Research Article Functional Epigenetic Analysis of …downloads.hindawi.com/journals/jbm/2014/362164.pdfResearch Article Functional Epigenetic Analysis of Prostate Carcinoma: A Role

Research ArticleFunctional Epigenetic Analysis of Prostate CarcinomaA Role for Seryl-tRNA Synthetase

Odiljon Ikromov1 Imad Alkamal1 Ahmed Magheli1 Nadine Ratert1 Mauricio Sendeski2

Kurt Miller1 Hans Krause1 and Carsten Kempkensteffen1

1 Klinik fur Urologie Charite-Universitatsmedizin Berlin Charite Campus Mitte (CCM) Chariteplatz 1 10117 Berlin Germany2 Institut fur Vegetative Physiologie Charite-Universitatsmedizin Berlin Chariteplatz 1 10117 Berlin Germany

Correspondence should be addressed to Hans Krause hanskrausecharitede

Received 24 October 2013 Accepted 21 February 2014 Published 27 March 2014

Academic Editor Ranju Ralhan

Copyright copy 2014 Odiljon Ikromov et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Transcriptional silencing as a result of aberrant promoter hypermethylation is a common mechanism through which genes incancer cells become inactive Functional epigenetic screens using demethylating agents to reexpress transcriptional silenced genesmay identify such inactivated genes for needing further evaluation We aimed to identify genes so far not known to be inactivatedby promoter hypermethylation in prostate cancer DU-145 and LNCaP cells were treated with the DNMT inhibitor zebularineExpression changes of total RNA from treated and untreated cells were compared using an RNA expression microarray Genesupregulated more than 2-fold were evaluated by RT-qPCR in 50 cases of paired normal and tumor tissues of prostate cancerpatients SARS was found to be downregulated in prostate cancer in 4250 cases (84) In addition GADD45A and SPRY4 showeda remarkable diminished expression (88 and 74 resp) The gold standard for promoter hypermethylation-inactivated genes inprostate cancer (GSTP1) was repressed in 90 of our patient samples ROC analyses reported statistically significant AUC curvesin SARS GADD45A and GSTP1 and positive Spearman correlations were found between these genes SARS was discovered to be anovel gene that is repressed in prostate cancer and could therefore be recommended for its involvement in prostate carcinogenesis

1 Introduction

Prostate cancer (PCa) is the second most frequently diag-nosed cancer and the fifth leading cause of cancer deathamong men worldwide with 1111689 new cases and 307471(66) deaths projected to occur in 2012 [1] In 2013 cancersof the prostate lung and bronchus and colorectum willaccount for about 50 of all newly diagnosed cancers inthe United States prostate cancer alone will account for 28(238500) of incident cases [2] In Germany prostate canceris already the third most cause of cancer death with morethan 68000 newly diagnosed cases (252) and a mortalityrate of approximately 12500 men (107) in 2012 [1] Despiteextensive scientific efforts and technological progress themolecular mechanisms of development and progression inparticular to lethal PCa are still elusive and need furtherinvestigation

Genetic and epigenetic alterations in prostate tumorige-nesis as in all other cancers accumulate within a multistep

transformation process on the cellular level [3 4] AberrantDNA promoter CpG islands hypermethylation of genes is awell-characterized mechanism for transcriptional silencingof tumour suppressor genes in various cancers [5] Epigeneticsilencing associated with promoter hypermethylation hasbeen well documented also in PCa including GSTP1 RARBAPC PYCARD PTGS2 ABCB1 and RASSF1 genes [6]

One way to identify epigenetically silenced genes intumor cells is based on reversal of epigenetic silencing byDNA methyltransferases (DNMT) inhibitors such as 5-aza-2-deoxycytidine and the structurally related compound zebu-larine [7] We used moderate concentrations of zebularine toreactivate epigenetically silenced genes bymeans of demethy-lating cytosine residues resulting in the ldquoreexpressionrdquo of therespective genes [8] Compared to other more frequentlyused drugs such as azacytidine and decitabine zebularineis a highly stable hydrophilic inhibitor of DNA methylationwith oral bioavailability and low toxicity even after prolonged

Hindawi Publishing CorporationJournal of BiomarkersVolume 2014 Article ID 362164 10 pageshttpdxdoiorg1011552014362164

2 Journal of Biomarkers

Table 1 Patientsrsquo clinical parameters

Characteristics Parameters Patients 119899 = 50 (100)

Age years Median 64Range 47ndash74

Preoperative PSA ngmL Median 87Range 106ndash78

T stage

pT2a 1 (2)pT2b 15 (30)pT2c 12 (24)pT3a 6 (12)pT3b 13 (26)pT3x 1 (2)pT4 2 (4)

N stage N0Nx 44 (88)N1 6 (12)

M stage M0 50 (100)

Gleason score

na 1 (2)3 1 (2)5 15 (30)6 12 (24)

7a (3 + 4) 9 (18)7b (4 + 3) 4 (8)

8 4 (8)9 4 (8)

administration [9 10] We treated androgen insensitive DU-145 and androgen sensitive LNCaP PCa cell lines withmoderate doses of zebularine and identified globally silencedgenes by RNA microarray analysis of the transcriptomeThrough intuitive selection of upregulated and validatedgenes we estimated relative gene expression (RGE) profilesfor selected candidate genes in 50 patients using pairedsamples of adjacent normal and tumour prostate tissueIn agreement with our working hypothesis of methylation-induced transcriptional inactivation we focused our attemptson genes that are exclusively downregulated in primaryPCa Eventually we identified for the first time seryl-tRNAsynthetase (SARS) to be downregulated in almost all PCacases analysed SARS was further characterized to qualify asa possible candidate for future epigenetic study approaches inPCa

2 Materials and Methods

21 Zebularine Treatment LNCaP prostate cancer cell lines(ATCC number CRL-1740) and DU-145 (ATCC numberHTB-81) were chosen for treatment experiments Both celllines were purchased from the American Type Culture Col-lection (ATCC Manassas VA USA) Cells were treated witha final concentration of 100120583Mof theDNAmethyltransferase(DNMT) inhibiting reagent zebularine Growth mediumcontaining zebularine was replaced regularly after 48ndash72hours and cells were split at a ratio of 1 3 In total cellswere exposed to zebularine for 216 hoursThree independent

treatment experiments including untreated controls wereperformed

22 Tissue Samples Tumor and adjacent normal tissues sam-ples of prostate cancer patients collected after radical prosta-tectomy between 2002 and 2004 at the Department ofUrology Charite-Universitatsmedizin Berlin Germany wereused in experimentsThe samples were collected immediatelyafter surgery in liquid nitrogen and stored at minus80∘C Thestudy was done according to the regulation of ethical boardof the hospital with informed consent of patients Sampleswere analyzed by a uropathologist for their tumor content Intotal 50 prostate adjacent normal and tumor tissue samplescontaining at least 60 tumor tissue with different stagewere included in this study Patientsrsquo clinical parameters aredescribed in Table 1

23 RNA Isolation and cDNA Synthesis Total RNA wasextracted from treated and untreated PCa cells as well asfrom prostate normal and tumor tissue samples by using theldquomiRNeasy Mini Kitrdquo (Qiagen Hilden Germany) accord-ing to manufacturerrsquos recommendations Approximately 4-5 times 106 cells and 20ndash30mg of tissue were used for RNAextraction RNA concentration and purity were determinedspectrophotometrically on a Nanodrop ND-1000 instrument(Thermo Scientific Germany) All RNA samples were freefrom remaining proteins (260280 nm ratio from sim18 to20) and other contaminations (260230 nm ratio = 20to 22) In addition integrity of RNA was assessed by

Journal of Biomarkers 3

capillary electrophoresis on the Bioanalyzer-2100 instru-ment (Agilent Technologies Waldbronn Germany) Onlycell line RNA samples with RIN numbers ge80 were usedfor RNA chip analysis For RT-qPCR analyses only tissueRNAs with RIN ge60 were considered Subsequently RNAwas reverse transcribed according to the manufacturerrsquosinstructions of the ldquoTranscriptor First Strand cDNA Syn-thesis Kitrdquo (Roche Diagnostics Mannheim Germany) Toincrease sensitivity a combination of anchored-oligo (dT)priming and random hexamer priming was used to tran-scribe 1 120583g RNA in a total volume of 10 120583L (SupplementaryFile S1) (see Supplementary Material available online athttpdxdoiorg1011552014362164)

24 Microarray Analyses RNA microchip analyses wereperformed at the core facility ldquoLabor fur funktionelleGenom-forschungrdquo (LFGC) of Charite-Universitatsmedizin BerlinTotally 12 Affymetrix 10 ST chip (Cat number 901086Affymetrix Inc Santa Clara CA USA) hybridization anal-yses for both PCa treated and untreated cells lines wereperformed in triplicateThis type of chip covers 36079 probesthat represent 21014 genes 300 ng of total RNA was used forfirst cycle cDNA synthesis from both treated and untreatedPCa cells Labeled cDNA was hybridized at 45∘C for 16 hrsStaining and washing were performed in a Fluidics Station450 (Affymetrix Inc Santa Clara CA USA) The scanningwas carried out on a GeneChip Scanner 3000 G7 system(Affymetrix Inc Santa Clara CA USA) The raw data werenormalized according to the log scale robust multiarray anal-ysis (RMA) [11] Briefly signal intensities were backgroundadjusted to the perfect match (PM) intensities and quantilenormalization approach was performed across all arrays ofthe experiment After log2 transformation data were globalmedian polished In order to control the false discovery rateat 120572 lt 005 for array data we applied the false discoveryrate multiple testing correction according to Benjamini andHochberg [12] All RNA chip data have been deposited inthe National Center for Biotechnology Information GEOdatabase under accession number GSE51629 (httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE51629)

25 Computational Analyses Differentially expressed geneswere identified using Microarray Suit 40 software (Affyme-trix Inc Santa Clara CA USA) Candidate genes wereselected according to their fold change upregulation witha cutoff of 2-fold In addition we applied the presence ofone or more CpG islands in the promoter region of a par-ticular gene as second criteria EMBOSS CpGPlot program(httpwwwebiacukToolsseqstatsemboss cpgplot) wasused to determine the presence of CpG islands of therespective genes Furthermore serial analysis of gene expres-sion (SAGE httpcgapncinihgovSAGE) was exploitedto check for candidates that show the same or even higherexpression in normal tissue when compared to tumor tissue

26 Quantitative Real-Time PCR (RT-qPCR) RT-qPCR forselected genes was performed on the LightCycler 480Instrument (Roche Diagnostics GmbH Mannheim Ger-many) in 96-well white plate format and analyzed using

proprietary software (v150) RT-qPCR was carried outaccording to the manufacturerrsquos protocol and the MIQEguidelines (Supplementary File S2) For relative quantifica-tion 110 of cDNA was amplified using the ldquoProbe Masterkitrdquo and proprietary ldquoUPL proberdquo PCR format (Roche)in a total reaction volume of 10 120583L Primer sequencesand appropriate probe sets for target and reference geneswere derived from the UPL website (httpwwwroche-applied-sciencecomsisrtpcruplezhomehtml) Amplifica-tion primers were synthesized by TIBMOLBIOL (TIBMOL-BIOL Berlin Germany) UPL hydrolysis-type probes werepurchased from Roche Diagnostics GmbH PCR was runwith a preincubation at 95∘C for 10 minutes followed bycycling (45x) at 95∘C10 sec and 59∘C20 sec (SupplementaryFile S1) All samples were measured in triplicate each PCRrun includes no-template control and interpolate calibra-tor mRNA expression levels were normalized for intra-and interassay variation by inclusion of a calibrator andPBGD as the reference gene PCR efficiency was determinedusing standard curves and ranged between 90 and 98(Supplementary File S3) Data were analyzed using GenExSoftware v 437 (MultiD Analyses AB Goteborg Swedenhttpmultidse)

27 Data Analyses Statistical analyses of gene expressiondata were performed using the statistical programsGraphPadPrism v601 (GraphPad Software Inc La Jolla CA) and SPSS190 (IBM Corporation Somers NY) Significant differencesbetween paired normal and tumor tissues were calculatedusing nonparametric Wilcoxon test Spearmanrsquos rank cor-relations were used to analyze the relationships betweenexpression levels of deregulated genes as well as betweenclinical variables To determine the discriminative potentialof deregulated genes between normal and tumor samples andthe diagnostic accuracy we used receiver operating curves(ROC) calculated by MedCalc Software v120 (MedCalcSoftware Mariakerke Belgium) Sample size calculation (120572= 5 power = 80) for the comparison of the area underthe receiver operating characteristics curve of 08 (takinginto account this value as appropriate discrimination power)with the null hypothesis value 05 was calculated to be 28in each group 119875 values lt005 (two-tailed) were consideredstatistically significant in all cases

3 Results and Discussion

Epigenetic screens exploiting the effects of demethylatingagents to reactivate transcriptionally silenced genes are suit-able tools for the discovery of new biomarkers and putativetherapeutic targets [13]

We performed an RNA microarray screen of zebularine-treated and zebularine-untreated prostate cancer cells linesDU-145 and LNCaP and discovered a total of 3449 genesexpressed at least ge15-fold in 3 independent experimentsThenumber of shared upregulated genes in both cell lines was85 and 31 respectively (Figure 1)

A total of 91 genes were at least 2-fold upregulated in thetwo prostate cancer cell lines By applying additional selectioncriteria like the presence of CpG islands in the promoter


1st experiment 2nd experiment

106

83

1357

85

24 76

235

3rd experiment

(a)

166

19

205

31

57 31

452


3rd experiment

(b)

Figure 1 Number of shared ge15-fold upregulated genes in Venn diagrams in three independent biological experiments in PCa cell linesDU-145 (a) and LNCaP (b) after treatment with the demethylating agent zebularine

region and SAGE database-derived expression data towardsan elevated intrinsic expression in normal prostate tissues thenumber of candidates was further reduced to 52 (Table 2)

Particular emphasis was added on the efficacy of zebu-larine action since this drug is known to be less activewhen compared to other demethylating agents Thereforewe measured the ldquodemethylating potentialrdquo of zebularineindirectly by measuring the reexpression of IFI6 gene thatis regulated by hypermethylation of its promoter [14] UsingRT-qPCR we measured a 194-fold (SD plusmn 46) upregulationof IFI6 after treatment with 100 120583M zebularine for 9 days

Next we aimed to validate our candidates in clinicalsamples to provide evidence that they are indeed dimin-ished in their expression in prostate carcinoma tissuesFor verification of our screening assay we were lookingspecifically for sprouty homolog 4 (SPRY4) and growth arrestand DNA-damage-inducible alpha (GADD45A) since thesegenes were already revealed by similar epigenetic reactivationscreens [15ndash17] Central to our measures of relative geneexpression (RGE) in 50 matched tumor and normal prostatetissues however was the gene for seryl-tRNA synthetase(SARS) so far not described for its diminished expressionin prostate cancer As a gold standard for subsequent com-parisons we used the frequently suppressed glutathione S-transferase 1205871 (GSTP1 Figure 2) [18] that was downregulatedin 45 of our prostate tumor tissues (90 119875 lt 00001) withmedian fold change of minus254 (Table 3)

SPRY4 was significantly downregulated in this study in37 tumor specimens (74 119875 = 00007 Figure 2) and mostof the samples (3137 84) were inhibited more than 15-fold (median = minus164 Table 3) Wang et al reported thatthe expression of this inhibitor of the growth factor-inducedcell responses was downregulated in approximately half ofprostate cancers due to promoter hypermethylation Thisregulation of SPRY4 by epigenetic inactivation was furthersubstantiated using 5 Aza-dC treatment of LNCaP cells thatrestored its expression [16]These results and the observation

that the close relative SPRY2 is also regulated by epigeneticmodification add further evidence on the role of membersof the SPRY family as putative tumor suppressors in prostatecancer [19]

Another gene that is repressed by promoter hyperme-thylation is the cell cycle regulator GADD45A (also knownas DDIT1) that causes cell cycle arrest by blocking G2-M transition in response to cellular DNA damage [20] Inagreement with findings by Lodygin et al [15] we foundthat this gene repressed in 44 tumor tissues (88 119875 lt00001) with a majority of samples downregulated more than15-fold (median = minus232 Table 3 and Figure 2) The roleof GADD45A as an epigenetically regulated and putativetherapeutic target is also emphasized by the observation thatDNMT inhibitors enhance sensitivity to docetaxel in DU-145and LNCaP cell lines [21]

Surprisingly expression of seryl-tRNA synthetase (SARSSERS or SERRS)was significantly repressed in themajority ofour prostate cancer specimens although we are aware of evi-dences for aminoacyl-tRNA synthetases in cancer progres-sion [22] SARS expression was significantly diminished in 42cases (84 119875 lt 00001) when compared to matched normaltissue and more than half of the downregulated samples(2342 55) were repressed more than 15-fold (median =minus144) (Table 3 and Figure 2) With regard to SARSs possibleinvolvement in prostate carcinogenesis recently a uniquedomain at the C-terminus of almost all vertebrate SARSs(named UNE-S) that links SARS to vascular developmentby its interaction with vascular endothelial growth factorA (VEGFA) was described [23] VEGFA itself is a keyregulator of angiogenesis that activates tyrosine kinase recep-tors VEGFR1 (Flt-1) and VEGFR2 (KDRFlk-1) respectively[24] Our own group recently aimed to link angiogenesis-related factors more closely to prostate cancer progressionby discovering decreased transcript levels of VEGFR2 andother endothelial factors such as CD34 CD146 and CAV1in prostate cancer [25] It is also noteworthy that SARS is


Table2Listof

upregu

latedgenesa

fterb

ioinform

aticsa

nalyses1

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ADRA

2Aadrenergicalpha-2A-

receptor

10q252

231

YES

DDDX6

0DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide6

04q

323

297

yes

SPO

TEF

POTE

ankyrin

domainfamilym

emberF

2q211

215

yes

DDDX5

8DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide5

89p

12375

yes

SIG

F1R

Insulin

-like

grow

thfactor

1receptor

15q263

272

yes

SIN

PP4B

Inosito

lpolypho

sphate-4-pho

sphatasetypeII

4q3121

263

yes

DIFI6

Interfe

ron

alph

a-indu

ciblep

rotein

61p35

486

yes

DTX

NIPlowast

Thioredo

xininteractingprotein

1q211

465

YES

DADAM32

ADAM

metallopeptidased

omain32

8p1122

373

yes

SST

C2Stanniocalcin2

5q351

368

YES

SBE

ST1

Bestr

ophin1

11q13

363

YES

SASN

SAs

paragine

synthetase

(glutamine-hydrolyzing)

7q213

339

YES

DCT

HCy

stathion

ase(cystathion

ineg

amma-lyase)

1p311

336

yes

SC1

2orf3

9Ch

romosom

e12op

enreadingfram

e39

12p121

319

YES

S

JHDM1D

Jumon

jiCdo

maincontaining

histo

nedemethylase

1hom

olog

D(Scerevisiae)

7q34

308

yes

S

PPP1R15A

Proteinph

osph

atase1regulatorysubu

nit15A

19q132

299

YES

SSP

RY4lowast

Sproutyho

molog

4(D

rosophila

)5q313

296

YES

SZC

3H6

Zinc

fingerC

CCH-ty

pecontaining

62q13

287

yes

STM

EM156

Transm

embranep

rotein

156

4p14

287

yes

SFA

M129A

Family

with

sequ

ence

similarity129mem

berA

1q25

280

yes

SCD

RT1

CMT1Adu

plicated

region

transcrip

t117p12

274

yes

SUPP

1Urid

inep

hospho

rylase

17p123

271

yes

DMAP2

Microtubu

le-associatedprotein2

2q34-q35

265

yes

SMOCO

SMolybdenu

mcofactor

sulfu

rase

18q12

259

yes

SC6

orf48

Chromosom

e6op

enreadingfram

e48

6p213

258

YES

SPY

ROXD

1Py

ridinen

ucleotide-disulphide

oxidoreductase

domain1

12p121

254

yes

SZN

F814

Zinc

fingerp

rotein

814

19q1343

252

yes

SCL

DN1

Claudin1

3q28-q29

252

yes

SABL

IM3

Actin

bind

ingLIM

proteinfamilym

ember3

5q32

252

yes

DDDIT4

DNA-

damage-indu

cibletranscript4

10q221

251

YES

STF

PI2

Tissue

factor

pathway

inhibitor2

7q22

251

YES

STU

BE1

Tubu

linepsilo

n1

6q21

245

yes

DGAD

D45

AlowastGrowth

arrestandDNA-

damage-indu

ciblealph

a1p312

236

YES

SFR

ZBFrizzle

d-related

protein

2qter

234

yes

SC5

orf28

Chromosom

e5op

enreadingfram

e28

5p12

233

yes

DSE

RPIN

B8Serpin

peptidaseinh

ibito

rcla

deB(ovalbum

in)mem

ber8

18q221

233

yes

SZN

F300

Zinc

fingerp

rotein

300

5q331

232

yes

S


Table2Con

tinued

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ZDHHC1

1Zinc

fingerDHHC-

type

containing

115p1533

229

yes

SGTP

BP2

GTP

bind

ingprotein2

6p21

227

YES

DMKX

Moh

awkho

meobo

x10p121

224

yes

SCD

274

CD274molecule

9p24

222

yes

SZN

F643

Zinc

fingerp

rotein

643

1p342

219

yes

SC9

orf150

Leuciner

ichadaptorp

rotein

1-like

9p23

219

yes

STE

STestisd

erived

transcrip

t(3LIM

domains)

7q312

217

yes

SPS

AT1

Phosph

oserinea

minotransferase

19q212

216

YES

SPA

X6Paire

dbo

x6

11p13

215

YES

SET

V5

ETSvaria

nt5

3q28

211

yes

SSA

RSSeryl-tRN

Asynthetase

1p133

210

YES

DCD

226

CD226molecule

18q223

204

yes

SGDPD

1Glyceroph

osph

odieste

rpho

spho

diesterase

domaincontaining

117q22

201

yes

SLE

TM2

Leucinez

ipper-EF

-handcontaining

transm

embranep

rotein

28p1123

201

YES

S1

Selected

52genesthatsho

wed

atleastge

2-fold

upregulationin

twoprostatecancer

celllin

es

2 Genen

ames

inbo

ldwerea

nalyzedby

RT-qPC

RGenen

ameinita

licwith

asteris

kindicatesg

enep

reviou

slyidentifi

edandhyperm

ethylatedin

PCab

utno

tanalyzedby

usG

enen

ames

inbo

ldita

licwith

asteris

kindicategenesp

reviou

slyidentifi

edandhyperm

ethylatedin

PCaa

ndanalyzed

inthispaper

3 Various

writingsty

lesind

icatethe

sizeo

fCpG

island(s)Y

ESthe

largestsizeyesthes

mallestsiz

e4 Sequ

allyexpressedin

norm

alandmalignant

tissueDincreased

expressio

nin

norm

alprostatetissue


03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100

Figure 2 Expression of downregulated candidate genes SPRY4 SARS GADD45A andGSTP1 in prostate nonmalignant andmalignant tissuesamples RT-qPCR was performed from 50 paired prostate tissue samples Values are given in boxes (white nonmalignant black malignant)that represent lower and upper quartiles with medians as horizontal line Whiskers depict the 10ndash90 percentiles Considered significances(119875 lt 005) are calculated with Wilcoxon signed rank test for all genes Data are given in Table 3

Table 3 mRNA expression changes of candidate genes represented by statistical analysislowast

Genes 119875 value Downregulation prevalence(normal versus tumor)

Fold changes(normal versus tumor)

geminus15-fold changes prevalence(normal versus tumor)

SPRY4 00007 74 (3750) minus164 84 (3137)SARS lt00001 84 (4250) minus144 55 (2342)GADD45A lt00001 88 (4450) minus232 84 (3744)GSTP1 lt00001 90 (4550) minus254 84 (4245)lowastConsidered significances (119875 lt 005) calculated with Wilcoxon signed rank test for all genes Normal versus tumor common downregulation and geminus15-foldchanges prevalence among downregulated samples


100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty

100 minus specifcity

SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty100 minus specifcity

GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1

Figure 3 Receiver operating characteristic (ROC) curve for the significantly downregulated candidate genes GADD45A SARS SPRY4 andGSTP1 to discriminate between tumor and adjacent normal samples Comparison of area under the receiver operating characteristic curve(AUC) of candidate genes with AUC of GSTP1 Data are given in Table 4

an essential regulatory component of selenium metabolismwhereby dietary selenium levels themselves are still a contro-versial issue in prostate cancer chemoprevention [26 27]

Next we performed receiver operating characteristic(ROC) curve analyses of SARS RGE data and compared themto SPRY4 GADD45A and the reference GSTP1 (Table 4)ROC area-under-curve (AUC) for SARS (0816) GADD45A(0841) and GSTP1 (0884) was almost the same with regardto value and shape (Table 4 and Figure 3)TheAUC of SPRY4

compared less well to the other genes (AUC = 0644) Inaddition it should be noted that at a sensitivity cutoff of 90SARS showed only moderate specificity (48)

Spearman correlation of ration of SARS expression datawith proven epigenetically regulated GADD45A and GSTP1resulted in a positive correlation among these genes (Table 5)However we did not find any significant correlation ofexpression data with pathological parameters like tumorstage grade and Gleason scores (data not shown) Since


Table 4 Performance of significantly downregulated candidate genes to discriminate between malignant and nonmalignant sampleslowast

Gene Sensitivity (95 CI) Specificity (95 CI) AUC 119875 value Standard errorGSTP1 90 (782ndash967) 78 (640ndash885) 0884 lt00001 00369GADD45A 90 (782ndash967) 58 (432ndash718) 0841 lt00001 00407SARS 90 (782ndash967) 48 (337ndash626) 0816 lt00001 0044SPRY4 90 (782ndash967) 16 (72ndash291) 0644 00085 00549lowastFive candidate genes that show significant downregulation on Wilcoxon signed rank test and further performance assessed by ROC curve analysis 90sensitivity and specificity with 95 confidence interval (95 CI)

Table 5 Spearman rank correlation coefficients by ration of expres-sion between downregulated candidate genes

Factorlowast SPRY4 SARS GADD45A GSTP1SPRY4 mdash 0236 0428b 0347a

SARS 0236 mdash 0591c 0685c

GADD45A 0428b 0591c mdash 0644c

GSTP1 0347a 0685c 0644c mdashlowastSignificantly downregulated candidate genes Correlation coefficient (119903

119904)

values and 119875 values are shown a119875 lt 005 b119875 lt 001 c119875 lt 0001

methylation events are known to be differentiation- andage-dependent and occur early in carcinogenesis [28] wespecifically tried to correlate SARS expression to the ageof our patients A Spearman correlation coefficient of 119903 =minus01626 (119875 = 02591) does not demonstrate any significantrelationships

4 Conclusion

An RNA microarray screen for epigenetically silenced genesin two prostate cancer cell lines revealed 52 candidatesthat were further analyzed in patient samples for theirinvolvement in cancer development We were able to verifyin the majority of our samples (88 and 74) a diminishedexpression of GADD45 and SPRY4 that were known to beinactivated by hypermethylation in prostate cancer Compa-rable results were obtained for a hitherto unknown transcriptthat now might be linked to prostate carcinogenesis tooTheenzyme SARS that interacts with components of VEGF andselenium pathways was found consistently downregulatedin more than 80 of tumor tissues Therefore we believethat SARS might be a promising putative target for further(epi)genetic studies in prostate cancer

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Odiljon Ikromov and Imad All-Kamal contributed equally tothis paper

Acknowledgments

This work was supported by the European Association ofUrology (EAU) Research fellowship and the Charite Promo-tionsstipendium for Odiljon Ikromov as part of his doctoralthesis The sponsors had no involvement in the study designthe collection analysis and interpretation of the data thewriting of the paper and the decision to submit the paperfor publication The authors thank U Ungethum for RNAchip analyses and processing data sets at the Charite LFGCcore facility They greatly acknowledge the help of WaltrautJekabsons Hellmuth-Alexander Meyer and Monika Jung inconducting these experiments

References

[1] J Ferlay I Soerjomataram M Ervik et al GLOBOCAN2012 V1 0 Cancer Incidence and Mortality Worldwide IARCCancerBase No 11 [Internet] International Agency for Researchon Cancer Lyon France 2013

[2] R Siegel DNaishadham andA Jemal ldquoCancer statistics 2013rdquoCAACancer Journal for Clinicians vol 63 no 1 pp 11ndash30 2013

[3] R Lister M Pelizzola R H Dowen et al ldquoHuman DNAmethylomes at base resolution show widespread epigenomicdifferencesrdquo Nature vol 462 no 7271 pp 315ndash322 2009

[4] M M Shen and C Abate-Shen ldquoMolecular genetics of prostatecancer new prospects for old challengesrdquo Genes and Develop-ment vol 24 no 18 pp 1967ndash2000 2010

[5] J G Herman and S B Baylin ldquoGene silencing in cancer inassociationwith promoter hypermethylationrdquoTheNewEnglandJournal of Medicine vol 349 no 21 pp 2042ndash2054 2003

[6] A S Perry RWGWatsonM Lawler andDHollywood ldquoTheepigenome as a therapeutic target in prostate cancerrdquo NatureReviews Urology vol 7 no 12 pp 668ndash680 2010

[7] C Mund B Brueckner and F Lyko ldquoReactivation of epigeneti-cally silenced genes by DNAmethyltransferase inhibitors basicconcepts and clinical applicationsrdquo Epigenetics vol 1 no 1 pp7ndash13 2006

[8] J C Cheng C B Yoo D J Weisenberger et al ldquoPreferentialresponse of cancer cells to zebularinerdquo Cancer Cell vol 6 no 2pp 151ndash158 2004

[9] J C Cheng C B Matsen F A Gonzales et al ldquoInhibitionof DNA methylation and reactivation of silenced genes byzebularinerdquo Journal of the National Cancer Institute vol 95 no5 pp 399ndash409 2003

[10] K Chiam M M Centenera L M Butler W D Tilley andT Bianco-Miotto ldquoGSTP1 DNA methylation and expressionstatus is indicative of 5-aza-21015840-deoxycytidine efficacy in human


prostate cancer cellsrdquoPLoSONE vol 6 no 9 Article ID e256342011

[11] R A Irizarry BM Bolstad F Collin LMCope BHobbs andT P Speed ldquoSummaries of Affymetrix GeneChip probe leveldatardquo Nucleic Acids Research vol 31 no 4 article e15 2003

[12] Y Benjamini and Y Hochberg ldquoControlling the false dicoveryrate a practical and powerful approach to multiple testingrdquoJournal of the Royal Statistical Society vol 57 no 1 pp 289ndash3001995

[13] H C Tsai and S B Baylin ldquoCancer epigenetics linking basicbiology to clinical medicinerdquo Cell Research vol 21 no 3 pp502ndash517 2011

[14] A R Karpf P W Peterson J T Rawlins et al ldquoInhibitionof DNA methyltransferase stimulates the expression of signaltransducer and activator of transcription 1 2 and 3 genes incolon tumor cellsrdquo Proceedings of the National Academy ofSciences of the United States of America vol 96 no 24 pp14007ndash14012 1999

[15] D Lodygin A Epanchintsev A Menssen J Diebold andH Hermeking ldquoFunctional epigenomics identifies genes fre-quently silenced in prostate cancerrdquo Cancer Research vol 65no 10 pp 4218ndash4227 2005

[16] JWang BThompson C RenM Ittmann and B Kwabi-AddoldquoSprouty4 a suppressor of tumor cell motility is downregulatedby DNA methylation in human prostate cancerrdquo Prostate vol66 no 6 pp 613ndash624 2006

[17] I Ibragimova I I de Caceres A M Hoffman et al ldquoGlobalreactivation of epigenetically silenced genes in prostate cancerrdquoCancer Prevention Research vol 3 no 9 pp 1084ndash1092 2010

[18] W H Lee R A Morton J I Epstein et al ldquoCytidine methy-lation of regulatory sequences near the 120587-class glutathione S-transferase gene accompanies human prostatic carcinogenesisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 91 no 24 pp 11733ndash11737 1994

[19] A B McKie D A Douglas S Olijslagers et al ldquoEpigeneticinactivation of the human sprouty (hSPRY2) homologue inprostate cancerrdquo Oncogene vol 24 no 13 pp 2166ndash2174 2005

[20] Q Zhan ldquoGadd45a a p53- and BRCA1-regulated stress proteinin cellular response to DNA damagerdquo Mutation ResearchmdashFundamental and Molecular Mechanisms of Mutagenesis vol569 no 1-2 pp 133ndash143 2005

[21] K Ramachandran G Gopisetty E Gordian et al ldquoMethyla-tion-mediated repression of GADD45120572 in prostate cancer andits role as a potential therapeutic targetrdquo Cancer Research vol69 no 4 pp 1527ndash1535 2009

[22] G P Sang P Schimmel and S Kim ldquoAminoacyl tRNA syn-thetases and their connections to diseaserdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 105 no 32 pp 11043ndash11049 2008

[23] X Xu Y Shi H Zhang et al ldquoUnique domain appended to ver-tebrate tRNA synthetase is essential for vascular developmentrdquoNature Communications vol 3 article 681 2012

[24] M Shibuya ldquoDifferential roles of vascular endothelial growthfactor receptor-1 and receptor-2 in angiogenesisrdquo Journal ofBiochemistry and Molecular Biology vol 39 no 5 pp 469ndash4782006

[25] I Steiner K Jung K Miller C Stephan and A ErbersdoblerldquoExpression of endothelial factors in prostate cancer a possiblerole of caveolin-1 for tumour progressionrdquo Oncology Reportsvol 27 no 2 pp 389ndash395 2012

[26] H Zeng and G F Combs Jr ldquoSelenium as an anticancernutrient roles in cell proliferation and tumor cell invasionrdquoJournal of Nutritional Biochemistry vol 19 no 1 pp 1ndash7 2008

[27] A M Algotar M S Stratton F R Ahmann et al ldquoPhase 3 clin-ical trial investigating the effect of selenium supplementation inmen at high-risk for prostate cancerrdquo vol 73 no 3 pp 328ndash3352013

[28] K Day L L Waite A Thalacker-Mercer et al ldquoDifferentialDNAmethylationwith age displays both common and dynamicfeatures across human tissues that are influenced by CpGlandscaperdquo Genome Biology vol 14 no 9 article R102 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Patientsrsquo clinical parameters

Characteristics Parameters Patients 119899 = 50 (100)

Age years Median 64Range 47ndash74

Preoperative PSA ngmL Median 87Range 106ndash78

T stage

pT2a 1 (2)pT2b 15 (30)pT2c 12 (24)pT3a 6 (12)pT3b 13 (26)pT3x 1 (2)pT4 2 (4)

N stage N0Nx 44 (88)N1 6 (12)

M stage M0 50 (100)

Gleason score

na 1 (2)3 1 (2)5 15 (30)6 12 (24)

7a (3 + 4) 9 (18)7b (4 + 3) 4 (8)

8 4 (8)9 4 (8)

administration [9 10] We treated androgen insensitive DU-145 and androgen sensitive LNCaP PCa cell lines withmoderate doses of zebularine and identified globally silencedgenes by RNA microarray analysis of the transcriptomeThrough intuitive selection of upregulated and validatedgenes we estimated relative gene expression (RGE) profilesfor selected candidate genes in 50 patients using pairedsamples of adjacent normal and tumour prostate tissueIn agreement with our working hypothesis of methylation-induced transcriptional inactivation we focused our attemptson genes that are exclusively downregulated in primaryPCa Eventually we identified for the first time seryl-tRNAsynthetase (SARS) to be downregulated in almost all PCacases analysed SARS was further characterized to qualify asa possible candidate for future epigenetic study approaches inPCa

2 Materials and Methods

21 Zebularine Treatment LNCaP prostate cancer cell lines(ATCC number CRL-1740) and DU-145 (ATCC numberHTB-81) were chosen for treatment experiments Both celllines were purchased from the American Type Culture Col-lection (ATCC Manassas VA USA) Cells were treated witha final concentration of 100120583Mof theDNAmethyltransferase(DNMT) inhibiting reagent zebularine Growth mediumcontaining zebularine was replaced regularly after 48ndash72hours and cells were split at a ratio of 1 3 In total cellswere exposed to zebularine for 216 hoursThree independent

treatment experiments including untreated controls wereperformed

22 Tissue Samples Tumor and adjacent normal tissues sam-ples of prostate cancer patients collected after radical prosta-tectomy between 2002 and 2004 at the Department ofUrology Charite-Universitatsmedizin Berlin Germany wereused in experimentsThe samples were collected immediatelyafter surgery in liquid nitrogen and stored at minus80∘C Thestudy was done according to the regulation of ethical boardof the hospital with informed consent of patients Sampleswere analyzed by a uropathologist for their tumor content Intotal 50 prostate adjacent normal and tumor tissue samplescontaining at least 60 tumor tissue with different stagewere included in this study Patientsrsquo clinical parameters aredescribed in Table 1

23 RNA Isolation and cDNA Synthesis Total RNA wasextracted from treated and untreated PCa cells as well asfrom prostate normal and tumor tissue samples by using theldquomiRNeasy Mini Kitrdquo (Qiagen Hilden Germany) accord-ing to manufacturerrsquos recommendations Approximately 4-5 times 106 cells and 20ndash30mg of tissue were used for RNAextraction RNA concentration and purity were determinedspectrophotometrically on a Nanodrop ND-1000 instrument(Thermo Scientific Germany) All RNA samples were freefrom remaining proteins (260280 nm ratio from sim18 to20) and other contaminations (260230 nm ratio = 20to 22) In addition integrity of RNA was assessed by














106

83

1357

85

24 76

235

3rd experiment

(a)

166

19

205

31

57 31

452


3rd experiment

(b)










Table2Listof

upregu

latedgenesa

fterb

ioinform

aticsa

nalyses1

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ADRA


receptor

10q252

231

YES

DDDX6

0DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide6

04q

323

297

yes

SPO

TEF

POTE

ankyrin

domainfamilym

emberF

2q211

215

yes

DDDX5

8DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide5

89p

12375

yes

SIG

F1R

Insulin

-like

grow

thfactor

1receptor

15q263

272

yes

SIN

PP4B

Inosito

lpolypho

sphate-4-pho

sphatasetypeII

4q3121

263

yes

DIFI6

Interfe

ron

alph

a-indu

ciblep

rotein

61p35

486

yes

DTX

NIPlowast

Thioredo


1q211

465

YES

DADAM32

ADAM

metallopeptidased

omain32

8p1122

373

yes

SST

C2Stanniocalcin2

5q351

368

YES

SBE

ST1

Bestr

ophin1

11q13

363

YES

SASN

SAs

paragine

synthetase


7q213

339

YES

DCT

HCy

stathion

ase(cystathion

ineg

amma-lyase)

1p311

336

yes

SC1

2orf3

9Ch

romosom

e12op

enreadingfram

e39

12p121

319

YES

S

JHDM1D

Jumon

jiCdo

maincontaining

histo

nedemethylase

1hom

olog

D(Scerevisiae)

7q34

308

yes

S

PPP1R15A

Proteinph

osph


nit15A

19q132

299

YES

SSP

RY4lowast

Sproutyho

molog

4(D

rosophila

)5q313

296

YES

SZC

3H6

Zinc

fingerC

CCH-ty

pecontaining

62q13

287

yes

STM

EM156

Transm

embranep

rotein

156

4p14

287

yes

SFA

M129A

Family

with

sequ

ence

similarity129mem

berA

1q25

280

yes

SCD

RT1

CMT1Adu

plicated

region

transcrip

t117p12

274

yes

SUPP

1Urid

inep

hospho

rylase

17p123

271

yes

DMAP2

Microtubu


2q34-q35

265

yes

SMOCO

SMolybdenu

mcofactor

sulfu

rase

18q12

259

yes

SC6

orf48

Chromosom

e6op

enreadingfram

e48

6p213

258

YES

SPY

ROXD

1Py

ridinen


oxidoreductase

domain1

12p121

254

yes

SZN

F814

Zinc

fingerp

rotein

814

19q1343

252

yes

SCL

DN1

Claudin1

3q28-q29

252

yes

SABL

IM3

Actin

bind

ingLIM

proteinfamilym

ember3

5q32

252

yes

DDDIT4

DNA-

damage-indu

cibletranscript4

10q221

251

YES

STF

PI2

Tissue

factor

pathway

inhibitor2

7q22

251

YES

STU

BE1

Tubu

linepsilo

n1

6q21

245

yes

DGAD

D45

AlowastGrowth

arrestandDNA-

damage-indu

ciblealph

a1p312

236

YES

SFR

ZBFrizzle

d-related

protein

2qter

234

yes

SC5

orf28

Chromosom

e5op

enreadingfram

e28

5p12

233

yes

DSE

RPIN

B8Serpin

peptidaseinh

ibito

rcla

deB(ovalbum

in)mem

ber8

18q221

233

yes

SZN

F300

Zinc

fingerp

rotein

300

5q331

232

yes

S


Table2Con

tinued

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ZDHHC1

1Zinc

fingerDHHC-

type

containing

115p1533

229

yes

SGTP

BP2

GTP

bind

ingprotein2

6p21

227

YES

DMKX

Moh

awkho

meobo

x10p121

224

yes

SCD

274

CD274molecule

9p24

222

yes

SZN

F643

Zinc

fingerp

rotein

643

1p342

219

yes

SC9

orf150

Leuciner

ichadaptorp

rotein

1-like

9p23

219

yes

STE

STestisd

erived

transcrip

t(3LIM

domains)

7q312

217

yes

SPS

AT1

Phosph

oserinea

minotransferase

19q212

216

YES

SPA

X6Paire

dbo

x6

11p13

215

YES

SET

V5

ETSvaria

nt5

3q28

211

yes

SSA

RSSeryl-tRN

Asynthetase

1p133

210

YES

DCD

226

CD226molecule

18q223

204

yes

SGDPD

1Glyceroph

osph

odieste

rpho

spho

diesterase

domaincontaining

117q22

201

yes

SLE

TM2

Leucinez

ipper-EF

-handcontaining

transm

embranep

rotein

28p1123

201

YES

S1

Selected

52genesthatsho

wed

atleastge

2-fold

upregulationin

twoprostatecancer

celllin

es

2 Genen

ames

inbo

ldwerea

nalyzedby

RT-qPC

RGenen

ameinita

licwith

asteris

kindicatesg

enep

reviou

slyidentifi

edandhyperm

ethylatedin

PCab

utno

tanalyzedby

usG

enen

ames

inbo

ldita

licwith

asteris

kindicategenesp

reviou

slyidentifi

edandhyperm

ethylatedin

PCaa

ndanalyzed

inthispaper

3 Various

writingsty

lesind

icatethe

sizeo

fCpG

island(s)Y

ESthe

largestsizeyesthes

mallestsiz

e4 Sequ

allyexpressedin

norm

alandmalignant

tissueDincreased

expressio

nin

norm

alprostatetissue


03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100








100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























106

83

1357

85

24 76

235

3rd experiment

(a)

166

19

205

31

57 31

452


3rd experiment

(b)










Table2Listof

upregu

latedgenesa

fterb

ioinform

aticsa

nalyses1

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ADRA


receptor

10q252

231

YES

DDDX6

0DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide6

04q

323

297

yes

SPO

TEF

POTE

ankyrin

domainfamilym

emberF

2q211

215

yes

DDDX5

8DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide5

89p

12375

yes

SIG

F1R

Insulin

-like

grow

thfactor

1receptor

15q263

272

yes

SIN

PP4B

Inosito

lpolypho

sphate-4-pho

sphatasetypeII

4q3121

263

yes

DIFI6

Interfe

ron

alph

a-indu

ciblep

rotein

61p35

486

yes

DTX

NIPlowast

Thioredo


1q211

465

YES

DADAM32

ADAM

metallopeptidased

omain32

8p1122

373

yes

SST

C2Stanniocalcin2

5q351

368

YES

SBE

ST1

Bestr

ophin1

11q13

363

YES

SASN

SAs

paragine

synthetase


7q213

339

YES

DCT

HCy

stathion

ase(cystathion

ineg

amma-lyase)

1p311

336

yes

SC1

2orf3

9Ch

romosom

e12op

enreadingfram

e39

12p121

319

YES

S

JHDM1D

Jumon

jiCdo

maincontaining

histo

nedemethylase

1hom

olog

D(Scerevisiae)

7q34

308

yes

S

PPP1R15A

Proteinph

osph


nit15A

19q132

299

YES

SSP

RY4lowast

Sproutyho

molog

4(D

rosophila

)5q313

296

YES

SZC

3H6

Zinc

fingerC

CCH-ty

pecontaining

62q13

287

yes

STM

EM156

Transm

embranep

rotein

156

4p14

287

yes

SFA

M129A

Family

with

sequ

ence

similarity129mem

berA

1q25

280

yes

SCD

RT1

CMT1Adu

plicated

region

transcrip

t117p12

274

yes

SUPP

1Urid

inep

hospho

rylase

17p123

271

yes

DMAP2

Microtubu


2q34-q35

265

yes

SMOCO

SMolybdenu

mcofactor

sulfu

rase

18q12

259

yes

SC6

orf48

Chromosom

e6op

enreadingfram

e48

6p213

258

YES

SPY

ROXD

1Py

ridinen


oxidoreductase

domain1

12p121

254

yes

SZN

F814

Zinc

fingerp

rotein

814

19q1343

252

yes

SCL

DN1

Claudin1

3q28-q29

252

yes

SABL

IM3

Actin

bind

ingLIM

proteinfamilym

ember3

5q32

252

yes

DDDIT4

DNA-

damage-indu

cibletranscript4

10q221

251

YES

STF

PI2

Tissue

factor

pathway

inhibitor2

7q22

251

YES

STU

BE1

Tubu

linepsilo

n1

6q21

245

yes

DGAD

D45

AlowastGrowth

arrestandDNA-

damage-indu

ciblealph

a1p312

236

YES

SFR

ZBFrizzle

d-related

protein

2qter

234

yes

SC5

orf28

Chromosom

e5op

enreadingfram

e28

5p12

233

yes

DSE

RPIN

B8Serpin

peptidaseinh

ibito

rcla

deB(ovalbum

in)mem

ber8

18q221

233

yes

SZN

F300

Zinc

fingerp

rotein

300

5q331

232

yes

S


Table2Con

tinued

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ZDHHC1

1Zinc

fingerDHHC-

type

containing

115p1533

229

yes

SGTP

BP2

GTP

bind

ingprotein2

6p21

227

YES

DMKX

Moh

awkho

meobo

x10p121

224

yes

SCD

274

CD274molecule

9p24

222

yes

SZN

F643

Zinc

fingerp

rotein

643

1p342

219

yes

SC9

orf150

Leuciner

ichadaptorp

rotein

1-like

9p23

219

yes

STE

STestisd

erived

transcrip

t(3LIM

domains)

7q312

217

yes

SPS

AT1

Phosph

oserinea

minotransferase

19q212

216

YES

SPA

X6Paire

dbo

x6

11p13

215

YES

SET

V5

ETSvaria

nt5

3q28

211

yes

SSA

RSSeryl-tRN

Asynthetase

1p133

210

YES

DCD

226

CD226molecule

18q223

204

yes

SGDPD

1Glyceroph

osph

odieste

rpho

spho

diesterase

domaincontaining

117q22

201

yes

SLE

TM2

Leucinez

ipper-EF

-handcontaining

transm

embranep

rotein

28p1123

201

YES

S1

Selected

52genesthatsho

wed

atleastge

2-fold

upregulationin

twoprostatecancer

celllin

es

2 Genen

ames

inbo

ldwerea

nalyzedby

RT-qPC

RGenen

ameinita

licwith

asteris

kindicatesg

enep

reviou

slyidentifi

edandhyperm

ethylatedin

PCab

utno

tanalyzedby

usG

enen

ames

inbo

ldita

licwith

asteris

kindicategenesp

reviou

slyidentifi

edandhyperm

ethylatedin

PCaa

ndanalyzed

inthispaper

3 Various

writingsty

lesind

icatethe

sizeo

fCpG

island(s)Y

ESthe

largestsizeyesthes

mallestsiz

e4 Sequ

allyexpressedin

norm

alandmalignant

tissueDincreased

expressio

nin

norm

alprostatetissue


03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100








100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















106

83

1357

85

24 76

235

3rd experiment

(a)

166

19

205

31

57 31

452


3rd experiment

(b)










Table2Listof

upregu

latedgenesa

fterb

ioinform

aticsa

nalyses1

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ADRA


receptor

10q252

231

YES

DDDX6

0DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide6

04q

323

297

yes

SPO

TEF

POTE

ankyrin

domainfamilym

emberF

2q211

215

yes

DDDX5

8DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide5

89p

12375

yes

SIG

F1R

Insulin

-like

grow

thfactor

1receptor

15q263

272

yes

SIN

PP4B

Inosito

lpolypho

sphate-4-pho

sphatasetypeII

4q3121

263

yes

DIFI6

Interfe

ron

alph

a-indu

ciblep

rotein

61p35

486

yes

DTX

NIPlowast

Thioredo


1q211

465

YES

DADAM32

ADAM

metallopeptidased

omain32

8p1122

373

yes

SST

C2Stanniocalcin2

5q351

368

YES

SBE

ST1

Bestr

ophin1

11q13

363

YES

SASN

SAs

paragine

synthetase


7q213

339

YES

DCT

HCy

stathion

ase(cystathion

ineg

amma-lyase)

1p311

336

yes

SC1

2orf3

9Ch

romosom

e12op

enreadingfram

e39

12p121

319

YES

S

JHDM1D

Jumon

jiCdo

maincontaining

histo

nedemethylase

1hom

olog

D(Scerevisiae)

7q34

308

yes

S

PPP1R15A

Proteinph

osph


nit15A

19q132

299

YES

SSP

RY4lowast

Sproutyho

molog

4(D

rosophila

)5q313

296

YES

SZC

3H6

Zinc

fingerC

CCH-ty

pecontaining

62q13

287

yes

STM

EM156

Transm

embranep

rotein

156

4p14

287

yes

SFA

M129A

Family

with

sequ

ence

similarity129mem

berA

1q25

280

yes

SCD

RT1

CMT1Adu

plicated

region

transcrip

t117p12

274

yes

SUPP

1Urid

inep

hospho

rylase

17p123

271

yes

DMAP2

Microtubu


2q34-q35

265

yes

SMOCO

SMolybdenu

mcofactor

sulfu

rase

18q12

259

yes

SC6

orf48

Chromosom

e6op

enreadingfram

e48

6p213

258

YES

SPY

ROXD

1Py

ridinen


oxidoreductase

domain1

12p121

254

yes

SZN

F814

Zinc

fingerp

rotein

814

19q1343

252

yes

SCL

DN1

Claudin1

3q28-q29

252

yes

SABL

IM3

Actin

bind

ingLIM

proteinfamilym

ember3

5q32

252

yes

DDDIT4

DNA-

damage-indu

cibletranscript4

10q221

251

YES

STF

PI2

Tissue

factor

pathway

inhibitor2

7q22

251

YES

STU

BE1

Tubu

linepsilo

n1

6q21

245

yes

DGAD

D45

AlowastGrowth

arrestandDNA-

damage-indu

ciblealph

a1p312

236

YES

SFR

ZBFrizzle

d-related

protein

2qter

234

yes

SC5

orf28

Chromosom

e5op

enreadingfram

e28

5p12

233

yes

DSE

RPIN

B8Serpin

peptidaseinh

ibito

rcla

deB(ovalbum

in)mem

ber8

18q221

233

yes

SZN

F300

Zinc

fingerp

rotein

300

5q331

232

yes

S


Table2Con

tinued

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ZDHHC1

1Zinc

fingerDHHC-

type

containing

115p1533

229

yes

SGTP

BP2

GTP

bind

ingprotein2

6p21

227

YES

DMKX

Moh

awkho

meobo

x10p121

224

yes

SCD

274

CD274molecule

9p24

222

yes

SZN

F643

Zinc

fingerp

rotein

643

1p342

219

yes

SC9

orf150

Leuciner

ichadaptorp

rotein

1-like

9p23

219

yes

STE

STestisd

erived

transcrip

t(3LIM

domains)

7q312

217

yes

SPS

AT1

Phosph

oserinea

minotransferase

19q212

216

YES

SPA

X6Paire

dbo

x6

11p13

215

YES

SET

V5

ETSvaria

nt5

3q28

211

yes

SSA

RSSeryl-tRN

Asynthetase

1p133

210

YES

DCD

226

CD226molecule

18q223

204

yes

SGDPD

1Glyceroph

osph

odieste

rpho

spho

diesterase

domaincontaining

117q22

201

yes

SLE

TM2

Leucinez

ipper-EF

-handcontaining

transm

embranep

rotein

28p1123

201

YES

S1

Selected

52genesthatsho

wed

atleastge

2-fold

upregulationin

twoprostatecancer

celllin

es

2 Genen

ames

inbo

ldwerea

nalyzedby

RT-qPC

RGenen

ameinita

licwith

asteris

kindicatesg

enep

reviou

slyidentifi

edandhyperm

ethylatedin

PCab

utno

tanalyzedby

usG

enen

ames

inbo

ldita

licwith

asteris

kindicategenesp

reviou

slyidentifi

edandhyperm

ethylatedin

PCaa

ndanalyzed

inthispaper

3 Various

writingsty

lesind

icatethe

sizeo

fCpG

island(s)Y

ESthe

largestsizeyesthes

mallestsiz

e4 Sequ

allyexpressedin

norm

alandmalignant

tissueDincreased

expressio

nin

norm

alprostatetissue


03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100








100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Listof

upregu

latedgenesa

fterb

ioinform

aticsa

nalyses1

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ADRA


receptor

10q252

231

YES

DDDX6

0DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide6

04q

323

297

yes

SPO

TEF

POTE

ankyrin

domainfamilym

emberF

2q211

215

yes

DDDX5

8DEA

D(A

sp-G

lu-Ala-Asp)b

oxpo

lypeptide5

89p

12375

yes

SIG

F1R

Insulin

-like

grow

thfactor

1receptor

15q263

272

yes

SIN

PP4B

Inosito

lpolypho

sphate-4-pho

sphatasetypeII

4q3121

263

yes

DIFI6

Interfe

ron

alph

a-indu

ciblep

rotein

61p35

486

yes

DTX

NIPlowast

Thioredo


1q211

465

YES

DADAM32

ADAM

metallopeptidased

omain32

8p1122

373

yes

SST

C2Stanniocalcin2

5q351

368

YES

SBE

ST1

Bestr

ophin1

11q13

363

YES

SASN

SAs

paragine

synthetase


7q213

339

YES

DCT

HCy

stathion

ase(cystathion

ineg

amma-lyase)

1p311

336

yes

SC1

2orf3

9Ch

romosom

e12op

enreadingfram

e39

12p121

319

YES

S

JHDM1D

Jumon

jiCdo

maincontaining

histo

nedemethylase

1hom

olog

D(Scerevisiae)

7q34

308

yes

S

PPP1R15A

Proteinph

osph


nit15A

19q132

299

YES

SSP

RY4lowast

Sproutyho

molog

4(D

rosophila

)5q313

296

YES

SZC

3H6

Zinc

fingerC

CCH-ty

pecontaining

62q13

287

yes

STM

EM156

Transm

embranep

rotein

156

4p14

287

yes

SFA

M129A

Family

with

sequ

ence

similarity129mem

berA

1q25

280

yes

SCD

RT1

CMT1Adu

plicated

region

transcrip

t117p12

274

yes

SUPP

1Urid

inep

hospho

rylase

17p123

271

yes

DMAP2

Microtubu


2q34-q35

265

yes

SMOCO

SMolybdenu

mcofactor

sulfu

rase

18q12

259

yes

SC6

orf48

Chromosom

e6op

enreadingfram

e48

6p213

258

YES

SPY

ROXD

1Py

ridinen


oxidoreductase

domain1

12p121

254

yes

SZN

F814

Zinc

fingerp

rotein

814

19q1343

252

yes

SCL

DN1

Claudin1

3q28-q29

252

yes

SABL

IM3

Actin

bind

ingLIM

proteinfamilym

ember3

5q32

252

yes

DDDIT4

DNA-

damage-indu

cibletranscript4

10q221

251

YES

STF

PI2

Tissue

factor

pathway

inhibitor2

7q22

251

YES

STU

BE1

Tubu

linepsilo

n1

6q21

245

yes

DGAD

D45

AlowastGrowth

arrestandDNA-

damage-indu

ciblealph

a1p312

236

YES

SFR

ZBFrizzle

d-related

protein

2qter

234

yes

SC5

orf28

Chromosom

e5op

enreadingfram

e28

5p12

233

yes

DSE

RPIN

B8Serpin

peptidaseinh

ibito

rcla

deB(ovalbum

in)mem

ber8

18q221

233

yes

SZN

F300

Zinc

fingerp

rotein

300

5q331

232

yes

S


Table2Con

tinued

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ZDHHC1

1Zinc

fingerDHHC-

type

containing

115p1533

229

yes

SGTP

BP2

GTP

bind

ingprotein2

6p21

227

YES

DMKX

Moh

awkho

meobo

x10p121

224

yes

SCD

274

CD274molecule

9p24

222

yes

SZN

F643

Zinc

fingerp

rotein

643

1p342

219

yes

SC9

orf150

Leuciner

ichadaptorp

rotein

1-like

9p23

219

yes

STE

STestisd

erived

transcrip

t(3LIM

domains)

7q312

217

yes

SPS

AT1

Phosph

oserinea

minotransferase

19q212

216

YES

SPA

X6Paire

dbo

x6

11p13

215

YES

SET

V5

ETSvaria

nt5

3q28

211

yes

SSA

RSSeryl-tRN

Asynthetase

1p133

210

YES

DCD

226

CD226molecule

18q223

204

yes

SGDPD

1Glyceroph

osph

odieste

rpho

spho

diesterase

domaincontaining

117q22

201

yes

SLE

TM2

Leucinez

ipper-EF

-handcontaining

transm

embranep

rotein

28p1123

201

YES

S1

Selected

52genesthatsho

wed

atleastge

2-fold

upregulationin

twoprostatecancer

celllin

es

2 Genen

ames

inbo

ldwerea

nalyzedby

RT-qPC

RGenen

ameinita

licwith

asteris

kindicatesg

enep

reviou

slyidentifi

edandhyperm

ethylatedin

PCab

utno

tanalyzedby

usG

enen

ames

inbo

ldita

licwith

asteris

kindicategenesp

reviou

slyidentifi

edandhyperm

ethylatedin

PCaa

ndanalyzed

inthispaper

3 Various

writingsty

lesind

icatethe

sizeo

fCpG

island(s)Y

ESthe

largestsizeyesthes

mallestsiz

e4 Sequ

allyexpressedin

norm

alandmalignant

tissueDincreased

expressio

nin

norm

alprostatetissue


03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100








100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Con

tinued

Genes

ymbo

l2Genen

ame

Locatio

nFo

ldchange

CpGisland3

Digita

lNorthern4

ZDHHC1

1Zinc

fingerDHHC-

type

containing

115p1533

229

yes

SGTP

BP2

GTP

bind

ingprotein2

6p21

227

YES

DMKX

Moh

awkho

meobo

x10p121

224

yes

SCD

274

CD274molecule

9p24

222

yes

SZN

F643

Zinc

fingerp

rotein

643

1p342

219

yes

SC9

orf150

Leuciner

ichadaptorp

rotein

1-like

9p23

219

yes

STE

STestisd

erived

transcrip

t(3LIM

domains)

7q312

217

yes

SPS

AT1

Phosph

oserinea

minotransferase

19q212

216

YES

SPA

X6Paire

dbo

x6

11p13

215

YES

SET

V5

ETSvaria

nt5

3q28

211

yes

SSA

RSSeryl-tRN

Asynthetase

1p133

210

YES

DCD

226

CD226molecule

18q223

204

yes

SGDPD

1Glyceroph

osph

odieste

rpho

spho

diesterase

domaincontaining

117q22

201

yes

SLE

TM2

Leucinez

ipper-EF

-handcontaining

transm

embranep

rotein

28p1123

201

YES

S1

Selected

52genesthatsho

wed

atleastge

2-fold

upregulationin

twoprostatecancer

celllin

es

2 Genen

ames

inbo

ldwerea

nalyzedby

RT-qPC

RGenen

ameinita

licwith

asteris

kindicatesg

enep

reviou

slyidentifi

edandhyperm

ethylatedin

PCab

utno

tanalyzedby

usG

enen

ames

inbo

ldita

licwith

asteris

kindicategenesp

reviou

slyidentifi

edandhyperm

ethylatedin

PCaa

ndanalyzed

inthispaper

3 Various

writingsty

lesind

icatethe

sizeo

fCpG

island(s)Y

ESthe

largestsizeyesthes

mallestsiz

e4 Sequ

allyexpressedin

norm

alandmalignant

tissueDincreased

expressio

nin

norm

alprostatetissue


03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100








100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















03

02

01

00

RGE

Normal Tumor

SPRY4

RGE

Normal Tumor

GADD45A80

60

40

20

0

RGE

Normal Tumor

SARS30

20

10

0

Normal Tumor

GSTP1RG

E

80

60

40

20

0

100








100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SPRY4

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi


GADD45A

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


SARS

100

100

80

80

60

60

40

40

20

20

0

0

Sens

itivi

ty


GSTP1














119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























119904)



4 Conclusion






Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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