Epigenome-wide DNA methylation profiling of periprostatic adipose tissue in prostate cancer patients with excess adiposity-a pilot study

RESEARCH Open Access Epigenome-wide DNA me hyla ion p o iling o pe ip os a ic adipose issue in p os a e cance pa ien s wi h excess adiposi y—a pilo s udy Yan Cheng 1,2 , Cá ia Mon ei o 3,4 , And eia Ma os 5,6 , Jiaying You 1 , A elino F aga 6,7 , Ca ina Pe ei a 3,8 , Vic o ia Ca alán 9,10 , Amaia Rod íguez 9,10 , Ja ie Gómez-Amb osi 9,10 , Gema F ühbeck 9,10,11 , Rica do Ribei o 3,5,6,12,13*† and Pingzhao Hu 1*† Abs ac Backg ound: Pe ip os a ic adipose issue (PPAT) has been ecognized o associa e wi h p os a e cance (PCa) agg essi eness and p og ession. He e, we sough o in es iga e whe he excess adiposi y modula es he me hylome o PPAT in PCa pa ien s. DNA me hyla ion p o iling was pe o med in PPAT om obese/o e weigh (OB/OW, BMI > 25 kg m −2 ) and no mal weigh (NW, BMI < 25 kg m −2 ) PCa pa ien s. Signi ican di e ences in me hyla ed CpGs be ween OB/OW and NW g oups we e in e ed by s a is ical modeling. Resul s: Fi e housand i e hund ed wen y-six di e en ially me hyla ed CpGs we e iden i ied be ween OB/OW and NW PCa pa ien s wi h 90.2% hype me hyla ed. Fou hund ed eigh y- h ee o hese CpGs we e ound o be loca ed a bo h p omo e s and CpG islands, whe eas he ep esen ing 412 genes we e ound o be in ol ed in plu ipo ency o s em cells, a y acid me abolism, and many o he biological p ocesses; 14 o hese genes, pa icula ly FADS1,MOGAT1, and PCYT2, wi h p omo e hype me hyla ion p esen ed wi h signi ican ly dec eased gene exp ession in ma ched samples. Addi ionally, 38 genes we e co ela ed wi h an igen p ocessing and p esen a ion o endogenous an igen ia MHC class I, which migh esul in a y acid accumula ion in PPAT and umo immune e asion. Conclusions: Resul s showed ha he whole epigenome me hyla ion p o iles o PPAT we e signi ican ly di e en in OB/OW compa ed o no mal weigh PCa pa ien s. The epigene ic a ia ion associa ed wi h excess adiposi y likely esul ed in al e ed lipid me abolism and immune dys egula ion, con ibu ing owa ds un a o able PCa mic oen i onmen , hus wa an ing u he alida ion s udies in la ge samples. Keywo ds: DNA me hyla ion, Pe ip os a ic adipose issue, Obesi y, P os a e cance , Mic oen i onmen Backg ound P os a e cance (PCa) is one o he mos equen malig- nancies in men and he second leading cause o cance - ela ed dea h in he No h Ame ica and mos wes e n Eu opean coun ies [1,2]. Epidemiological s udies suppo obesi y o excess adiposi y as an impo an en i onmen al isk ac o o PCa, being p ima ily associa ed wi h ad anced disease and dea h [3]. Pe ip os a ic adipose issue (PPAT), a whi e a depo su ounding he p os a e capsula -like s uc u e, has been ecognized o ha e he po en ial o exe p o- umo al endoc ine and pa ac ine in luences on p os a e cance cell’s biological pheno ypes [4]. The e is now e idence ha obesi y and o e weigh esul in excess a deposi a PPAT [5], al e ed a y acid p o ile [6], mig a ion o umo cells [7], sec e ion o a a ie y o adipokines, such as in e leukin-1 be a (IL-1b), os eopon in, lep in, umo nec osis ac o alpha (TNF-a), * Co espondence: [email p o ec ed];[email p o ec ed] † Equal con ibu o s 3 Molecula Oncology G oup, Po uguese Ins i u e o Oncology, Po o, Po ugal 1 Depa men o Biochemis y and Medical Gene ics & Depa men o Elec ical and Compu e Enginee ing, Uni e si y o Mani oba, Winnipeg, Canada Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Cheng e al. Clinical Epigene ics (2018) 10:54 h ps://doi.o g/10.1186/s13148-018-0490-3 and dec eased adiponec in, hus con ibu ing o a umo mic oen i onmen ha ul ima ely acili a es PCa agg es- si eness [7,8]. DNA me hyla ion is a well-known epigene ic mechan- ism esul ing om he in e ac ion be ween en i onmen- al ac o s and he genome [9]. DNA me hyla ion wi h a ia ion o CpG si es is associa ed wi h issue-speci ic gene modula ion and in ol ed in pheno ype ansmis- sion and in he de elopmen o diseases [10]. Excess adiposi y, as a consequence o en i onmen al ac o s such as excessi e ood consump ion o inac i e li es yle, has been iden i ied as a egula o o epigene ic modi ica- ion in adipose issue. Recen indings om expe imen al s udies sugges ed ha modi ica ion o DNA me hyla ion pa e n in adipose issue and adipocy es was ela ed wi h de elopmen o cance , ype 2 diabe es, and ca dio ascu- la diseases h ough in luencing me abolism and in lam- ma ion [11–13]. Addi ionally, se e al s udies epo ed al e ed DNA me hyla ion in PCa cells as compa ed wi h adjacen benign issue, and some signi ican ly me hyl- a ed CpG si es and genes we e ound o be esponsible o he occu ence and p og ession o PCa [14–16]. Ne e heless, he epigenome-wide DNA me hyla ion p o ile o PPAT om excess adiposi y PCa pa ien s is cu en ly unknown despi e i s po en ial mechanis ic in ol emen in obesi y associa ion wi h PCa. The aim o his s udy was o pe o m a epigene ic- wide associa ion s udy (EWAS) in o de o e alua e DNA me hyla ion p o ile o PPAT ob ained om obese/ o e weigh (OB/OW) in compa ison wi h no mal weigh (NW) PCa pa ien s and iden i y di e en ially me hyla ed si es. We also explo ed he consequen ial po en ial bio- logical unc ions ha accoun o he e ec o PPAT om OB/OW subjec s in PCa molecula mechanisms. Me hods S udy samples This s udy included en p os a e cance pa ien s om he Po uguese Ins i u e o Oncology, Po o Cen e. In- clusion c i e ia and condi ions o his s udy ha e been p e iously epo ed, including he p ocedu es o PPAT collec ion, handling, and s o age [4]. B ie ly, PPAT was collec ed and immedia ely p ocessed in he ope a ing oom and anspo ed o he labo a o y wi hin 2 h in app op ia e cul u e media and empe a u e condi ions, in o de o minimize p e-analy ical e o s. Pa ien s’ signed in o med consen and esea ch p ocedu es we e app o ed by he ins i u e’s e hics commi ee. The clinical and pa hological cha ac e is ics o pa ici- pan s a e p esen ed in Table 1. The en subjec s we e selec ed om a la ge g oup o pa ien s unde going p os a e su ge y (n= 51) [4,17] ha i ed he s ic inclusion and exclusion c i e ia, in o de o con ol o a iables ha migh in luence adipose issue gene exp ession o me hyla ion (e.g., an i-diabe ic o an i- dyslipidemia d ugs, s age o disease and PSA, concomi an diseases such as diabe es, o he neoplasia o me abolic synd ome). Subjec s we e ma ched o age a diagnosis, PSA alue, Gleason g ade, and s age o disease, which di e ed in body mass index (BMI). BMI was calcula ed by di iding weigh in kilog ams by he squa ed heigh in me e s and ca ego ized using he WHO (Wo ld Heal h O ganiza ion) c i e ia: no mal weigh , BMI < 25 kg m −2 , o e weigh , 25 ≤BMI < 30 kg m −2 , and obese, BMI ≥ 30 kg m −2 . Obese and o e weigh we e combined in o one excess adiposi y g oup (n=5, BMI≥25 kg m −2 ) e sus no mal weigh g oup (n= 5, BMI < 25 kg m −2 ). The e o e, he wo g oups we e selec ed o di e only by BMI, in o de o e lec ou objec i e o assessing whe he excess adiposi y (BMI) in luences PPAT me hyla ion p o ile. Epigenome-wide DNA me hyla ion analysis DNA was isola ed om PPAT using Pu egene hisal ex ac ion me hod (Qiagen/Gen a). B ie ly, he issue was minced wi h scalpels in a s e ile pe i dish on ice and hen ans e ed o Pu egene Cell Ki o o e nigh P o einase K diges a 55 °C. A second P o einase K diges was done he nex mo ning o 5 h. DNA om he diges ed issue was pu i ied using Pu egene ex ac- ion p o ocol (Qiagen/Gen a). Pu i ied DNA was washed 2× wi h 70% e hanol and DNA pelle ai d ied and ehyd a ed in TE (10 mM T is-Cl, 1 mM EDTA pH 7.5). Epigenome-wide DNA me hyla ion was ana- lyzed using he In inium Human Me hyla ion450 (HM450) BeadChip (Illumina, San Diego, CA, USA) in he Cen e o Applied Genomics (To on o). This a ay Table 1 Clinicopa hological cha ac e is ics o PCa pa ien s by BMI ca ego y Cha ac e NW (n= 5) OB/OW (n=5) P alue Age (yea s) 65.2 ± 3.8 63.2 ± 2.5 0.67 a BMI (kg/m 2 ) 23.0 ± 0.3 29.0 ± 0.9 0.0003 a Gleason sco e < 7 2 (40%) 1 (20%) ≥7 3 (60%) 4 (80%) 1.00 b S age OCPCa 2 (40%) 2 (40%) EPCa 3 (60%) 3 (60%) 1.00 b Smoking s a us Yes 1 5 No 4 0 0.05 b PSA (ng/ml) 10.7 ± 2.7 12.1 ± 3.23 0.74 a Da a a e p esen ed as mean ± SD o numbe (%). Signi ican di e ence be ween OB/OW and NW was e alua ed using a es and b Fishe ’s exac es OB/OW obese/o e weigh , NW no mal weigh , BMI body mass index; PSA, p os a e speci ic an igen; PCa, p os a e cance ; OCPCa, o gan-con ined p os a e cance ; EPCa, ex a-p os a ic PCa Cheng e al. Clinical Epigene ics (2018) 10:54 Page 2 o 15 con ains 485,577 p obes, which co e 21,231 (99%) Re Seq genes. B ie ly, DNA was bisul i e-con e ed using he EZ DNA me hyla ion ki (Zymo Resea ch, O ange, CA, USA) and hen used on he In inium Assay® ollowed by he In inium HD Assay Me hyla ion P o ocol (Illumina). The imaging da a on he BeadChips was cap u ed by Illumina iScan sys em. Da a il e ing and no maliza ion Raw me hyla ion le el o each p obe was ep esen ed by me hyla ion β alue, which was calcula ed based on β= in ensi y o he me hyla ed allele/(in ensi y o he unme hyla ed allele + in ensi y o he me hyla ed allele + 100). M alues we e he logi ans o ma ion o β alues based on M=log 2 (β/(1 −β)), which makes he da a mo e homoscedas ic and app op ia e o u he bioin o ma ic and s a is ical analysis. Me hyla ion alues we e no malized using he unc- ional no maliza ion algo i hm implemen ed in Min i R package [18]. Quali y con ol was pe o med by exclud- ing CpG p obes, which a e ound by Chen e al. o be c oss- eac i e wi h a eas o he genome no a he si e o in e es [19], as well as con ol p obes and p obes on sex ch omosomes. We analyzed a o al o 438,458 CpG si es om he PPAT o 5 OB/OW PCa pa ien s and 5 NW PCa pa ien s. Di e en ial me hyla ion analysis A s a is ical linea modeling app oach was applied o he de ec ed di e en ially me hyla ed CpG si es (DMCs) asso- cia ed wi h obesi y in PPAT using he Bioconduc o “limma”package [20]. Hype - o hypome hyla ion was de e mined when me hyla ion le els o CpGs inc eased o dec eased be ween he OB/OW PCa g oup and he NW PCa g oup based on mean di e en β> 0 o < 0. False disco e y a e (FDR)-co ec ed P alues we e de e mined acco ding o he me hod o Benjamin and Hochbe g’s (BH me hod) mul iple es ing p ocedu e [21]. Di e en ially me hyla ed egions (DMRs) we e iden i ied using he “Bumphun e ”me hod implemen ed in he “chAMP”Rpackage wi h he pa ame e s (B= 1000, useWeigh s = TRUE, minP obes = 10, pickCu o = TRUE, and o he se ings wi h de aul alues) [22]. The p opo ions o signi ican hype - o hypome hy- la ed CpGs we e calcula ed and isualized acco ding o hei ela ion o he nea es genes o o he CpG islands, sepa a ely. Gene p omo e egion was de ined as 1500 base pai s (bp) and 200 bp ups eam o he ansc ip ion s a si e (TSS) (TSS1500 and TSS200) [23]. Iden i ied genes we e selec ed when mo e han wo signi ican ly hype me hyla ed CpGs we e simul aneously loca ed in he p omo e egion. Func ions, pa hway, and ne wo k en ichmen analysis Gene on ology (GO) and KEGG pa hway en ichmen ana- lyses we e pe o med o explo e he biological unc ions o signi ican ly me hyla ed genes using he online bioin o - ma ic ool En ich [24]. P o ein-p o ein in e ac ion (PPI) analysis o all DMC- ela ed genes was pe o med using Ne wo kAnalys acco ding o STRING da abase [25]. Associa ion analysis be ween DNA me hyla ion and gene exp ession We ha e p e iously pe o med gene exp ession expe i- men o he PPAT o he 5 OB/OW PCa pa ien s and he 5 NW PCa pa ien s using he HG-U133 Plus 2.0 A yme ix GeneChip A ay (A yme ix, San a Cla a, CA, USA) [4]. Di e en ial gene exp ession (DGE) ana- lysis be ween he OB/OW PCa pa ien s and he NW PCa pa ien s was e-pe o med using he Bioconduc o “limma”package as p e iously desc ibed [4]. Spea man’s ank co ela ion analysis was pe o med be ween he me hyla ion p o iles o he hype me hyla ed CpGs and he gene exp ession p o iles o he genes in PPAT. Resul s Clinical cha ac e is ics Clinical cha ac e is ics o PCa pa ien s in his s udy we e s a i ied acco ding o obesi y classi ica ion g oups and a e p esen ed in Table 1. Mean age, PSA le el, Gleason sum sco e, and cance s age in subjec s wi h PCa we e simila (P alue > 0.05) be ween OB/OW and NW g oups. As expec ed, he mean BMI o he OB/OW g oup was signi ican ly highe han ha o he NW sub- jec s (P alue < 0.01). All he pa ien s in he OB/OW g oup a e ex-smoke s o ac i e smoke s, while only one pa ien in he NW g oup is a smoke (P alue = 0.05). Epigenome-wide DNA me hyla ion p o iling o PPAT To s udy he impac o obesi y s a us on DNA me hyla- ion p o iles and o iden i y di e en ially me hyla ed CpG si es in PPAT om OB/OW and NW p os a e cance pa ien s, we conduc ed epigenome-wide DNA me hyla- ion analyses. A lowcha o he da a analysis is depic ed in Addi ional ile 1: Figu e S1. A e quali y con ol and il e ing, he In inium a ay gene a ed me hyla ion da a o 438,458 CpG si es, om which 5526 we e di e en ially me hyla ed a e FDR con ol in he PPAT o OB/OW PCa pa ien s compa ed o NW (adjus ed P alue < 0.25; Addi ional ile 2: Table S1 and Table 2). The unsupe ised hie a chical clus e ing o DMCs showed di e en ial DNA me hyla ion pa e ns in PPAT be ween OB/OW and NW samples (Addi ional ile 3: Figu e S2). The majo i y o DMCs we e hype me hyla ed (n= 4985, 90.2%), wi h 9.8% hypome hyla ed CpG si es (n= 541) in OB/OW e sus NW p os a e cance pa ien s (Fig. 1a, b, c). Cheng e al. Clinical Epigene ics (2018) 10:54 Page 3 o 15 Ch omosomal dis ibu ion o he DMCs To u he explo e he me hyla ion p o ile, we in es i- ga ed he ch omosome dis ibu ion o DMCs. Resul s showed ha hype me hyla ed CpG si es we e loca ed a ch omosomes 1, 6, 11, and 17 (p opo ion > 6%, Fig. 1d) and hypome hyla ed CpG si es we e loca ed a ch omo- somes 1, 2, 6, 7, and 11 (p opo ion > 6%, Fig. 1e). Me hyla ion a ia ions o hype me hyla ed DMCs and hypome hyla ed DMCs we e ound mainly dis ibu ed on ch omosomes 1, 6, and 11, sugges ing ha he DNA me hyla ion al e a ions in hese ch omosomes we e co ela ed wi h he body weigh changes in p os a e pa ien s. Fu he mo e, we compa ed he dis ibu ion o he DMCs (hype - and hypome hyla ed, sepa a ely) wi h he dis ibu ion o all e alua ed CpG si es based on hei ela ion o nea es gene egions (Fig. 2 , Addi ional ile 4: Table S2) o hei ela ion o CpG islands (Fig. 2g, Addi ional ile 5: Table S3) using χ 2 es . The esul s showed ha hype me hyla ed CpGs a e mainly loca ed a TSS1500 ( ansc ip ion s a si es 1500), IGR (in e genic Table 2 Di e en ially me hyla ed CpG si es in PPAT be ween obese/o e weigh PCa pa ien s and no mal weigh con ols P obe ID Ch omosome and coo dina e (GRCh37) Nea es gene Rela ion o gene egion Rela ion o CpG island DNAm β di e ence (%) P alue Adjus ed P alue (< 0.25) Hype me hyla ed CpG si es cg09476130 ch 1:159870086 CCDC19 TSS200 Island 12.1 1.87E−03 0.213 cg21293934 ch 18:14748230 ANKRD30B TSS200 Island 11.2 1.83E−03 0.212 cg16925210 ch 2:216946718 PECR TSS200 Island 11.2 2.44E−03 0.226 cg11625005 ch 5:1295737 TERT TSS1500 Island 11.1 1.38E−03 0.196 cg07039560 ch 5:140683681 SLC25A2 TSS200 Island 10.5 2.24E−03 0.222 cg00329447 ch 8:145028170 PLEC1 TSS200 Island 10.1 3.41E−03 0.244 cg24463471 ch 1:25257978 RUNX3 TSS1500 Island 9.9 3.58E−04 0.155 cg26149485 ch 19:2428350 TIMM13 TSS1500 Island 9.7 3.36E−04 0.154 cg05156901 ch 22:51016646 CPT1B TSS200 Island 9.3 3.05E−03 0.238 cg18689454 ch 21:45705694 AIRE TSS200 Island 9.3 7.33E−04 0.174 cg01454592 ch 3:49236800 CCDC36 TSS200 Island 9.3 2.89E−03 0.236 cg24041556 ch 19:10736059 SLC44A2 TSS200 Island 9.1 2.15E−05 0.110 cg22257574 ch 9:135754383 C9o 98 TSS200 Island 9.0 1.97E−05 0.110 cg23005885 ch 15:90543450 ZNF710 TSS1500 Island 8.9 6.20E−04 0.169 cg05726756 ch 17:46608288 HOXB1 TSS200 Island 8.6 1.67E−03 0.206 cg12782180 ch 7:127880932 LEP TSS1500 Island 8.5 9.78E−04 0.184 cg04675542 ch 5:150284416 ZNF300 TSS200 Island 8.4 3.42E−03 0.244 cg10134527 ch 6:33283015 TAPBP TSS1500 Island 8.4 3.19E−03 0.241 cg23387569 ch 12:58120011 LOC100130776 TSS200 Island 8.4 3.39E−04 0.155 cg17205324 ch 14:23835595 EFS TSS1500 Island 8.3 1.46E−04 0.133 cg24402300 ch 19:55591437 EPS8L1 TSS1500 Island 8.2 2.02E−03 0.216 cg18081258 ch 14:21494161 NDRG2 TSS1500 Island 8.2 1.05E−03 0.187 cg00730561 ch 10:102279703 SEC31B TSS200 Island 8.1 4.82E−04 0.162 cg17791651 ch 1:38513489 POU3F1 TSS1500 Island 8.0 1.31E−04 0.133 Hypome hyla ed CpG si es cg03462171 ch 16:1664488 CRAMP1L TSS200 Island −8.2 2.30E−03 0.223 cg11648730 ch 5:92907151 FLJ42709 TSS1500 Island −6.4 1.72E−03 0.207 cg04558166 ch 1:210001279 C1o 107 TSS200 Island −4.0 1.75E−03 0.209 cg25472897 ch 8:145560555 SCRT1 TSS1500 Island −3.1 1.53E−03 0.201 cg17612948 ch 5:110427863 WDR36 TSS200 Island −3.0 1.70E−03 0.207 cg21665057 ch 3:196295764 WDR53 TSS1500 Island −2.4 2.42E−03 0.225 cg12683173 ch 7:69063404 AUTS2 TSS1500 Island −1.4 3.30E−03 0.242 cg04872557 ch 1:76190008 ACADM TSS200 Island −1.0 1.96E−03 0.215 Cheng e al. Clinical Epigene ics (2018) 10:54 Page 4 o 15 egion), N-sho e, and S-sho e, and hypome hyla ed CpGs a e mos ly loca ed a he gene body and open sea. Func ional en ichmen analysis o signi ican ly obesi y-associa ed DMCs To in es iga e he po en ial biological ele ance o he sig- ni ican DMCs, we u he il e ed 483 DMCs (dis ibu ed wi hin 413 genes) om a o al o 5526 DMCs acco ding o hei loca ions a bo h he gene p omo e and CpG island (Addi ional ile 6: Table S4). Fou hund ed se en y- i e o he 483 DMCs ( ep esen ing 404 genes) we e hype me hyla ed. Func ional en ichmen analysis o he hype me hyla ed genes showed ha hese genes we e en iched o biological p ocesses, such as pa e n speci ica ion p ocess, neu on di e en ia ion, neu on a e speci ica ion, and nega i e egula ion o phospha e me abolic p ocess (adjus ed P alue < 0.05, Addi ional ile 7: Table S5), as well as molecula unc ions, such as Fig. 1 Epigene ic p o iles o di e en ially me hyla ed CpGs o PPAT be ween OB/OW and NW g oups. Manha an plo s show epigene ic p o iles o all inc eased me hyla ed CpGs (a) and all dec eased me hyla ed CpGs (b). The X-axis shows ch omosomes, and he Y-axisisa−log10 (P alue). The black line ep esen s he h eshold o adjus ed P alue = 0.25. CpGs abo e he black line a e signi ican ly hype - o hypome hyla ed. The olcano plo o DNA me hyla ion (c) shows a signi ican di e ence in PPAT be ween he OB/OW and NW g oups. Fou housand nine hund ed eigh y- i e hype me hyla ed CpGs a e labeled in ed, and 541 hypome hyla ed CpGs a e labeled in g een (adjus ed P alue > 0.25). The p opo ions o hype - and hypome hyla ed CpGs on each ch omosome a e shown in (d)and(e). The black line indica es i he p opo ions o hype -andhypome hyla edCpGs on a ch omosome a e highe han 6%. The dis ibu ion o signi ican DMCs (hype - o hypome hyla ed CpGs) and globe DNA me hyla ion CpGs in loca ions ela ed o he nea es gene egions and CpG islands a e shown in and g. Hype me hyla ed CpGs a e mainly loca ed a TSS1500 ( ansc ip ion s a si es 1500), IGR (in e genic egion), N-sho e ( he 2 kb egions ups eam o he CpG islandbounda ies),andS-sho e( he2kb egions downs eam o he CpG island bounda ies), and hypome hyla ed CpGs a e mos ly loca ed a he gene body and open sea. The di e ence o he p opo ion o CpGs among he h ee CpG g oups was calcula ed based on he χ 2 es (*P< 0.05, **P< 0.01, ***P< 0.001, ns no signi ican ). CpG islands we e de ined as DNA sequences (500 base windows; excluding mos epe i i e Alu-elemen s) wi h a GC base composi ion g ea e han 50% and a CpG obse ed/expec ed a io o mo e han 0.6. The 2 kb egions immedia ely ups eam (N_Sho e) and downs eam (S_Sho e) o he CpG island bounda ies we e de ined as “CpG island sho es,”and he 2 kb egions ups eam (N_Shel ) and downs eam (S_Shel ) o he CpG island sho es we e e e ed as “CpG island shel es.”Open seas we e he egions mo e han 4 kb om CpG islands Cheng e al. Clinical Epigene ics (2018) 10:54 Page 5 o 15 neu opep ide ecep o ac i i y and sequence-speci ic DNA-binding RNA polyme ase II ansc ip ion ac o ac- i i y (adjus ed P alue < 0.1, Addi ional ile 8: Table S6). KEGG pa hway en ichmen analysis showed ha hype - me hyla ed genes we e in ol ed in signaling pa hways egula ing plu ipo ency o s em cells, a y acid me abol- ism, basal cell ca cinoma, non-alcoholic a y li e disease (NAFLD), and AMPK signaling pa hway (P alue < 0.05, Addi ional ile 9: Table S7). We mapped he 404 hype me hyla ed genes o he STRING da abase and gene a ed a p o ein-p o ein in e - ac ion (PPI) ne wo k by he Ne wo kAnalys . The la ges subne wo k was iden i ied o include 247 nodes (genes) and 403 edges (Fig. 2a). In he ne wo k, he size o he nodes was based on hei deg ee alues and he colo o nodes was based on hei P alues. This ne wo k con- ained 118 seed genes om he DMCs, and he en ich- men pa hway analysis showed ha he genes o he Fig. 2 P o ein-p o ein in e ac ion analysis. aA subne wo k composing o 247 nodes and 403 edges was gene a ed using me hyla ed genes. Blue do s ep esen he genes in ol ed in p os a e cance ; ed and pink do s ep esen he seeds (me hyla ed genes) acco ding o he di e en P alues; he g ay do s ep esen he p o eins which we e closely in e ac ed wi h he seeds, and he ci cle size ep esen s he node deg ee. bThe pa hway en ichmen analysis shows he subne wo k is mainly en iched in cance pa hways (P<0.0001) Cheng e al. Clinical Epigene ics (2018) 10:54 Page 6 o 15 subne wo k we e mos ly in ol ed in he pa hways o p os- a e cance and o he cance s (Fig. 2b,Addi ional ile10: Table S8, adjus ed P alue < 0.05). Pa icula ly, he gene UBC (ubiqui in C) was ound o be a hub connec ing wi h many o he nodes in he ne wo k, sugges ing ha he gene may play impo an biological oles in he PPAT o obese PCa pa ien s. Selec ed genes wi h mul iple me hyla ed CpG si es In o de o explo e ep ession o genes by DNA me hy- la ion modi ica ions, we selec ed genes which had mul- iple hype me hyla ed CpG si es ( he numbe o me hyla ed CpG si es ≥2, in a leas one o he si es wi h ameandi e enceo β> 3% and an adjus ed P alue < 0.25) (Addi ional ile 1: Figu e S1 and Addi ional ile 11:TableS9). A o al o 38 genes wi h 100 di e en ially me hyla ed CpG si es we e selec ed, which included TAPBP,RUNX3,CPT1B, CPT1C,MOGAT3,WNT2,andAIRE (Addi ional ile 11: Table S9). No ably, he p omo e egion o TAPBP (TAP- binding p o ein) had eigh hype me hyla ed CpG si es in he p omo e (Fig. 3a), which we e signi ican ly mo e me hyla ed in he OB/OW han hose in he NW g oups (Fig. 3b), wi h ameandi e enceo β alue g ea e han 5% (Addi ional ile 10: Table S8). Spea man’s ank co ela ion showed s ong associa ion ( 2 =0.73–0.97) o he eigh hype me hyla ed CpGs in he TAPBP p omo e wi h hei me hyla ion le els (Fig. 3c). Pa hway analysis o hese genes e ealed en ichmen o a y acid me abolism, PPAR signaling pa hway, glucagon signaling pa hway, AMPK signaling pa hway, glyce olipid me abolism, basal cell ca cinoma, an igen p ocessing and p esen a ion, ECM ecep o in e ac ion, and insulin esis ance (adjus ed P alue < 0.25) (Addi ional ile 12: Table S10). Di e en ial me hyla ed egions analysis Ten DMRs we e iden i ied (P< 0.01) in obesi y PPAT samples compa ed o no mal weigh con ols (Table 3). The size o he DMRs a ied om 161 o 1287 bp. No e- wo hy, ou ou o he en DMRs we e disco e ed on ch omosome 6. Eigh egions we e loca ed in genes, and wo we e in he in e genic egion. Fou egions we e in he gene p omo e o FAM104A,C17o 80,HOXA4A, and TAPBP. Associa ion analysis be ween DNA me hyla ion and mRNA exp ession Inc eased DNA me hyla ion o p omo e in CpG islands was ob iously linked o gene ansc ip ional silencing [26]. The e o e, we ela ed hype me hyla ed CpG si es in PPAT wi h genes showed dec eased gene exp ession le el om ou p e iously gene a ed mRNA exp ession da a [4]. DNA me hyla ion o 16 CpG si es, co esponding o 14 genes, was associa ed wi h signi ican ly dec eased ansc ip s in OB/OW g oup (P alue < 0.05) (Table 4). The Spea man’s ank co ela ion analysis showed ha eigh o he 14 genes ha e signi ican ly nega i e associa ion (P alue < 0.05) be- ween he me hyla ion p o iles and he gene exp ession p o ileso hesegenes(Table4). The ep ession genes we e mainly in ol ed in me abolic pa hways (Addi ional ile 13: Table S11, adjus ed P alue < 0.25), such as MOGAT1 (glyce olipid me abolism), FADS1 ( a y acid me abolism and biosyn hesis o unsa u a ed a y acids), and PCYT2 (glyce ophospholipid me abolism). The mRNA exp ession le el o FADS1 was signi ican ly dec eased in he PPAT o obese wi h p os a e cance s in ou p e ious s udy using qRT-PCR [4]. Besides hese, GO en ichmen analysis showed ha hese genes a e unc ionally ela ed o ecep o binding (neu opep ide ecep o binding, dopamine ecep o binding, and insulin ecep o binding) and enzyme ac i i y (acid phospha ase ac i i y, me alloca boxypep idase ac i i y, and acylglyce ol O-acyl ans e ase ac i i y) (Addi ional ile 14: Table S12, adjus ed P alue < 0.25). Discussion This pilo s udy e ealed signi ican di e ences o DNA me hyla ion p o iles be ween he PPATs om OB/OW e sus NW PCa pa ien s. Va ia ions in global DNA me hyla ion demons a ed ha excess adiposi y played an impo an ole in DNA me hyla ion le el o PPAT issues in p os a e cance pa ien s, which p o ide an oppo uni y o explo e he e ec o obesi y on PPAT epigene ic modi ica ion and subsequen ly on p os a e cance . These indings epo ed o he i s ime in PPAT depo a e in conco dance wi h p e ious wo ks epo ing ha excess adiposi y and BMI ac i a e DNA me hyla ion in adipose issue [27–29]. Thus, conside ing he p esen unde s anding o he po en ial causal ela ionship be ween excess adiposi y and cance [30], diabe es [11], and ca dio ascula disease [31], ou esul s p o ide me hyla ed candida e genes, which migh os e esea ch on he po en ial biological mechanisms unde - lying epigene ic egula ion o PPAT by excess adiposi y and p os a e cance . Gi en ha DNA me hyla ion o CpGs loca ed a p o- mo e s and islands a e associa ed wi h gene ansc ip- ion silencing, we pe o med a s ic il e ing o DMCs and explo ed he biological unc ions o all p omo e hype me hyla ed genes, aiming o ind he c i ical me h- yla ed CpGs in he PPAT be ween he obese and no mal weigh PCa pa ien s. Bioin o ma ic analysis showed ha he en iched pa hways we e mos ly in ol ed in me a- bolic diso de s, pa icula ly a y acid deg ada ion and glyce olipid and choline me abolism. These pa hways a e known o media e he p o- umo al e ec o whi e adipose issue in umo s, hus con ibu ing o umo i- genesis and me as asis [32,33], pa icula ly in p os a e cance [5]. Findings om o he oncological models high- ligh excess adiposi y-associa ed impac in me hyla ion Cheng e al. Clinical Epigene ics (2018) 10:54 Page 7 o 15 Fig. 3 Visualiza ion and analysis o hype me hyla ed CpG si es in TAPBP p omo e .aThe ch omosome posi ions o hype me hyla ed CpG si es show ha DMCs a e loca ed a ch 6 (p21.32), which is in he egion o TSS1500 (pink squa e) o TAPBP and a he loca ion o CpG island 31. bMe hyla ion le els o eigh CpG si es in PPAT om OB/OW and NW PCa pa ien s ha e shown a signi ican di e ence. cCo ela ion analysis shows s ong co ela ion (Spea man co ela ion coe icien 2 0.73~ 0.97) be ween he eigh hype me hyla ed CpG si es based on he mean β alue di e ence o indi idual p obes Cheng e al. Clinical Epigene ics (2018) 10:54 Page 8 o 15 ma ke s known o associa e wi h po en ial e ec in he cance mic oen i onmen (e.g., a oma ase, p os aglandin E 2 ecep o in b eas cance ) [34,35]. Obesi y has also been shown o associa e wi h me hyla ion o cance - ela ed genes (E-cadhe in, p16, and RAR-β(2)) di ec ly in malignan b eas cells [36,37]. Pa hway en ichmen analysis showed a s ong associ- a ion be ween p omo e hype me hyla ion o CPT1B, CPT1C,ACADM, and FADS1, wi h a y acid me abol- ism. CPT1B (ca ni ine palmi oyl ans e ase 1B) and CPT1C (ca ni ine palmi oyl ans e ase 1C) genes encode a e-limi ing enzymes in a y acid deg ada ion and play c i ical oles in long-chain a y acid (LCFA) β-oxida ion by con olling anspo a ion o long-chain a y acyl- CoAs om he cy oplasm ac oss he ou e mi ochond ia memb ane [38]. Maple e al. epo ed ha inc eased me hyla ion o speci ic CpGs in he CPT1B p omo e was co ela ed wi h dec eased CPT1B ansc ip s in he skele al muscle a e lipid o e supply in se e e obesi y, which esul ed in obese indi idual’s incapaci y o inc ease a oxida ion, con ibu ing o me abolic in lex- ibili y [39]. Al hough he biochemical unc ion o CPT1C has been e i ied o be necessa y o he egula ion o ene gy homeos asis in CPT1C knockou mouse b ain [40], he s udy o CPT1C me hyla ion was absen . CPT1B and CPT1C we e p e iously epo ed o be highly exp essed in he muscle, b ain, and many o he no mal issues including adipocy es [41]. Taken oge he , hese indings sugges ha me hyla ion o speci ic CpG si es in he CPT1B and CPT1C p omo e s likely esul in gene Table 3 Di e en ially me hyla ed egions (DMR) in PPAT o obese/o e weigh PCa pa ien s compa ed o no mal weigh con ols DMR Ch S a –end (bp) Size (bp) P alue FDR Loca ed gene DMCs* Rela ion o CpG island 1 6 30,038,791–30,039,801 1010 5.11E−05 2.07E−02 RNF39 37(0) Island 2 6 29,648,161–29,649,084 923 1.54E−03 2.08E−01 ZFP57 # 22(0) Open sea 3 17 71,228,123–71,228,832 709 3.10E−03 2.86E−01 FAM104A 4(3) Island 4 17 71,228,123–71,228,832 709 3.10E−03 2.86E−01 C17o 80 10(8) Island 5 12 42,720,006–42,720,167 161 5.56E−03 2.86E−01 PPHLN1 4(0) Island 6 6 31,650,735–31,651,158 423 5.59E−03 2.86E−01 MIR4646 # 16(0) Island 7 7 27,169,674–27,170,961 1287 5.66E−03 2.86E−01 HOXA4 17(11) Island 8 6 33,282,736–33,283,145 409 5.87E−03 2.86E−01 TAPBP 18(18) Island 9 20 57,463,763–57,464,129 366 6.74E−03 2.86E−01 GNAS 15(0) Island 10 16 86,546,938–86,547,322 384 7.03E−03 2.86E−01 FOXF1 4(0) Sho e #The DMR is loca ed a he in e genic egion Ch ch omosome *The numbe in he b acke is he quan i ies o DMCs loca ed a he p omo e (TSS200 and TSS1500) egions Table 4 Genes hype me hyla ed in p omo e s wi h signi ican ly dec eased gene exp ession Gene symbol DNA me hyla ion Gene exp ession Co ela ion analysis P obe ID DNAm β di . (%) Adjus ed P alue (< 0.25) P obe ID FC P alue (< 0.05) Spea man’s ank co ela ion coe icien P alue (< 0.05) UCN cg20442078 5.6 0.17 8051061 −1.12 3.61E−02 −8.42E−01 2.23E−03 CCHCR1 cg00160818 1.9 0.17 8124868 −1.14 1.73E−02 −7.45E−01 9.21E−03 CRB3 cg14782015 4.3 0.20 8025041 −1.13 1.84E−02 −7.21E−01 1.21E−02 AGBL4 cg21834207 3.2 0.13 7915971 −1.17 1.29E−02 −6.73E−01 1.97E−02 INSL3 cg10174482 4.2 0.13 8035345 −1.13 4.94E−02 −6.36E−01 2.72E−02 ANKRD30B cg21293934 11.2 0.21 8069499 −1.17 2.24E−02 −6.08E−01 3.11E−02 FADS1 cg16213375 3.6 0.16 7948612 −1.8 9.55E−04 −5.88E−01 4.01E−02 PAPL cg18481683 2.3 0.24 8028570 −1.19 1.45E−02 −5.52E−01 5.21E−02 MOGAT1 cg12678667 4 0.15 8048725 −1.28 3.87E−02 −4.67E−01 8.91E−02 PPP1R1B cg09762778 5 0.12 8006865 −1.27 4.74E−02 −4.67E−01 8.91E−02 PRUNE2 cg00390775 4.4 0.15 8161884 −1.31 1.91E−02 −3.82E−01 1.39E−01 CIDEA cg18309817 1.8 0.18 8020211 −1.32 3.21E−02 −2.97E−01 2.03E−01 PCYT2 cg19583655 6.2 0.21 8019280 −1.26 6.38E−04 −1.88E−01 3.04E−01 SCUBE1 cg07697597 1.7 0.23 8076586 −1.23 9.03E−03 −4.24E−02 4.59E−01 FC old change, DNAm βdi . DNAm βdi e ence Cheng e al. Clinical Epigene ics (2018) 10:54 Page 9 o 15