MiRNA profiles in lymphoblastoid cell lines of Finnish prostate cancer families

RESEARCH ARTICLE MiRNA P o iles in Lymphoblas oid Cell Lines o Finnish P os a e Cance Families Daniel Fische 1 , Tiina Wahl o s 2 , Henna Ma ila 2 , Hannu Oja 3 , Teu o L. J. Tammela 4 , Johanna Schleu ke 5 * 1School o Heal h Sciences, Uni e si y o Tampe e, 33014 Tampe e, Finland, 2BioMediTech, Uni e si y o Tampe e, and Fimlab Labo a o ies, Tampe e, Finland, 3Depa men o Ma hema ics and S a is ics, Uni e si y o Tu ku, 20014 Tu ku, Finland, 4Depa men o U ology, Tampe e Uni e si y Hospi al and Medical School, Uni e si y o Tampe e, Tampe e, Finland, 5Medical Biochemis y and Gene ics, Ins i u e o Biomedicine, Uni e si y o Tu ku, Tu ku, Finland *johanna.schleu ke @u u. i Abs ac Backg ound He i able ac o s a e e iden ly in ol ed in p os a e cance (P Ca) ca cinogenesis, bu cu - en ly, gene ic ma ke s a e no ou inely used in sc eening o diagnos ics o he disease. Mo e p ecise in o ma ion is needed o making ea men decisions o dis inguish agg es- si e cases om indolen disease, o which he i able ac o s could be a use ul ool. The ge- ne ic makeup o P Ca has only ecen ly begun o be un a elled h ough la ge-scale genome-wide associa ion s udies (GWAS). The hus a iden i ied Single Nucleo ide Poly- mo phisms (SNPs) explain, howe e , only a ac ion o amilial clus e ing. Mo eo e , he known isk SNPs a e no associa ed wi h he clinical ou come o he disease, such as ag- g essi e o me as asised disease, and he e o e canno be used o p edic he p ognosis. Anno a ing he SNPs wi h deep clinical da a oge he wi h miRNA exp ession p o iles can imp o e he unde s anding o he unde lying mechanisms o di e en pheno ypes o p os a e cance . Resul s In his s udy mic oRNA (miRNA) p o iles we e s udied as po en ial bioma ke s o p edic he disease ou come. The s udy subjec s we e om Finnish high isk p os a e cance amilies. To iden i y po en ial bioma ke s we combined a no el non-pa ame ical es wi h an impo - ance measu e p o ided om a Random Fo es classi ie . This combina ion deli e ed a se o nine miRNAs ha was able o sepa a e cases om con ols. The de ec ed miRNA ex- p ession p o iles could p edic he de elopmen o he disease yea s be o e he ac ual P Ca diagnosis o de ec he exis ence o o he cance s in he s udied indi iduals. Fu he mo e, using an exp ession Quan i a i e T ai Loci (eQTL) analysis, egula o y SNPs o miRNA miR-483-3p ha we e also di ec ly associa ed wi h P Ca we e ound. PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 1/17 a11111 OPEN ACCESS Ci a ion: Fische D, Wahl o s T, Ma ila H, Oja H, Tammela TLJ, Schleu ke J (2015) MiRNA P o iles in Lymphoblas oid Cell Lines o Finnish P os a e Cance Families. PLoS ONE 10(5): e0127427. doi:10.1371/ jou nal.pone.0127427 Academic Edi o : Xin-Yuan Guan, The Uni e si y o Hong Kong, CHINA Recei ed: Decembe 19, 2014 Accep ed: Ap il 15, 2015 Published: May 28, 2015 Copy igh : © 2015 Fische e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All ele an da a a e a ailable om EBI (accession numbe E-MTAB- 3397). Funding: This wo k was suppo ed by Medical Resea ch Fund o Tampe e Uni e si y Hospi al (9L091, 9M094, and 9N069), he Finnish Cance O ganiza ions, he Sig id Juselius Founda ion, and he Academy o Finland (g an s 116437 and 251074) o JS. This wo k was also suppo ed by The Finnish Doc o al P og amme in S ochas ics and S a is ics o DF. Conclusion Based on ou indings, we sugges ha blood-based miRNA exp ession p o iling can be used in he diagnosis and maybe e en p ognosis o he disease. In he u u e, miRNA p o il- ing could possibly be used in a ge ed sc eening, oge he wi h P os a e Speci ic An igene (PSA) es ing, o iden i y men wi h an ele a ed P Ca isk. In oduc ion P os a e cance (P Ca) is he mos common noncu aneous malignancy and he second leading cause o cance - ela ed dea hs among men in indus ialised coun ies [1]. In Finland, 4604 new p os a e cance cases we e diagnosed in 2012 (Finnish Cance Regis y, h p://www.cance . i/ syopa ekis e i/). Aging and PSA es ing may be he mos e iden easons o he inc eased numbe o new cases. The g owing incidence c ea es p essu e on he heal h ca e sys em as he conce n ega ding o e ea men is conside able. The e o e, one o he majo challenges is o imp o e he diagnos ic and p ognos ic ools o be able o dis inguish le hal om indolen dis- ease a a cu able s a e o he disease. The con ibu ion o gene ic a ian s has been s udied widely in associa ion wi h p os a e cance p edisposi ion. Bo h linkage and GWAS oge he wi h he ew examples a ising om candida e gene app oaches ha e led o he iden i ica ion o abou 100 gene ic loci ha explain only app oxima ely 30% o he gene ic isk o he disease [2][3][4][5]. Howe e , he e is no ob ious molecula o unc ional e idence indica ing how he a ia ions in hese candida e si es o hei co-inhe i ed neighbou ing a ian s could cause P Ca. In ac , mos o he single nucleo- ide a ian s (SNPs) ound by GWAS a e unlikely o a ec he coding sequence o any gene bu a he eside in in e genic egions. These indings sugges ha hey ha e a egula o y ole, such as in ansc ip ion, splicing o mRNA s abili y, ins ead o a di ec e ec on he unc ion o he gene p oduc [6]. In ecen yea s, he impo ance o he non-p o ein coding genome in he unc ional egula- ion o no mal de elopmen and disease de elopmen has become e iden . MiRNAs a e sho non-coding RNAs ha egula e hei a ge gene exp ession ypically by binding o he 3’un- ansla ed egion (UTR) o he a ge mRNA [7]. Indi idual a ia ion o he miRNA exp ession le els can in luence he exp ession o he mRNA a ge gene, causing pheno ypic di e ences. Se e al s udies ha e shown ha miRNA exp ession le els a e p edic i e o he ou come o solid umou s and leukaemias, bu he con ibu ion o al e ed miRNA exp ession le els o ge- ne ic cance suscep ibili y is no known. The ansc ip ional ac i i y o p o ein coding genes is inhe i ed as a quan i a i e ai , and egula o y polymo phisms associa ed wi h he a iabili y in he le els o mRNA a e conside ed o be eQTL. Despi e he demons a ed impo ance, knowledge o he gene ic egula ion o miRNA exp ession is s ill in i s in ancy. In a ecen pub- lica ion, o e one hund ed eQTLs in p ima y ib oblas s we e desc ibed, indica ing a leas a pa ial ole o gene ic a ia ion in al e ed miRNA exp ession [8]. Combined analyses o com- mon SNPs and a ia ions in miRNA exp ession p o iles migh se e as one way o elucida e he biological unc ions o SNPs iden i ied om GWAS in common diseases. The objec i e o his s udy was o e alua e he miRNA exp ession p o iles o lymphoblas oid cell lines (LCL) de i ed om membe s o high isk P Ca amilies. Al e ed miRNA exp ession in pa ien LCLs compa ed wi h hose om heal hy amily membe s p o ided an oppo uni y o iden i y ge mline a ian s in p omo e o o he egula o y egions o p o ein coding genes as a conside able amoun o miRNA exp ession is co ela ed o hos and a ge gene exp ession MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 2/17 Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . [9]. The la ge amoun o signi ican miRNA-wise es esul s wi hin he da a also equi ed he de elopmen o a new ype o di e en ially exp ession analysis pipeline. To de elop such a pipeline, di e en ially exp ession es ing has been combined wi h he impo ance measu es o he machine lea ning algo i hm, Random Fo es [10]. Ma e ials and Me hods E hics S a emen This s udy has been app o ed by he espec i e IRB boa ds o The Minis y o Social A ai s and Heal h (SMT), Na ional Supe iso y Au ho i y o Wel a e and Heal h (Val i a) and E h- ics Commi ee o Tampe e Uni e si y Hospi al. E e y indi idual pa icipa ing in he s udy has gi en w i en in o med consen . S udy popula ion All samples a e o Finnish o igin and he collec ion o he amilies has been epo ed p e iously [11]. Fo he miRNA mic oa ay s udy, 115 cases om 70 P Ca amilies we e used. The selec - ed amilies had a leas wo i s -deg ee ela i es diagnosed wi h p os a e cance a any age. Heal hy (= no diagnosed p os a e cance ) indi iduals (n = 78) om 47 amilies we e used as he con ols. The median age a diagnosis o he cases was 65 (44–86.2) yea s and he con ols had a median age o 57.5 (35.2–83.3) yea s a he ime he samples we e ob ained. A subse o indi iduals (n = 54) om he mic oa ay expe imen we e geno yped wi h Illu- mina’s HumanOmniExp ess a ay o ano he expe imen , and he esul s a e published else- whe e [12]. Hence, hose 54 samples could be used he e o an eQTL analysis (39 P Ca cases and 15 con ols). Addi ional 83 indi iduals could be used o alida ion pu poses. Al oge he , he e we e 137 geno yped pe sons om 33 amilies (20 o e lapping amilies wi h he mic oa - ay pa o he s udy). The clinical ou come o p os a e cance can oughly be classi ied in o agg essi e and non- agg essi e cance , based on PSA, Gleason sco e and o he clinical e alua ions [13]. Based on hese guidelines, he p os a e cance pa ien s om he wo expe imen s we e g ouped in o 36 (36) agg essi e and 79 (66) non-agg essi e p os a e cance s. The maximum numbe o agg es- si e cases pe amily was 3, and he minimum was 1. A de ailed o e iew o he indi iduals in he s udy is gi en in Fig 1. RNA ex ac ion om lymphoblas oid cell lines LCLs we e de i ed by he Eps ein-Ba i us ans o ma ion o pe iphe al mononuclea leuko- cy es om pa ien s and hei heal hy ela i es. The lymphoblas oid cell lines we e g own in RPMI-1640 medium (Lonza, Walke s ille, MD, USA) supplemen ed wi h 10% e al bo ine se um (Sigma-Ald ich, S . Louis, MO, USA) and an ibio ics a 37°C, 5% CO2 and 95% hu- midi y. The cell pelle s we e snap- ozen, and o al RNA was ex ac ed wi h T izol acco ding o he manu ac u e ’s ins uc ions (In i ogen, Ca lsbad, CA, USA). The RNA yields we e quan i- ied using an ND-1000 spec opho ome e (Nanod op Technologies, Wilming on, DE, USA) and Agilen 2100 Bioanalyze (Agilen Technologies, San a Cla a, CA, USA). Mic oRNA mic oa ay analysis The mic oRNA exp ession le els in LCLs we e de ec ed using Agilen Human miRNA V2 Oligo Mic oa ay Ki (Agilen Technologies). Fi s , 100 ng o o al RNA was used as he s a - ing ma e ial, and miRNAs we e labeled using he Agilen miRNA Labelling Ki . Labelled RNA was hyb idised o Agilen miRNA mic oa ays ha ha e eigh iden ical a ays pe slide, wi h MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 3/17 each a ay con aining p obes di ec ed agains 817 miRNAs (719 human, 76 non-human i al miRNAs and 22 con ol miRNAs). In o al, 26 slides we e used, and he da a we e ex ac ed using Agilen ’s Fea u e Ex ac ion so wa e (FES), e sion 10.7.1.1 wi h he g id layou D_F_20091030. Fo he da a analysis, low quali y samples we e i s emo ed, esul ing in 193 indi iduals. Each indi idual Agilen mic oa ay V2 measu es 13,737 ea u es, and he FES hen used hese ea u es o calcula e he exp ession alues o 2,466 (2,125 human) p obes; based on hose p obes he 817 miRNA exp ession alues we e calcula ed. The da a can be accessed ia A ayExp ess accession E-MTAB-3397. The miRNA exp ession alues a e ypically calcula ed wi h he algo i hm gTo alGeneSignal as implemen ed in FES, bu in his s udy, howe e , p obe-wise, backg ound sub ac ed median alues we e used ins ead. The analysis o di e en p obes o he same miRNA as a single miRNA exp ession alue did no appea o be eliable enough, and an analysis a he p obe le el was mo e easible. A e calcula ing he exp ession alues a he p obe le el, all non- human p obes and hose no de ec ed by he FES we e emo ed. Only hose p obes ha we e de ec ed o a leas 50% o he samples in a leas one heal h s a us g oup we e used o u he analysis. Addi ionally, non-human con ol ea u es we e emo ed be o e he analysis. In o al, 547 p obes, ep esen ing 211 miRNAs, ul illed hese c i e ia. The echnical a iabili y o he da a was educed by applying a quan ile no malisa ion [14]. Geno yping Da a Analysis The single nucleo ide polymo phism (SNP) geno ype da a we e gene a ed using Illumina’s HumanOmniExp ess a ay in collabo a ion wi h he Ins i u e o Molecula Medicine Finland (FIMM). The chosen a ay enabled he geno yping o app oxima ely 700k SNPs. To p oduce he geno ype da a, he aw da a we e analysed wi h Genome S udio acco ding o he manu ac- u e ’s ins uc ions (Illumina, San Diego, USA). In o al, he geno ype in o ma ion o 137 indi iduals was a ailable, wi h he miRNA ex- p ession le els also measu ed in 54 o hese indi iduals. Hence, he eQTL analysis was based on hese 54 pe sons. The emaining 83 indi iduals we e used o alida ion o he esul s. Fig 1. uppe : Popula ion quan i ies, isualisa ion o how he 277 indi iduals in his s udy a e dis ibu ed among he h ee heal h-s a us g oups. Fo each heal h g oup, he numbe o indi iduals om he di e en expe imen s is shown. The o e all numbe om an expe imen is hen indica ed by he espec i e colou ed box plus he ed box (o e lap). lowe : Visualisa ion o he amilial backg ound. The h ee op ions ‘P Ca only’,‘Heal hy only’o ‘P Ca/ Heal hy’a e shown and g ouped acco dingly. Addi ionally, in ol emen o di e en amilies in he wo expe imen s is shown. O de ing is acco ding o an in e nal amily code. doi:10.1371/jou nal.pone.0127427.g001 MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 4/17 Iden i ica ion o di e en ially exp essed p obes using di ec ional es ing P Ca pa ien s we e di ided in o agg essi e (A) and non-agg essi e/mild (M) P Ca g oups and compa ed wi h heal hy con ols (H). A new gene alisa ion o Mann-Whi ney ype es s was ap- plied o iden i y di e en ially exp essed p obes in he h ee-g oup compa ison. The same gen- e alisa ion was used o he eQTL analysis ( o de ails see [15] and [16]). Fo a gene al de ini ion, le he sample sizes o he h ee g oups be N H ,N M and N A which e- sul s in a o al sample size o N H +N M +N A =N. The gene alised Mann-Whi ney es is based on p obabilis ic indices calcula ed wi h iple sums o co esponding indica o unc ions. Le x p;H =(x 1,p;H ,x 2,p;H ,...,x N H ,p;H ) T ,x p;M =(x 1,p;M ,x 2,p;M ,...,x N M ,p;M ) T and x p;A =(x 1,p;A ,x 2,p;A , ...,x N A ,p;A ) T be he exp ession alues o a p obe pin each heal h g oup wi h unde lying cd ’s F p;H ,F p;M and F p;A . The p obabilis ic index ^ PH;M;A;p o p obe pused in his app oach can hen be calcula ed by ^ PH;M;A;p¼1 NHNMNA X NH i¼1 X NM j¼1 X NA k¼1 Iðxi;p;H<xj;p;M<xk;p;AÞ; and I() is he indica o unc ion ha is 1 i condi ion () is ue and 0 i no . Please no ice ha he o de in he index o ^ PH;M;A;p e e s o he o de used in he indica o unc ion. Fu he mo e, he p obabilis ic index ^ PH;M;A;pcan hen be used o es he di ec ional hypo h- esis H0:Fp;H¼Fp;M¼Fp;A s:H1:Fp;HFp;MFp;A; whe e  e e s o he s ochas ic o de ing o cd ’s. Na u ally, di e en o de s in he condi ion () o he indica o unc ion can be used o es o di e en al e na i es. In addi ion, when ex- p ession alues a e assigned o geno ype g oups ins ead o heal h s a us, his es p ocedu e is ideal o eQTL es ing as i es s o he di ec ional al e na i es ha a e clea ly p esen in he con ex o an eQTL analysis. The wo p obabilis ic indices ^ PH;M;A;pand ^ PA;M;H;pwe e used o es ing p obes p=1,..., 547, and p- alues o he pe mu a ion es e sion we e calcula ed based on 5000 pe mu a ions. Tes esul s wi h p- alue less han 0.01 we e conside ed o be signi ican . The es me hod is implemen ed in he R-package gMWT [16], and he package Gene icTools exploi s his es me hod o eQTL es ing. Bo h packages a e eely a ailable om he Comp ehensi e R A - chi e Ne wo k (CRAN). The Benjamini-Hochbe g mul iple es ing p ocedu e o con ol he alse disco e y a e is i- sualised using ejec ion plo s and lines. The a io o expec ed ejec ions unde he null hypo he- sis is plo ed agains he obse ed a io o ejec ions. I his cu e is abo e he (0, 1)-line, we ha e mo e ejec ions han expec ed unde he null hypo hesis. The ejec ions o a ixed es size can be isualised wi h a e ical line, and he ejec ions o di e en mul iple es ing adjus - men s can be isualised by lines wi h a ce ain slope. The numbe o ejec ed null hypo heses is hen de e mined by he c ossing poin o he cu e and he line. Fo de ails, see [15]. Classi ica ion, Impo ance Measu e and Clus e ing The machine lea ning classi ie Random Fo es [10], as implemen ed in he R-package an- domFo es [17], was applied o he exp ession da a, such ha he da ase was spli in o he aining (75%) and es (25%) da a. The aining da a we e used o c ea e an ensemble o 2500 decision ees, and hese ees we e hen used o classi y he es da a. The di ision be ween he aining and alida ion da a was hen epea ed 2000 imes, and a e wa ds he classi ica ion MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 5/17 esul s o all es da a uns we e e alua ed. The Gini impo ance measu e was also ex ac ed o e e y single Random Fo es , and he a e age impo ance o each p obe was combined wi h he co esponding p- alue om he di ec ional es . P obes ha had a p- alue less han 0.01 and ha belonged o he 10% mos impo an p obes o e all Random Fo es uns we e conside ed o be o high in e es (HI p obes) and we e hen used in he clus e ing s ep and in he eQTL analysis. The Random Fo es s we e ained o he h ee possible ou come classes heal hy (H), mild P Ca (M) and agg essi e P Ca (A). Le L i, ;H ,L i, ;M and L i, ;A be he class likelihoods p o ided by he Random Fo es classi ie un o indi idual iwi h L i, ;H +L i, ;M +L i, ;A = 1. These likeli- hoods we e hen combined in o a single P Ca se e eness alue Si; ¼1 2Li; ;MþLi; ;A. The se e - ness alue S i, was chosen in such a way ha S i, = 0 in case ha L i, ;H =1,S i, = 0.5 o L i, ;M =1 and S i, =1i L i, ;A =1. In a 2-way Random Fo es un, he classi ica ion was pe o med only be ween he heal hy and P Ca classes, wi h same se up as ha o he 3-way Random Fo es desc ibed abo e. To calcula e he A ea Unde he Cu e (AUC) o he Recei e Ope a ing Cha ac e is ic (ROC) cu e in he Random Fo es case, wo di e en app oaches we e chosen. Fi s , he wo likelihoods L i, ;M and L i, ;A we e added o e alua e he Random Fo es ’s capabili y o classi y P Ca in gene al. Then, in he second compa ison, he likelihoods L i, ;H and L i, ;M we e added o e alua e i s ap i ude o iden i y agg essi e P Ca. E en ually, o plo he ROC a con inuous cu - o alue in [0, 1] was applied on o he likelihood o classi y indi iduals in o ue/ alse posi i es. Fo he clus e ing in he hea map, he Kendall au co ela ion ma ix Samong all samples was calcula ed based on he exp ession alues o he HI p obes. Kendall’ au be ween wo a i- ables is a measu e o posi i e/nega i e dependence and is in a ian unde any s ic ly inc eas- ing ans o ma ion o he ma ginal a iables. The co esponding dis ance be ween he a iables is hen de ined as D=(1−S)/2. Le hen Dbe he ma ix o dis ances used o he hie achical clus e ing. eQTL Analysis The geno ype in o ma ion om he 700k a ay was combined wi h he exp ession alues o he HI p obes using an eQTL analysis. The ch omosomal loca ions o he miRNA p obes we e iden i ied and all SNPs wi hin a window o 1Mb a ound he p obe’s cen al loca ion we e linked o his p obe. The p obe exp ession alues we e hen assigned o he geno ype g oups o e e y linked SNP (Fig 2 shows a sys ema ic ske ch o his s ep). In an eQTL app oach, h ee cases a e possible, depending on whe he he exp ession alues ha e been assigned o one, wo o all h ee possible geno ype g oups. Monomo phic a ian s we e no u he conside ed in he analysis, and in he wo-g oup case, a wo-sided Mann- Whi ney es was applied. In he h ee-g oup case, he gene alised Mann-Whi ney es o di- ec ional al e na i es was used o he wo di e en al e na i es whe he he highe exp ession alues we e linked o he wild- ype o he homozygous mu a ion. This ype o di ec ional es was used in he h ee-g oup case as an o de o he exp ession alues wi h espec o he geno- ype g oups is clea ly expec ed. Compa a i e Analysis The he e used wo-s age app oach was compa ed wi h wo o he commonly used me hods. The i s me hod was a classical Analysis o Va iance (ANOVA), es ing he al e na i e hypo h- esis ha he e is a di e ence be ween a leas wo ou o he h ee g oups. Le μ p,H ,μ p,M and μ p,A be he a e age exp ession alues o p obe p o he h ee g oups, hen is he p obe-wise MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 6/17 hypo hesis o he one-way ANOVA H0:mp;H¼mp;M¼mp;A s:H1:No all mp;: a e equal Resul ing p- alues we e hen adjus ed o mul iple es ing using a bon e oni co ec ion. The second me hod ha was used as compa ison was a wo-s aged logis ic eg ession wi h lasso (LRL). Fi s , LRL was applied on o he ull da ase wi h he wo classes heal hy/diseased. The uning pa ame e λwas chosen such ha he amoun o selec ed a iables we e in he same le el o magni ude as he he e p oposed me hod iden i ies. The second LRL un was hen applied on o he cance cases only and aimed o he sepa a ion o mild and agg essi e P Ca. Finally he esul ing p obes we e me ged o one esul ma ix om he LRL analysis. To compa e he esul s o he ANOVA and he LRL wi h he he e p oposed app oach, a hi- e a chical clus e ing was applied on o he iden i ied p obes using also a Kendall’s au based dis- ance ma ix. Then, he adjus ed Rand Index was calcula ed be ween he classi ica ion o he h ee di e en clus e ings and he ue cance s a us o he indi iduals o de e mine he le el o ag eemen . Resul s Using he di ec ional es ing p ocedu e, 146 (87 wi h highe exp ession in agg essi e P Ca and 59 wi h highe exp ession in con ols) ou o a o al o 547 p obes we e iden i ied ha ing di e - en exp ession p o iles. The ch omosomal loca ion o he signi ican p obes and he ype o es ing al e na i e a e isualised in Fig 3. To iden i y HI p obes om his unexpec edly la ge amoun o di e en ially exp essed p obes, a Random Fo es classi ie was also applied o he exp ession da a. Signi ican p obes ha we e wi hin 10% o he mos impo an p obes in he Random Fo es , measu ed as Gini Index, we e called HI p obes and a e highligh ed in Fig 3. The 13 iden i ied p obes ep esen eigh di e en miRNAs and one spliceosomal RNA. Mo e de ails abou he 13 iden i ied p obes a e lis ed in Table 1. The o e all classi ica ion esul based on he se e eness alues S i, o he Random Fo es is isualised in Fig 4. Heal hy indi iduals (g een) clea ly ended o be in he lowe isk a ea, bu Fig 2. Each line ep esen s an indi idual, ha ing a ce ain exp ession alue o miRNA X. Independen o he heal h s a us o each indi idual, he exp ession alues a e g ouped acco ding o he geno ype g oups o he su ounding SNPs and hen es ed o di e en ial exp ession be ween hose g oups. (Figu e aken om [16]) doi:10.1371/jou nal.pone.0127427.g002 MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 7/17 agg essi e P Ca pa ien s ( ed) did no end o ha e la ge alues han non-agg essi e P Ca pa- ien s (yellow). In addi ion, an a e age classi ica ion a e o e all classi ica ion uns was de e - mined sepa a ely o he compa isons be ween heal hy and P Ca and be ween agg essi e P Ca and combined heal hy and non-agg essi e P Ca. The Random Fo es was able o classi y P Ca wi h an a e age AUC o he ROC o app oxima ely 0.89 and agg essi e P Ca e sus he com- bined samples o non-agg essi e P Ca and con ols o 0.68 (Fig 5). The classi ica ion esul s a he indi idual le el a e isualised in he suppo ing in o ma ion (S1 and S2 Figs). A hie a chical clus e ing shows he impo ance o he HI p obes. Clus e ing he da ase based on all p obes esul ed in only a sligh ly be e classi ica ion han he clus e ing based on Fig 3. Loca ion o he di ec ional es esul s o he wo p obabilis ic indicies ^ PH;M;Aand ^ PA;M;Hdeno ed by H<M<A espec i e A<M<H. Signi ican es esul s ha also belong o he 10% mos impo an (Gini Index) miRNAs in he Random Fo es un a e deno ed as HI p obes. doi:10.1371/jou nal.pone.0127427.g003 Table 1. O e iew o he HI P obes, hei a ge miRNAs wi h co esponding median exp ession alues and ch omosomal posi ion. P obeID Ta ge ID Ch omosomalLoca ion ~ xH~ xM~ xA A_25_P00010263 mi |hsa-miR-328 Ch 16:67,236,292—67,236,276 20.81 27.15 25.75 A_25_P00011068 mi |hsa-miR-107 Ch 10:91,352,575—91,352,557 405.20 483.35 483.35 A_25_P00011440 mi |hsa-miR-801_ 10.1 Ch 1:28,847,749—28,847,763 50.77 41.10 34.29 A_25_P00011476 mi |hsa-miR-770-5p Ch 14:101,318,754—101,318,768 28.59 24.52 24.13 A_25_P00011477 mi |hsa-miR-770-5p Ch 14:101,318,755—101,318,768 24.59 20.52 20.53 A_25_P00011979 mi |hsa-miR-770-5p Ch 14:101,318,752—101,318,768 31.16 26.31 26.08 A_25_P00012461 mi |hsa-miR-483-3p Ch 11:2,155,431—2,155,415 24.72 29.56 29.94 A_25_P00012462 mi |hsa-miR-483-3p Ch 11:2,155,431—2,155,414 23.46 29.28 29.35 A_25_P00012991 mi |hsa-miR-885-5p Ch 3:10,436,204—10,436,189 22.43 33.34 32.14 A_25_P00013086 mi |hsa-miR-939 Ch 8:145,619,401—145,619,390 152.93 71.98 72.08 A_25_P00013207 mi |hsa-miR-29a*Ch 7:13,0561,530—130,561,511 18.89 21.36 21.52 A_25_P00014864 mi |hsa-miR-202 Ch 10:135,061,097—135,061,083 25.92 21.29 20.68 A_25_P00014914 mi |hsa-miR-885-5p Ch 3:10,436,204—10,436,188 21.25 30.92 29.61 doi:10.1371/jou nal.pone.0127427. 001 MiRNA P o iles in Lymphoblas oid Cell Lines PLOS ONE | DOI:10.1371/jou nal.pone.0127427 May 28, 2015 8/17 he 13 HI p obes. The dend og am o clus e ing indi iduals based on he 13 HI p obes oge h- e wi h he co esponding hea map is shown in Fig 6. He e, he abili y o sepa a e clea ly be- ween agg essi e and non-agg essi e P Ca was limi ed, bu in e es ingly only i e o he 78 heal hy indi iduals we e clus e ed closely oge he wi h P Ca indi iduals. In con as , 46 o 115 P Ca cases we e inside he clus e ha con ained mos o he heal hy indi iduals. In addi ion, a cis-eQTL (0.5Mb up/downs eam window) o he HI p obes was pe o med. In o al, 3863 SNP-miRNA associa ions we e es ed, and 79 had a p- alue o 0.01, (S3 Fig in he suppo ing in o ma ion). All SNPs ha we e ound o ha e a possible egula o y e ec on an HI p obe we e hen es ed o a di ec P Ca associa ion by applying a Fishe - es on he 2 × 3 able be ween geno ype and heal h s a us g oups. Fo ou SNPs, a signi ican associa ion was ound o he 53 geno ypes o he eQTL samples ( es size 0.05). Fig 4. 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