, CA, USA). Briefly, 1 ng of totl RNA were mplified nd lbeled using Low-input Quick AmpLbeling Kit One-color (Agilent Technologies), fter which the synthesized crna ws hybridized to SurePrint G Humn GE microrry (G4851F; Agilent Technologies). The microrry dt were then nlyzed using GeneSpring GX version 11 (Agilent Technologies). The Gene Expression Omnibus ccession number for the mirna microrry dt is GSE Sttisticl nlysis All dt were nlyzed using GrphPd Prism 4. sttisticl softwre (GrphPd Softwre, L Joll, CA, USA). Quntittive vribles were nlyzed using Student t test nd one-wy nlysis of vrince (ANOVA) with post hoc Tukey test. Fisher exct test ws used for nlysis of ctegoricl dt. The Person correltion coefficient ws used to evlute correltions between continuous dt. Receiver operting chrcteristic (ROC) curves for the dignosis of BC were constructed on the bsis of the methyltion levels, followed by clcultion of the re under the curve (AUC). The best cut-off vlue for ech mirna gene ws defined s the point on the ROC curve closest to the upper left corner. A dignostic scoring system using urinry DNA methyltion ws constructed by nlyzing the trining set using the following threestep lgorithm: (1) The methyltion sttus of mirna genes ws ssessed using the respective cut-off vlues; () the number of methyltionpositive genes ws determined, which we termed the mir-methyltion score (M-score); nd () the smples were clssified into five groups bsed on the M-score. The vlue of p <.5 (two-sided) ws regrded s significnt. [(Fig._1)TD$FIG] A UMUMUMUMUM UMUM mir-9-1 mir-9- mir-1b mir-4b mir-14-1 mir-14- mir-14- mir-17 mir-b mir- mir-49 mir-675. Results.1. Identifiction of epigeneticlly silenced microrna genes in bldder cncer To identify epigeneticlly silenced mirnas in BC, we performed TqMn rry nlysis using two BC cell lines (T4 nd UM-UC-) treted with 1 mm 5-z-dC plus mm PBA. Of the 664 mirnas exmined, the drug tretment induced upregultion (more thn five-fold) of 8 mirnas in T4 cells nd mirnas in UM-UC- cells. Of those, 146 mirnas were upregulted in both cell lines (Supplementry Fig. nd ; Supplementry Tble 4). We selected mirna genes tht hrbored CGIs in the proximl upstrem (<5 kb) of their coding regions (Supplementry Tble 5), nd subsequent MSP nlysis reveled tht the CGIs of 1 were hypermethylted in multiple BC cell lines (Fig. 1A). These mirnas were lso induced by low dose (.1 mm) of 5-z-dC plus PBA, mking it unlikely tht the observed induction ws secondry effect of DNA dmge (Supplementry Fig. 4). We next used bisulfite pyrosequencing to quntittively nlyze the methyltion of the 1 mirna genes showing CGI methyltion in series of BC tissues (n = 6), smple of norml urothelium tissue, nd norml urothelil cell line (SV-HUC-1). We found tht four mirna genes (mir- 17, mir14-, mir-14-, nd mir-9-) were frequently methylted in primry tumors, though their methyltion levels were limited in norml urothelium (Fig. 1B; Supplementry Fig. 5 nd 6). In ddition, we observed mrked B Cncer cell lines Primry tumors mir-9- mir-14- mir-14- mir-17 T stge is T4 UM-UC- HT-1197 HT-176 SW 78 Norml tissue SV-HUC-1 Grde (%) Fig. 1 Methyltion nlysis of microrna (mirna) genes in bldder cncer (BC). (A) Methyltion-specific polymerse chin rection (PCR) nlysis of the CpG islnds of 1 mirna genes in the indicted cell lines. In vitro methylted DNA nd DNA methyltrnsferse knockout cells served s positive nd negtive controls, respectively. Bnds in the M lnes re PCR products obtined with methyltion-specific primers; those in the U lnes re products obtined with unmethylted-specific primers. (B) Summrized results for the bisulfite pyrosequencing of mirna genes in BC cell lines, smple of norml urothelil tissue, norml urothelil cell line SV-HUC-1, nd set of primry BC tissues (n = 6). Tumor stges nd grdes re indicted on the right. DNMTs KO = DNA methyltrnsferse knockout cells; IVD = in vitro methylted DNA.

4 194 EUROPEAN UROLOGY 6 (1) Tble 1 Correltion between microrna gene methyltion nd the clinicopthologic fetures of bldder cncer (n = 8) mir-17 met (%) mir-14- met (%) mir-14- met (%) mir-9- met (%) Men SD p * Men SD p * Men SD p * Men SD p * Age, yr: Medin (rnge) 7 (4 9) Gender: Mle Femle T stge: T Tis T T Grde: LN metstsis: N N1 N SD = stndrd devition; LN = lymph node; ANOVA = nlysis of vrince. * Person correltion coefficient, student t test, or ANOVA. reduction in the methyltion levels in BC cells treted with 5-z-dC plus PBA, which is consistent with the upregultion of mirnas (Supplementry Fig. 7)... Methyltion of microrna genes in primry bldder cncer We next exmined the methyltion levels of mir-17, mir- 14-, mir-14-, nd mir-9- in lrger set of primry tumors (n = 8), long with djcent nd distnt nontumorous bldder tissues from the sme ptients (Tble 1). Elevted levels of mirna gene methyltion (>15.%) were frequently detected in primry BC tissues (mir-17: 57 of 8, 68.7%; mir-14-: 4 of 8, 5.6%; mir-14-: 54 of 8, 65.1%; mir-9-: 8 of 8, 45.8%), nd the tumor tissues exhibited significntly higher methyltion levels thn their nontumorous counterprts (Fig. A). In ddition, we found tht levels of mirna gene methyltion were more frequently elevted in djcent nontumorous bldder tissues (AN; mir-17: 6 of 74, 5.1%; mir-14-: 19 of 74, 5.7%; mir-14-: 15 of 74,.%; mir-9-: 1 of 74, 16.%) thn in more distnt nontumorous tissues (DN; mir- 17: 18 of 8, 1.7%; mir-14-: 6 of 8, 7.%; mir-14-: 11 of 8, 1.%; mir-9-: 9 of 8, 1.8%). No significnt correltion ws found between the levels of mirna gene methyltion nd the clinicopthologic chrcteristics of the ptients (Tble 1). When we exmined the methyltion sttus of mir-17 in selected tissue specimens in more detil, we observed dense methyltion in tumor tissues but only scttered methyltion in nontumorous tissues (Fig. B). We then compred the levels of mir-17 expression determined in TqMn ssys with the methyltion levels obtined by bisulfite pyrosequencing in selected pirs of tumors nd corresponding distnt nontumorous tissues (Fig. C). We found tht there ws n inverse reltionship between the expression of mir-17 nd its methyltion, which suggests tht CGI methyltion is ssocited with the downregultion of mir-17 in BC tissues... Detection of microrna gene methyltion in urine smples To ssess the usefulness of mirna gene methyltion, we collected voided urine specimens from 86 BC ptients (Tble ) nd cncer-free individuls. Upon performing bisulfite pyrosequencing, we observed elevted methyltion of mir-17, mir-14-, mir-14-, nd mir-9- in the urine smples from the cncer ptients (Fig. A) but only limited methyltion of the genes in cncer-free individuls (Fig. B). Moreover, the methyltion levels in the urine smples correlted positively with those in the corresponding tumor tissues (Supplementry Fig. 8). Notbly, when we then collected postopertive voided urine smples from 6 of the 86 ptients fter surgicl resection of their tumors, we observed drmticlly reduced methyltion levels (Fig. A; Supplementry Fig. 9). To further evlute the clinicl usefulness of the mirna gene methyltion in urine smples, we crried out ROC curve nlysis to ssess its bility to distinguish preopertive from postopertive smples (Fig. C). The most discriminting cut-offs for mir-17, mir-14-, mir-14-, nd mir-9- were 5.% (sensitivity, 77.9%; specificity, 77.8%), 5.% (sensitivity, 69.8%; specificity, 88.9%), 1.% (sensitivity, 65.1%; specificity, 97.%), nd 7.% (sensitivity, 69.4%; specificity, 86.1%), respectively (Tble ). We next compred these results with those obtined with urine cytology. Bsed on the urinry cytology using Ppnicolou s clssifiction of the 86 ptients, 55 (64%) were dignosed s clss I or II, 15 (17%) were clss III, nd only 16 (19%) were clss IV or V (strongly suggestive or conclusive of mlignncy), suggesting tht the sensitivity of urinry methyltion for detection of BC is significntly greter thn tht of conventionl cytology (Supplementry Tble 6).

5 [(Fig._)TD$FIG] EUROPEAN UROLOGY 6 (1) A 1 mir-17 mir-14- p <.1 p <.1 p <.1 p =.7 p <.1 p = T AN DN T AN DN mir-14- p <.1 mir-9- p <.1 1 p <.1 p =.46 p <.1 p = T AN DN T AN DN T DN N B C Reltive expression (mir-17/u6) cse 1 cse cse T DN T DN bp cse 1 cse cse Fig. Anlysis of microrna (mirna) gene methyltion in primry bldder cncer. (A) Summrized results of bisulfite pyrosequencing of the indicted mirna genes in primry tumors (T; n = 8), nontumorous bldder tissues djcent to the tumors (AN; n = 74), nd nontumorous bldder tissues distnt from the tumors (DN; n = 8). p <.5. (B) Bisulfite sequencing nlysis of the mir-17 CpG islnd (CGI) in pir of tumor (T) nd distnt nontumorous tissues (DN). (C) Inverse reltionship between the expression nd methyltion of mir-17 in three pirs of tumor (T) nd distnt nontumorous tissues (DN). Expression ws ssessed in TqMn ssys (upper pnel), nd methyltion ws determined by bisulfite pyrosequencing (lower pnel). To develop more efficient dignostic method for detecting BC, we constructed scoring system using the urinry methyltion of the four methylted mirna genes (Fig. 4). Using the cut-off vlue for ech gene (Tble ), we clssified the smples into five groups bsed on the M-score. A ROC curve ws then constructed to evlute the bility of the scoring system to distinguish preopertive from postopertive urine smples by plotting the sensitivity over 1-specificity t ech point (Fig. 4B). We then vlidted the dignostic system by nlyzing n independent test set

6 196 EUROPEAN UROLOGY 6 (1) Tble Clinicopthologic chrcteristics of the ptients in the trining nd test sets Trining set Test set (n = 86) (n = 4) Age, yr: Medin (rnge) 7 (4 9) 71 (58 9) Gender, no.: Mle 69 5 Femle 17 9 T stge, no.: T 4 16 Tis 7 5 T1 1 8 T 5 Grde, no.: Lymph node metstsis, no.: N 81 N1 N 5 Tretment, no.: TURBT 64 RC 4 TURBT = trnsurethrl resection of bldder tumor; RC = rdicl cystectomy. prolifertion, wheres mir-9 exerted no significnt suppressive effect on growth (Supplementry Fig. 11 nd 1). We then crried out Mtrigel invsion ssys to test the effect of the mirnas on cell invsion. Although we detected no effect of mir-17 nd mir-14 on cell invsion, ectopic expression of mir-9 suppressed the invsiveness of BC cells (Supplementry Fig. 1). Finlly, to further clrify the effect of mirnas, we crried out gene expression microrry nlysis of SW78 cells trnsfected with mir-17 precursor or negtive control. We found tht 16 probe sets (116 unique genes) were downregulted (more thn two-fold) by ectopic mir-17 expression, including the previously reported mir-17 trget genes cyclin-dependent kinse 6 (CDK6), cell division cycle 4 (CDC4), nd uror kinse A (AURKA) [14,15]. Among the 116 downregulted genes, the TrgetScn progrm predicted tht 144 genes re potentil trgets of mir-17 (Supplementry Tble 7). Moreover, Gene Ontology nlysis reveled tht genes relted to the cell cycle were significntly enriched mong the ffected genes (Supplementry Tble 8). Our results strongly suggest tht the mirnas in question ct s tumor suppressors in BC. 4. Discussion (Tble ). AUCs in both sets were high (trining set:.916; test set:.91), confirming the ccurcy of our system for detecting BC using urinry mirna gene methyltion (Fig. 4). We lso found tht our scoring system could effectively detect erly-stge T nd low-grde (grdes 1 nd ) BC (sensitivity:.679; specificity:.889; AUC =.86), which ws undetectble using urinry cytology (Supplementry Fig. 1)..4. Functionl nlysis of micrornas To test whether ny of the mirnas could ct s tumor suppressors, we trnsfected BC cells with n mirna precursor molecule or negtive control, nd then crried out cell vibility ssys. The ssys showed tht ectopic expression of mir-17 or mir-14 suppressed BC cell We identified four mirna genes (mir-17, mir-14-, mir- 14-, nd mir-9-) tht were frequently methylted in both cultured nd primry BC cells. Erlier studies hve shown tht these mirnas re tumor-suppressive or tumorrelted nd tht they re epigeneticlly silenced in cncers of vrious origins. Hypermethyltion of mir-17 ws first discovered in orl cncer [16] nd hs since been noted in other mlignncies, including cncers of the colon [14] nd stomch []. Within cncer cells, mir-17 trgets CDK6, CDC4, nd AURKA, which is indictive of its tumorsuppressive properties [14 16], wheres in norml cells, mir-17 regultes neuronl differentition through trgeting enhncer of zeste homolog (EZH) nd mindbomb E ubiquitin protein ligse 1 (MIB1) [17,18]. Methyltion of mir-14 fmily genes (mir-14-1, mir-14-, nd mir- 14-) ws identified in colorectl cncer [19] nd ws lso Tble Receiver operting chrcteristic nlysis of microrna gene methyltion to detect bldder cncer Gene nme Cut-off, % Trining set AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) mir (.71.86) ( ) ( ) mir ( ) ( ) ( ) mir (.7.88) 65.1 ( ) 97. ( ) mir ( ) ( ) ( ) Gene nme Cut-off, % Test set AUC (95% CI) Sensitivity (95% CI) Specificity (95% CI) mir (.69.98) ( ) 6.64 ( ) mir ( ) ( ) 9.91 ( ) mir ( ) 58.8 ( ) 1. ( ) mir (.66.94) ( ) 7.7 ( ) AUC = re under the curve; CI = confidence intervl.

7 [(Fig._)TD$FIG] EUROPEAN UROLOGY 6 (1) A mir-17 p < mir-14- p <.1 Pre Post Pre Post Pre mir-14- p <.1 Post Pre mir-9- p <.1 Post B mir-17 mir-14- mir-14- mir-9- C Sensitivity (%) mir-17 Sensitivity (%) 1 mir AUC =.78 4 AUC = specificity (%) 1 mir specificity (%) mir-9- Sensitivity (%) AUC =.85 Sensitivity (%) AUC = specificity (%) specificity (%) Fig. Detection of microrna (mirna) gene methyltion in urine specimens from bldder cncer (BC) ptients. (A) Summry of bisulfite pyrosequencing nlysis of the indicted mirna genes in voided urine smples collected from BC ptients before (Pre: n = 86) nd fter surgicl tretment (Post: n = 6). p <.1. (B) Bisulfite pyrosequencing results for mir-17, mir-14-, mir-14-, nd mir-9- in voided urine smples from cncer-free individuls (n = ). (C) Receiver operting chrcteristics curve nlysis of the bility of mirna gene methyltion to distinguish preopertive nd postopertive urine smples. AUC = re under the curve. found in gstric cncer [], hemtologic mlignncies [1], nd heptocellulr crcinom []. In ddition, screening for methylted mirna genes in metsttic cncer cell lines lso identified mir-9 fmily genes (mir-9-1, mir-9-, nd mir-9-) []. Cumultive evidence suggests tht mirnas ply importnt roles in the pthogenesis of BC, nd previous studies demonstrted their epigenetic silencing in the disese. For exmple, mir-4, which is direct trget of p5 nd cndidte tumor suppressor gene, is frequently methylted nd silenced in mny types of cncer, including BC [4].In ddition, Wiklund et l. found tht the silencing of mir- fmily genes nd mir-5 is ssocited with DNA methyltion in invsive BC [1]. They lso showed tht reduced expression of mir-c is ssocited with disese progression nd poor outcome, suggesting tht epigenetic silencing of mir- fmily genes could be prognostic mrker in BC. Recently, Dudziec et l. crried out n mirna microrry nlysis fter treting norml urothelium nd urothelil cncer cell lines with 5-zcytidine. They identified 4 mirtrons nd 16 mirnas whose silencing ws ssocited with DNA methyltion [1]. Some of those

8 198 [(Fig._4)TD$FIG] EUROPEAN UROLOGY 6 (1) A Test Cut-off Positive: Pre/Post Negtive: Pre/Post mir-17 >5.% Positive: 67/8 Negtive: 19/8 mir-14- >1.% Positive: 56/1 Negtive: /5 Trining set mir-14- >5.% Positive: 6/4 Negtive: 6/ mir-9- >7.% Positive: 59/5 Negtive: 6/1 mir-17 >5.% Positive: 7/ Negtive: 7/8 mir-14- >1.% Positive: / Negtive: 14/11 Test set mir-14- >5.% Positive: 6/1 Negtive: 8/1 mir-9- >7.% Positive: 6/ Negtive: 8/9 M-score M-score Se: 94% Sp: 64% Se: 81% Sp: 89% Se: 65% Sp: 97% Se: 41% Sp: 1% Se: 94% Sp: 64% Se: 8% Sp: 91% Se: 71% Sp: 91% Se: 41% Sp: 1% Higher sensitivity Higher specificity Higher sensitivity Higher specificity Trining set Test set B 1 1 sensitivity (%) sensitivity (%) AUC =.916 AUC = specificity (%) specificity (%) Fig. 4 Dignostic system for detecting bldder cncer (BC) using urinry microrna (mirna) gene methyltion. (A) Workflow of system estblished bsed on the bility to distinguish preopertive from postopertive urine. Results of the trining set re shown on the left; those of test set re on the right. The methyltion sttus of mirna genes in preopertive (trining set: n = 86; test set: n = 6) nd postopertive urine (trining set: n = 4; test set: n = 11) ws determined using the cut-off vlues in the respective boxes. A mir-methyltion score (M-score) ws determined from the number of methyltion-positive genes, nd smples were clssified into five groups bsed on the M-score. The sensitivity (Se) nd specificity (Sp) t ech point re indicted below. (B) Receiver operting chrcteristic curve nlysis of the trining nd test sets. Ares under the curve re shown in the grph. M-score = mir-methyltion score; AUC = re under the curve. mirtrons nd mirnas, including mir-9 fmily genes, more frequently exhibited CpG shore methyltion thn CGI methyltion, suggesting tht methyltion in both the CpG shore nd CGI is relted to epigenetic silencing of mirna in BC. Interestingly, mir-9-1 nd -9- were ssocited with both CGI nd CpG shore methyltion, wheres mir-9- showed only CGI methyltion [1]. Consistent with those findings, we observed tht mong the mir-9 fmily genes, mir-9- most frequently showed CGI methyltion. Methyltion of severl mirna genes is strongly relted to the clinicl chrcteristics of cncer, suggesting its potentil usefulness s biomrker. For instnce, methyltion of mir-9-1 nd -9- is reportedly ssocited with metsttic recurrence of RCC, which is indictive of the possible role of mir-9 in cncer metstsis [5]. Despite this report, however, we did not find significnt difference in the levels of mir-9- methyltion between noninvsive nd invsive BC tissues. Further study to clrify the functions of these mirnas in BC will be needed. Recent studies hve shown tht mirna levels in urine could serve s moleculr mrker for detection of BC. For instnce, expression of mir-96 nd mir-18 is reportedly upregulted in urothelil cncer, nd their detection in urine strongly distinguished cncer ptients from cncerfree ptients [6]. Mih et l. lso showed tht evlution of pnel of 1 mirnas in urine is highly sensitive method of detecting BC [7]. DNA methyltion is nother potentil moleculr mrker detectble in urine specimens. Severl

9 EUROPEAN UROLOGY 6 (1) protein-coding genes re trgets of DNA methyltion in BC, nd their urinry methyltion ppers to be useful biomrker [8,9]. For instnce, methyltion of 11 proteincoding genes found in urine sediments reveled the presence of BC with high sensitivity nd specificity [], nd in nother study pnel of three genes (growth differentition fctor 15 [GDF15], trnsmembrne protein with EGF-like nd two follisttin-like domins [TMEFF], nd vimentin [VIM]) in urine could be used to ccurtely detect BC [1]. In the present study, we show for the first time tht methyltion of mirna genes could serve s biomrker for detection of BC. Methyltion of mirna genes ws redily detectble in voided urine from cncer ptients, nd its levels were drmticlly reduced fter tumor resection, confirming its tumor specificity. We lso showed tht combintion of multiple mirna genes could ccurtely distinguish between preopertive nd postopertive urine smples. Our study hs severl limittions. The prognostic vlue of mirna gene methyltion remins uncler, becuse the prognosis of the ptients in this study is not yet vilble. A follow-up study in post-tretment ptients will be needed to test whether urinry methyltion cn predict outcome or detect BC recurrence. In ddition, urinry methyltion in non-bc ptients (eg, ptients with other types of cncer) should be tested to evlute the specificity of our method. Further studies to ddress these issues would contribute to overcoming the difficulties in trnslting our present findings into clinicl prctice. 5. Conclusions We identified four mirna genes tht re frequent trgets of epigenetic silencing in BC. Although their specific functions in bldder crcinogenesis remin unknown, it is evident tht restortion of these mirnas my be n effective nticncer therpy. Furthermore, methyltion of these mirna genes in urine specimens could serve s useful nd noninvsive biomrker for ccurte detection of BC. Author contributions: Hiromu Suzuki hd full ccess to ll the dt in the study nd tkes responsibility for the integrity of the dt nd the ccurcy of the dt nlysis. Study concept nd design: Suzuki, Toyot. Acquisition of dt: Shimizu, Ashid, Hthir, Ymmoto, Mruym, Ki. Anlysis nd interprettion of dt: Shimizu, Suzuki, Nojim. Drfting of the mnuscript: Shimizu, Suzuki. Criticl revision of the mnuscript for importnt intellectul content: Tiji Tsukmoto. Sttisticl nlysis: Nojim. Obtining funding: Suzuki, Toyot, Tsukmoto. Administrtive, technicl, or mteril support: Kitmur, Msumori, Tokino, Imi, Tsukmoto. Supervision: Minoru Toyot, Tiji Tsukmoto. Other (specify): None. Finncil disclosures: Hiromu Suzuki certifies tht ll conflicts of interest, including specific finncil interests nd reltionships nd ffilitions relevnt to the subject mtter or mterils discussed in the mnuscript (eg, employment/ffilition, grnts or funding, consultncies, honorri, stock ownership oroptions, experttestimony, roylties, orptents filed, received, or pending), re the following: None. Funding/Support nd role of the sponsor: This study ws supported in prt by Progrm for Developing Supporting System for Upgrding Eduction nd Reserch from the Ministry of Eduction, Culture, Sports, Science, nd Technology (T. Tsukmoto, M. Toyot); Grnt-in-Aid for the Third-term Comprehensive 1-yer Strtegy of Cncer Control from the Ministry of Helth, Lbor, nd Welfre, Jpn (M. Toyot, H. Suzuki); the A Foresight Progrm from the Jpn Society for Promotion of Science (H. Suzuki); nd the Project for Developing Innovtive Reserch on Cncer Therpeutics (P-DIRECT; H. Suzuki). Acknowledgement sttement: The uthors thnk the stff of the Deprtments of Urology t Spporo Medicl University Hospitl nd NTT Est Spporo Hospitl for their kind ssistnce in the collection of specimens nd Dr Willim Goldmn for editing the mnuscript. Appendix A. Supplementry dt Supplementry dt ssocited with this rticle cn be found, in the online version, t j.eururo References [1] He L, Hnnon GJ. MicroRNAs: smll RNAs with big role in gene regultion. Nt Rev Genet 4;5:5 1. [] Croce CM. Cuses nd consequences of microrna dysregultion in cncer. Nt Rev Genet 9;1: [] Chen Q, Chen X, Zhng M, Fn Q, Luo S, Co X. mir-17 is frequently down-regulted in gstric cncer nd is negtive regultor of Cdc4. Dig Dis Sci 11;56:9 16. [4] Ctto JW, Mih S, Owen HC, et l. Distinct microrna ltertions chrcterize high- nd low-grde bldder cncer. Cncer Res 9;69: [5] Neely LA, Rieger-Christ KM, Neto BS, et l. A microrna expression rtio defining the invsive phenotype in bldder tumors. Urol Oncol 1;8:9 48. [6] Adm L, Zhong M, Choi W, et l. mir- expression regultes epithelil-to-mesenchyml trnsition in bldder cncer cells nd reverses resistnce to epiderml growth fctor receptor therpy. Clin Cncer Res 9;15:56 7. [7] Sito Y, Ling G, Egger G, et l. Specific ctivtion of microrna- 17 with downregultion of the proto-oncogene BCL6 by chromtin-modifying drugs in humn cncer cells. Cncer Cell 6;9:45 4. [8] Suzuki H, Tktsuk S, Akshi H, et l. Genome-wide profiling of chromtin signtures revels epigenetic regultion of microrna genes in colorectl cncer. Cncer Res 11;71: [9] Suzuki H, Ymmoto E, Nojim M, et l. Methyltion-ssocited silencing of microrna-4b/c in gstric cncer nd its involvement in n epigenetic field defect. Crcinogenesis 1;1:66 7. [1] Mruym R, Suzuki H, Ymmoto E, Imi K, Shinomur Y. Emerging links between epigenetic ltertions nd dysregultion of noncoding RNAs in cncer. Tumour Biol 1;: [11] Esteller M. Non-coding RNAs in humn disese. Nt Rev Genet 11;1: [1] Wiklund ED, Brmsen JB, Hulf T, et l. Coordinted epigenetic repression of the mir- fmily nd mir-5 in invsive bldder cncer. Int J Cncer 11;18:17 4. [1] Dudziec E, Mih S, Choudhry HM, et l. Hypermethyltion of CpG islnds nd shores round specific micrornas nd mirtrons is ssocited with the phenotype nd presence of bldder cncer. Clin Cncer Res 11;17:

10 11 EUROPEAN UROLOGY 6 (1) [14] Blguer F, Link A, Lozno JJ, et l. Epigenetic silencing of mir-17 is [] Lujmbio A, Clin GA, Villnuev A, et l. A microrna DNA meth- n erly event in colorectl crcinogenesis. Cncer Res 1;7: yltion signture for humn cncer metstsis. Proc Ntl Acd Sci U S A 8;15: [15] Liu M, Lng N, Qiu M, et l. mir-17 trgets Cdc4 expression, [4] Lodygin D, Trsov V, Epnchintsev A, et l. Inctivtion of mir-4 induces cell cycle G1 rrest nd inhibits invsion in colorectl by berrnt CpG methyltion in multiple types of cncer. Cell Cycle cncer cells. Int J Cncer 11;18: ;7: [16] Kozki K, Imoto I, Mogi S, Omur K, Inzw J. Explortion of tumor- [5] Hildebrndt MA, Gu J, Lin J, et l. Hs-miR-9 methyltion sttus is suppressive micrornas silenced by DNA hypermethyltion in orl ssocited with cncer development nd metsttic recurrence in cncer. Cncer Res 8;68: ptients with cler cell renl cell crcinom. Oncogene 1;9: [17] Szulwch KE, Li X, Smrt RD, et l. Cross tlk between microrna nd epigenetic regultion in dult neurogenesis. J Cell Biol 1;189: [18] Smrt RD, Szulwch KE, Pfeiffer RL, et l. MicroRNA mir-17 regultes neuronl mturtion by trgeting ubiquitin ligse mind bomb-1. Stem Cells 1;8:16 7. [19] Lujmbio A, Ropero S, Bllestr E, et l. Genetic unmsking of n epigeneticlly silenced microrna in humn cncer cells. Cncer Res 7;67: [] Ando T, Yoshid T, Enomoto S, et l. DNA methyltion of microrna genes in gstric mucose of gstric cncer ptients: its possible involvement in the formtion of epigenetic field defect. Int J Cncer 9;14: [6] Ymd Y, Enokid H, Kojim S, et l. MiR-96 nd mir-18 detection in urine serve s potentil tumor mrkers of urothelil crcinom: correltion with stge nd grde, nd comprison with urinry cytology. Cncer Sci 11;1:5 9. [7] Mih S, Dudziec E, Dryton RM, et l. An evlution of urinry microrna revels high sensitivity for bldder cncer. Br J Cncer 1;17:1 8. [8] Nishiym N, Ari E, Chihr Y, et l. Genome-wide DNA methyltion profiles in urothelil crcinoms nd urotheli t the precncerous stge. Cncer Sci 1;11:1 4. [9] Cirns P. Gene methyltion nd erly detection of genitourinry cncer: the rod hed. Nt Rev Cncer 7;7:51 4. [1] Agirre X, Vils-Zornoz A, Jimenez-Velsco A, et l. Epigenetic [] Yu J, Zhu T, Wng Z, et l. A novel set of DNA methyltion mrkers in silencing of the tumor suppressor microrna Hs-miR-14 reg- urine sediments for sensitive/specific detection of bldder cncer. ultes CDK6 expression nd confers poor prognosis in cute lymphoblstic leukemi. Cncer Res 9;69: Clin Cncer Res 7;1: [1] Cost VL, Henrique R, Dnielsen SA, et l. Three epigenetic bio- [] Furut M, Kozki KI, Tnk S, Arii S, Imoto I, Inzw J. mir-14 nd mrkers, GDF15, TMEFF, nd VIM, ccurtely predict bldder mir- re epigeneticlly silenced tumor-suppressive micrornas cncer from DNA-bsed nlyses of urine smples. Clin Cncer in heptocellulr crcinom. Crcinogenesis 1;1: Res 1;16: