research article genetic variations in inflammatory response...

Research ArticleGenetic Variations in Inflammatory Response Genes andTheir Association with the Risk of Prostate Cancer

Xin Cui Hao Yan Tong-Wen Ou Chun-Song Jia Qi Wang and Jian-Jun Xu

Department of Urology Xuanwu Hospital Capital Medical University Beijing 100053 China

Correspondence should be addressed to Tong-Wen Ou outongwensohucom

Received 1 April 2015 Revised 25 May 2015 Accepted 10 June 2015

Academic Editor Sabine Rohrmann

Copyright copy 2015 Xin Cui et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Prostate cancer is a common cancer in men Genetic variations in inflammatory response genes can potentially influence the riskof prostate cancer We aimed to examine the association between PPARG Pro12Ala NFKB1 -94 insdel NFKBIA -826CT COX-1(50CgtT) and COX-2 (-1195GgtA) polymorphisms on prostate cancer risk The genotypes of the polymorphisms were ascertainedin 543 prostate cancer patients and 753 controls through PCR-RFLP and the risk association was evaluated statistically usinglogistic regression analysis The NFKB1 -94 polymorphism was shown to decrease prostate cancer risk in both heterozygous andhomozygous comparison models (odds ratios of 074 (95 CI = 058ndash096) (119875 = 002) and 057 (95 CI = 042ndash078) (119875 lt 001)resp) An opposite finding was observed for COX-2 (-1195) polymorphism (odds ratios of 158 (95 CI = 115ndash218) (119875 lt 001) forheterozygous comparisonmodel and 208 (95CI = 148ndash292) (119875 lt 001) for homozygous comparisonmodel) No association wasobserved for other polymorphisms In conclusionNFKB1 -94 insdel and COX-2 (-1195GgtA) polymorphisms may be respectivelyassociated with decreased and increased prostate cancer risk in the Chinese population

1 Introduction

Prostate cancer is a form of cancer in which malignant cellsdevelop in the prostate gland of themale reproductive systemProstate cancer is the second leading type of cancer in menglobally with an age-standardized incidence rate of 311 per100000 men [1] In 2012 alone a total of 1111689 cases ofprostate cancers were diagnosed and approximately 307471deaths were attended [1] In comparison the estimatednumbers of incidence and mortality of the cancer were679000 and 221000 respectively in 2002 [2]Thus amarkedincrease in the incidence andmortality of prostate cancer hasbeen observed during the past decade and development ofan effective screening strategy for identifying individuals atrisk of prostate cancer is greatly needed This is especiallytrue for the Chinese population in which the mortality-to-incidence rate ratio (MRIR) has been consistently higherin comparison to the average MRIR of Asian countries andWestern populations [3 4]

Established etiological factors of prostate cancer includeage ethnicity diet and environmental carcinogen expo-sure [5] The interactions among the above elements are

believed to play significant roles in contributing to prostatecarcinogenesis although the precise mechanisms of thishave not been fully understood [5] In addition in recentyears chronic inflammation of the prostate and importantlygenetic profile of an individual have been shown to playan equally important role in oncogenesis of the prostate[6 7] Epidemiological and molecular evidences implied thatdysregulation of inflammatory response genes may promotethe development and progression of approximately 20 ofprostate malignancies by inducing DNA damage cell prolif-eration enhancement apoptosis inhibition and angiogenesissimulation [8]

The most common cause of inflammatory response genedysregulations is mutations in high penetrance genes [9]Despite the fact that suchmutations can strongly increase therisk of prostate cancer they are only present in a small fre-quency in the general population [9] In comparison geneticvariations in the form of single nucleotide polymorphisms(SNPs) in low penetrance genes can increase the risk ofcancer modestly but they are more prevalent in the generalpopulation and thus their overall impact could be substan-tial [10] SNPs may alter the expression and functionality

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 674039 7 pageshttpdxdoiorg1011552015674039

2 BioMed Research International

of the protein products [11] and many studies have showeda relationship between inherited variations in inflammatoryresponse genes and the risk of prostate carcinogenesis [12ndash16] However the influence of several common SNPs inthe inflammatory response genes either individually or incombinations on prostate cancer risk has been understudiedin theChinese populationThis includes thePPARGPro12AlapolymorphismNFKB1 -94 insdel polymorphismNFKBIA -826CT polymorphism COX-1 (50CgtT) polymorphism andCOX-2 (-1195GgtA) polymorphism The present study aimedto investigate the association of the above polymorphismswith the risk of prostate cancer in the Chinese population

2 Materials and Methods

21 Study Subjects The study was approved by the MedicalEthics Committee of the Xuanwu Hospital Beijing ChinaAll subjects were recruited from Xuanwu Hospital BeijingChina between October 2010 and August 2014 A total of 543males who were diagnosed by qualified physicians as prostatecancer patients based on clinical pathological and laboratorytests were recruited into the study To ensure consistencyonly patients with high grade prostate cancer (Gleason scoregt 7) who had a PSA level of gt10 ngmL were included inthe study At the same period of time a total of 753 healthycontrols were enrolled into this study Eligibility criteria forcontrol included no individual history of cancer and noknown genetic syndromes associated with prostate cancerand a PSA level of lt2 ngmL Blood samples were collectedfrom the subjectswhomeet the criteriamentioned above afterobtaining written informed consent

22 SNP Genotyping Genomic DNA extraction was doneby using EasyPure Blood Genomic DNA Kit (TransGenBiotech Beijing China) after the blood samples were takenfrom peripheral blood of all subjects The DNA was usedfor genotyping of the five SNPs by using the PCR-RFLPmethod All PCRwere performed in a 25 120583L reactionmixturecontaining standard PCR buffer 15mM MgCl

2 025mM

dNTP 1 unit Taq polymerase and 04 120583Mof each primer Forall polymorphisms approximately 10 of the samples werechosen at random and sequenced to confirm the genotypesfrom the amplified PCR product The laboratory personnelperforming the genotyping were blinded to the identity thecase-control status and source of the samples

221 PPARG Pro12Ala Polymorphism The PCR primersused for amplification of the region containing the PPARGPro12Ala polymorphism were 51015840-GCC AAT TCA AGC CCAGTC-31015840 and 51015840-GAT ATG TTT GCA GAC AGT GTATCA GTG AAG GAA TCG CTT TCC-31015840 The annealingtemperature of the reaction was 58∘CThe PCR amplificationproduced a fragment of 270 bp in size which was thendigested using BstUI restriction enzyme The fragment wascleaved into a 227 bp and a 43 bp fragment for the AlaAlagenotype while the ProPro genotype was uncut Apart fromthat heterozygous genotype (ProAla) was detected by thepresence of all the above bands in agarose gel

222 NFKB1 -94 insdel Polymorphism The PCR for ampli-fication of the region containing the NFKB1 -94 insdelpolymorphism was performed by using 51015840-TGG GCA CAAGTC GTT TAT GA-31015840 and 51015840-CTG GAG CCG GTA GGGAAG-31015840 primers which were annealed to the genomic DNAat 61∘C PflMI restriction enzyme was subsequently usedto digest the amplified PCR product of 281 bp in size Forinsins genotype the absence of restriction site resulted inthe undigested fragment of 281 bp after the reaction On theother hand a 240 bp and a 45 bp fragment were obtained forthe deldel genotype while a 281 bp a 240 bp and a 45 bpfragment were found in the insdel genotype

223 NFKBIA -826CT Polymorphism The NFKBIA -826CT polymorphismwas genotyped by first amplifying theregion of interest by using the following PCR primers 51015840-GGT CCT TAA GGT CCA ATC G-31015840 and 51015840-GTT GTGGAT ACC TTG CAC TA-31015840 at an annealing temperature of595∘CThis produced an amplicon of 200 bp in size Restric-tion enzyme digestion was performed with BfaI restrictionenzyme The TT genotype was uncut by the enzyme andremained 200 bp but the CC genotype was cleaved into180 bp and 20 bp bandsHeterozygotes showed all three bandson agarose gel

224 COX-1 (50CgtT) Polymorphism ThePCRprimers usedwere 51015840-GGT GCC CGG TGG GGA ATT TTC-31015840 and 51015840-GAG GGG AAA GGA GGG GGT TG-3 The annealingtemperature used was 60∘C The PCR reaction generated aproduct of 245 bp Then the fragment was digested by usingSmuI restriction enzymeTheTT genotype was not cut by theenzyme hence remaining as a 245 bp band on agarose gelsThe CC genotyped on the other hand was cleaved into twofragments of 120 bp and 125 bp in size Heterozygotes showedthe presence of all the above bands in agarose gel

225 COX-2 (-1195GgtA) Polymorphism The PCR primersused for amplification of the region containing the COX-2 (1195GgtA) polymorphism were 51015840-CCC TGA GCA CTACCCATGAT-31015840 and 51015840-GCCTTCATAGGAGATACTGG-31015840The annealing temperature of the reaction was 62∘C ThePCR amplification gave a product of 273 bp in size whichwas then digested using PvuII restriction enzyme The AAgenotype was undigested while the PCR product was cleavedinto a 220 bp and a 53 bp fragment for theGGgenotype Apartfrom that heterozygous genotype (AG) was detected by thepresence of all the above bands on the agarose gel

23 Statistical Analysis Statistical analyses were carried outusing the SPSS software package 180 (SPSS Inc ChicagoIL) Mean age for cases and controls was evaluated by usingStudentrsquos 119905-test On the other hand chi square test (1205942 test)was used to determine the significance difference in thesmoking status and the distribution of genotypes of thecases and controls Odds ratios (ORs) and the corresponding95 confidence intervals (CI) calculated by using logisticregression analysis were used to analyze the associationbetween the polymorphic genotypes and prostate risk based

BioMed Research International 3

Table 1 Difference between cases and controls characteristics

Characteristics Case (119899 = 543) Control (119899 = 753) 119875

Age 029Mean plusmn SD 6990 plusmn 843 6938 plusmn 876Range 48ndash87 46ndash85

Smoking status 052Ever smokers 247 329Never smokers 296 424

on heterozygous and homozygous comparison models withthe wild type genotype served as the reference Odds ratios(ORs) of gt100 indicated a positive risk association and viceversa Interaction between the polymorphisms and smokingstatus as well as between polymorphisms in related genes(NFKB1 and NFKBIA COX-2 and COX-1) was tested byusing the same logistic regression model as above by usingSPSS software119875 values oflt005were significant For analysesinvolving gene-gene and gene-environment interaction Bon-ferroni correction was applied for multiple testing in whichcase the statistical significance was assumed at 119875 lt 0025

3 Results

31 General Characteristics Table 1 shows the differencebetween cases and controls in terms of age and smokingstatusThe ages of cases ranged from 48 to 87 years and thoseof controls ranged from 46 to 85 years The mean age was6990 plusmn 843 for cases while the mean age was 6938 plusmn 876for controls No significant difference was observed betweenthe mean ages of both groups (119875 = 029) For both cases andcontrols the frequency of never smokers was slightly higherthan ever smokers In cases 247 (4549) were ever smokersand 296 (5451) were never smokers while in controls 329(4369) were ever smokers and 424 (5631) were neversmokers The difference in the distribution of ever and neversmokers among cases and controls was not significant (119875 =052)

32 Distribution of Genotypes The distribution of genotypesof the five polymorphisms in cases and controls is shown inTable 2 For the NFKB1 -94 insdel polymorphism the fre-quency of insins insdel and deldel genotypes was 198 246and 99 in cases and 212 355 and 186 in controls Significantdifference was observed between cases and controls in thedistribution of genotypes (1205942 = 1312 119875 lt 001)

Apart from that another polymorphism which showedsignificant difference between cases and controls in the geno-typic distributionwas theCOX-2 (-1195GgtA) polymorphismAmong the cases 71 269 and 203 subjects had GG GAand AA genotypes respectively On the other hand thedistribution of the genotypes among the controls was 158 378and 217 respectively (1205942 = 1834 119875 lt 001)

For the three other polymorphisms no statistically sig-nificant difference was observed between cases and controlsin the genotypic distribution (119875 gt 005) For NFKBIA -826CT polymorphism the distribution of the CC CT and

Table 2 Distribution of genotypes

Genotype Case(119899 = 543)

Control(119899 = 753) 120594

2119875

NFKB1 -94 Insdel 1312 lt001InsIns 198 212InsDel 246 355DelDel 99 186

NFKBIA -826CT 051 077CC 382 541CT 152 198TT 9 14

COX-2 (-1195GgtA) 1834 lt001GG 71 158GA 269 378AA 203 217

COX-1 (50CgtT) 405 013CC 452 655CT 83 92TT 8 6

PPARG Pro12Ala 023 089ProPro 483 676ProAla 57 73AlaAla 3 4

TT genotypes was 382 152 and 9 in cases and 541 198and 14 in controls (1205942 = 051 119875 = 077) For COX-1(50CgtT) polymorphism theCCCT andTT genotypeswererespectively present in 452 83 and 8 cases as well as 65592 and 6 controls (1205942 = 405 119875 = 013) On the otherhand for PPARG Pro12Ala polymorphism 483 57 and 3cases had ProPro ProAla and AlaAla genotypes while thedistribution of the same genotypes in controls was 676 73and 4 respectively (1205942 = 023 119875 = 089)

For all polymorphisms the genotype distribution did notdeviate significantly fromHardyWeinberg equilibrium bothamong cases and among controls (119875 gt 005)

33 Risk Association Overall Table 3 shows the associationbetween the five polymorphisms and prostate cancer riskin the population studied For NFKB1 -94 insdel polymor-phism a significantly decreased prostate cancer risk wasobserved in the heterozygous comparison model (insdelversus insins) and homozygous comparison model (deldelversus insins) In heterozygous comparison model the oddsratio was 074 (95 CI = 058ndash096) with a 119875 value of 002On the other hand in homozygous comparison model theodds ratio was 057 (95 CI = 042ndash078 119875 lt 001)

For COX-2 (-1195GgtA) polymorphism a significantlyincreased prostate cancer risk was observed in the heterozy-gous comparison model (GA versus GG) and homozygouscomparison model (AA versus GG) In heterozygous com-parison model the odds ratio was 158 (95 CI = 115ndash218)with a 119875 value of 001 On the other hand in homozygous


Table 3 Association between polymorphisms and prostate cancer risk

Genotype Case (119899 = 543) Control (119899 = 753) OR (95 CI) 119875

NFKB1 -94 insdelInsIns 198 212 Ref mdashInsDel 246 355 074 (058 to 096) 002DelDel 99 186 057 (042 to 078) lt001

NFKBIA -826CTCC 382 541 Ref mdashCT 152 198 109 (085 to 139) 051TT 9 14 091 (039 to 213) 083

COX-2 (-1195GgtA)GG 71 158 Ref mdashGA 269 378 158 (115 to 218) 001AA 203 217 208 (148 to 292) lt001

COX-1 (50CgtT)CC 452 655 Ref mdashCT 83 92 131 (095 to 180) 010TT 8 6 193 (067 to 561) 023

PPARG Pro12AlaPropro 483 676 Ref mdashProala 57 73 109 (076 to 158) 063Alaala 3 4 105 (023 to 471) 095

comparison model the odds ratio was 208 (95 CI = 148ndash292 119875 lt 001)

For NFKBIA -826CT polymorphism statistically notsignificant risk association was observed The heterozygouscomparison model (CT versus CC) showed an odds ratio of109 (95 CI = 085ndash139) and a 119875 value of 051 On contrarythe homozygous comparison model (TT versus CC) showedan odds ratio of 09104 (95 Cl = 039ndash213) and a 119875 value of083

Similarly lack of significant association was observedfor COX-1 (50CgtT) polymorphism and PPARG Pro12Alapolymorphism Both the polymorphisms showed positiveodds ratio values for both heterozygous and homozygouscomparison models For COX-1 (50CgtT) polymorphism theodds ratio obtained in the heterozygous comparison modelwas 131 (95 CI = 095ndash180 119875 = 010) while that in thehomozygous comparison model was 193 (95 CI = 067ndash561 119875 = 023) On the other hand for PPARG Pro12Alapolymorphism the odds ratio was 109 (95 CI = 076ndash158)in the heterozygous comparison model (119875 = 063) and 105(95 CI = 023ndash5471) in the homozygous comparisonmodel(119875 = 095)

34 Risk Association Interaction of Polymorphisms withSmoking Status The interaction between smoking statusand the polymorphisms in modulating prostate cancer riskwas also formally investigated and the results are shownin Table 4 Significant interaction with smoking status wasobserved only for theCOX-2 (-1195GgtA) polymorphism (119875 lt001) in which a significant risk association was observedbetween the AA genotype and ever smokers (OR = 176 95CI = 128ndash241 119875 lt 001)

35 Risk Association Gene-Gene Interactions The effectof interactions between polymorphisms in related genes(NFKB1 and NFKBIA COX-2 and COX-1) on the risk ofprostate cancer was also examined (Table 5) BothNFKB1 andNFKBIA combination and COX-2 and COX-1 combinationshowed statistically significant interactions (119875 = 003 and119875 =002 resp) For the interactions between COX-2 -1195GgtAand COX-1 (50CgtT) polymorphisms interaction betweenCOX-2 (-1195AA) lowast COX-1 (50CT) genotypes conferred arisk increment with OR = 183 (95 CI = 106ndash317 119875 =003) However this was deemed not statistically significantafter application of Bonferroni correction where 119875 lt 0025was considered as significant For the interactions betweenNFKB1 -94 insdel and NFKBIA -826CT polymorphismsstatistically significant prostate cancer risk association wasnot observed for any polymorphic combination investigatedNo other genotypic interactions yielded a statistically signifi-cant association

4 Discussion

In this study we investigated and established the associationof five inflammation-related genetic polymorphisms namelyPPARG Pro12Ala NFKB1 -94 insdel NFKBIA -826CTCOX-1 (50CgtT) and COX-2 (-1195GgtA) polymorphismswith prostate cancer risk in a Chinese population Thesepolymorphisms have been widely investigated for their asso-ciations with other cancers or in other populations butreport is scarce or unavailable for prostate cancer in theChinese population This novelty of data represents one ofthe strengths of the present study Additionally our studyinvolved a considerably large number of subjects which


Table 4 Gene-environment interaction between the polymorphisms and smoking status

Interaction 119875 for interaction OR (95 CI) 119875

NFKB1 lowast smoking status 031NFKB1 -94 insins lowast nonsmokers Ref mdashNFKB1 -94 insdel lowast smokers 101 (076ndash133) 096NFKB1 -94 deldel lowast smokers 074 (050ndash110) 014NFKBIA lowast smoking status 071NFKBIA -826CC lowast nonsmokers Ref mdashNFKBIA -826CT lowast smokers 113 (080ndash160) 049NFKBIA -826TT lowast smokers 141 (035ndash566) 063COX-2 lowast smoking status lt001COX-2 (-1195GG) lowast nonsmokers Ref mdashCOX-2 (-1195GA) lowast smokers 129 (098ndash169) 007COX-2 (-1195AA) lowast smokers 176 (128ndash241) lt001COX-1 lowast smoking status 013COX-1 (50CC) lowast nonsmokers Ref mdashCOX-1 (50CT) lowast smokers 146 (092ndash232) 011COX-1 (50TT) lowast smokers 238 (057ndash1000) 024PPARG lowast smoking status 023PPARG ProPro lowast nonsmokers Ref mdashPPARG ProAla lowast smokers 092 (050ndash168) 011PPARG AlaAla lowast smokers NA NAThere were two factors involved (polymorphism and smoking status) in each testing hence significance is assumed at119875 lt 0025 after application of Bonferronicorrection

Table 5 Combination of polymorphisms and prostate cancer risk


NFKB1 lowast NFKBIA polymorphisms 003NFKB1 -94 insins lowast NFKBIA -826CC Ref mdashNFKB1 -94 insdel lowast NFKBIA -826CT 099 (071ndash138) 094NFKB1 -94 deldel lowast NFKBIA -826TT 316 (081ndash1228) 010NFKB1 -94 insdel lowast NFKBIA -826CT 065 (039ndash107) 009NFKB1 -94 deldel lowast NFKBIA -826TT NA mdashCOX-2 (-1195GG) lowast COX-1 (50CC) 002 Ref mdashCOX-2 (-1195GA) lowast COX-1 (50CT) 134 (087ndash207) 019COX-2 (-1195AA) lowast COX-1 (50CT) 183 (106ndash317) 003COX-2 (-1195GA) lowast COX-1 (50TT) NA mdashCOX-2 (-1195AA) lowast COX-1 (50TT) 147 (030ndash729) 064There were two polymorphisms involved in each testing hence significance is assumed at 119875 lt 0025 after application of Bonferroni correction

increases the reliability of the results obtained Apart fromthat our study analyzed the combined effect of polymor-phisms in a similar pathway (ieNFKB1 andNFKBIACOX-2and COX-1) an approach which has been neglected in manyprevious reports

PPARG gene encodes for peroxisome proliferator-activated receptor-120574 (PPAR-120574) which is a ligand-activatedtranscription factor that is pleiotropic in nature PPAR-120574 is best known for its function in the regulation ofadipogenesis and fatty acid metabolism but it appearsthat the protein also plays a role in several carcinogenicprocesses including apoptosis cell cycle control andnotably inflammation [17] The Pro12Ala polymorphism has

been shown previously to affect the binding affinity of PPAR-120574 towards its target molecules [18] Therefore we postulatedthat this polymorphism could influence the risk of prostatecancer However we observed a lack of association betweenthe PPARG Pro12Ala polymorphism and prostate cancer riskin our population We propose that this observation couldbe due to the fact that although different genotypes of thepolymorphism have different binding affinity to its targetmolecules the difference is subtle and does not influence theability of PPAR-120574 in activating the transcription of its targetmolecules Our result was in agreement with a study fromFinland [19] and in partial agreement with another studyfrom USA [20] which showed that the polymorphism was


associated with an increased prostate cancer risk only amongindividuals with high body mass index

On the other hand NFKB1 and NFKBIA encode forNF-120581B1 and its inhibitor I120581B120572 respectively Both NF-120581B1and I120581B120572 are part of the NF-120581B pathway which playsan important role in inflammation and other carcinogenicprocesses The -94 insdel polymorphism of NFKB1 and-826CT polymorphism of NFKBIA have been shown toaffect the transcriptional activity of the respective genes asthe polymorphisms were located in the promoter regionof the genes [10 12] We observed that the NFKB1 -94insdel polymorphism was associated with decreased riskof prostate cancer in both heterozygous and homozygouscomparisonmodels suggesting that the variant del allele maybe linked to a reduced inflammatory status among its carriersand therefore a reduced risk This risk reduction was inagreement with an in vitro assay which showed that the delallele decreased the transcriptional activity ofNFKB1 therebytruncating its role in promoting inflammation which in turnled to the decreased prostate cancer risk [21] Our finding wasalso in agreement with two other previous studies on prostatecancers namely Zhang et al [22] in China and Kopp et al[13] in Denmark For NFKBIA -826CT polymorphism nosignificant associationwith prostate cancer riskwas observedDespite many previous studies reported on the association(or lack of association) between the NFKBIA polymorphismand risk of various cancers [10 23ndash25] we believe that thisis the first work which examined its association with prostatecancer risk

Cyclooxygenases are enzymes which catalyze the con-version of arachidonic acid to prostaglandins which pro-mote and regulate the process of inflammation There aretwo isoforms of cyclooxygenases namely the constitu-tively expressed cyclooxygenase-1 and inducibly expressedcyclooxygenase-2 which are encoded by COX-1 and COX-2 genes respectively The COX-1 (50CgtT) polymorphism islocated within the coding region of the gene and can poten-tially influence the functional importance of the enzymeproduced [26] On the other hand the promoter COX-2 (-1195GgtA) polymorphism can alter the transcriptionalactivity of the gene [27] Our result showed the absenceof prostate cancer risk association for the COX-1 (50CgtT)polymorphism This indicates that despite its functionalimportance the polymorphism is not sufficiently strong toresult in significant risk alteration among its carriers We alsoshowed the presence of an association between COX-2 (-1195GgtA) polymorphism and increased prostate cancer riskin both heterozygous and homozygous comparison modelsThis suggests that the variant A allele of the COX-2 polymor-phism could contribute to an increased prostate cancer riskamong its carriersThe observation that the A allele increasesprostate cancer risk can be explained by the fact that the Aallele creates a c-Myb transcription factor binding site in thegene resulting in an increased transcription of COX-2 [27]This in turn leads to the greater promotion of inflammationwhich mediates the process of carcinogenesis Our study isthe first to evaluate the association between COX-1 (50CgtT)polymorphism and prostate cancer risk whereas severalprevious reports were available for the association between

COX-2 (-1195GgtA) polymorphism and prostate cancer riskOur study results were in agreement with Sugie et al [28] ina Japanese population but Wu et al (Taiwan) [29] Chenget al (USA) [30] and Kopp et al (Denmark) [13] showedthe absence of risk association between COX-2 (-1195GgtA)polymorphism and prostate cancer risk

In summary we have demonstrated the presence ofa positive prostate cancer risk association for COX-2 (-1195GgtA) polymorphism a negative risk association forNFKB1 -94 insdel polymorphism and absence of associa-tion for PPARG Pro12Ala polymorphism NFKBIA -826CTpolymorphism and COX-1 (50CgtT) polymorphism Thereare several limitations in this study First although the overallsample size was large the sample size became small whenanalysis was performed separately between smokers andnonsmokers and when the genotypes were combined Inaddition we only included five polymorphisms which wereconsidered more well established based on our literaturereviewTherefore a more extensive examination for a greaternumber of polymorphisms in a larger sample size is neededand recommended for future work for reconfirmation of ourfindings

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Xin Cui and Hao Yan contributed equally to this work

References

[1] J Ferlay I Soerjomataram M Ervik et al GLOBOCAN 2012v10 Cancer Incidence and Mortality Worldwide IARC Cancer-Base No 11 International Agency for Research on Cancer LyonFrance 2013 httpglobocaniarcfr

[2] D M Parkin F Bray J Ferlay and P Pisani ldquoGlobal cancerstatistics 2002rdquo Ca A Cancer Journal for Clinicians vol 55 no2 pp 74ndash108 2005

[3] L Zhang S Wu L-R Guo and X-J Zhao ldquoDiagnosticstrategies and the incidence of prostate cancer reasons forthe low reported incidence of prostate cancer in Chinardquo AsianJournal of Andrology vol 11 no 1 pp 9ndash13 2009

[4] P D Baade D R Youlden S M Cramb J Dunn and R AGardiner ldquoEpidemiology of prostate cancer in the Asia-Pacificregionrdquo Prostate International vol 1 no 2 pp 47ndash58 2013

[5] M F Leitzmann and S Rohrmann ldquoRisk factors for the onsetof prostatic cancer age location and behavioral correlatesrdquoClinical Epidemiology vol 4 no 1 pp 1ndash11 2012

[6] O N Kryvenko M Jankowski D A Chitale et al ldquoInflamma-tion and preneoplastic lesions in benign prostate as risk factorsfor prostate cancerrdquo Modern Pathology vol 25 no 7 pp 1023ndash1032 2012

[7] 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

[8] A M De Marzo E A Platz S Sutcliffe et al ldquoInflammationin prostate carcinogenesisrdquoNature Reviews Cancer vol 7 no 4pp 256ndash269 2007


[9] M J Alvarez-Cubero M Saiz L J Martinez-Gonzalez J CAlvarez J A Lorente and J M Cozar ldquoGenetic analysis of theprincipal genes related to prostate cancer a reviewrdquo UrologicOncology Seminars andOriginal Investigations vol 31 no 8 pp1419ndash1429 2013

[10] S C TanM SM Suzairi A A A Aizat et al ldquoGender-specificassociation of NFKBIA promoter polymorphisms with the riskof sporadic colorectal cancerrdquoMedical Oncology vol 30 no 4article 693 2013

[11] X Li S-J Wang S C Tan et al ldquoThe A55T and K153Rpolymorphisms of MSTN gene are associated with the strengthtraining-induced muscle hypertrophy among Han Chinesemenrdquo Journal of Sports Sciences vol 32 no 9 pp 883ndash891 2014

[12] M S Mohd Suzairi S C Tan A A Ahmad Aizat et al ldquoThefunctional -94 insertiondeletion ATTG polymorphism in thepromoter region of NFKB1 gene increases the risk of sporadiccolorectal cancerrdquo Cancer Epidemiology vol 37 no 5 pp 634ndash638 2013

[13] T I Kopp S Friis J Christensen A Tjoslashnneland and U VogelldquoPolymorphisms in genes related to inflammation NSAID useand the risk of prostate cancer among Danish menrdquo CancerGenetics vol 206 no 7-8 pp 266ndash278 2013

[14] R Kazma J A Mefford I Cheng et al ldquoAssociation of theinnate immunity and inflammation pathway with advancedprostate cancer riskrdquo PLoS ONE vol 7 no 12 Article ID e516802012

[15] M Ianni E Porcellini I Carbone et al ldquoGenetic factorsregulating inflammation and DNAmethylation associated withprostate cancerrdquo Prostate Cancer and Prostatic Diseases vol 16no 1 pp 56ndash60 2013

[16] E M Kwon C A Salinas S Kolb et al ldquoGenetic polymor-phisms in inflammation pathway genes and prostate cancerriskrdquoCancer Epidemiology BiomarkersampPrevention vol 20 no5 pp 923ndash933 2011

[17] H Martin ldquoRole of PPAR-gamma in inflammation Prospectsfor therapeutic intervention by food componentsrdquo MutationResearch vol 690 no 1-2 pp 57ndash63 2010

[18] S S Deeb L Fajas M Nemoto et al ldquoA Pro12Ala substitutionin PPARgamma2 associated with decreased receptor activitylower bodymass index and improved insulin sensitivityrdquoNatureGenetics vol 20 no 3 pp 284ndash287 1998

[19] D Paltoo K Woodson P Taylor D Albanes J Virtamoand J Tangrea ldquoPro12Ala polymorphism in the peroxisomeproliferator-activated receptor-gamma (PPAR-gamma) geneand risk of prostate cancer among men in a large cancerprevention studyrdquoCancer Letters vol 191 no 1 pp 67ndash74 2003

[20] J M Zmuda F Modugno J L Weissfeld et al ldquoPerox-isome proliferator-activated receptor-120574 polymorphism bodymass and prostate cancer risk evidence for gene-environmentinteractionrdquo Oncology vol 70 no 3 pp 185ndash189 2006

[21] A S Karban T Okazaki C I M Panhuysen et al ldquoFunctionalannotation of a novel NFKB1 promoter polymorphism thatincreases risk for ulcerative colitisrdquoHuman Molecular Geneticsvol 13 no 1 pp 35ndash45 2004

[22] P Zhang Q Wei X Li et al ldquoA functional insertiondeletionpolymorphism in the promoter region of the NFKB1 geneincreases susceptibility for prostate cancerrdquoCancer Genetics andCytogenetics vol 191 no 2 pp 73ndash77 2009

[23] C-W Cheng J-L Su C-W Lin et al ldquoEffects of NFKB1andNFKBIA gene polymorphisms on hepatocellular carcinomasusceptibility and clinicopathological featuresrdquo PLoS ONE vol8 no 2 Article ID e56130 2013

[24] C-W Lin Y-S Hsieh C-H Hsin et al ldquoEffects of NFKB1andNFKBIA gene polymorphisms on susceptibility to environ-mental factors and the clinicopathologic development of oralcancerrdquo PLoS ONE vol 7 no 4 Article ID e35078 2012

[25] M Umar R Upadhyay S Kumar U C Ghoshal and B MittalldquoAssociation of common polymorphisms in TNFA NFkB1 andNFKBIA with risk and prognosis of esophageal squamous cellcarcinomardquo PLoS ONE vol 8 no 12 Article ID e81999 2013

[26] J Shi N L A Misso D L Duffy et al ldquoCyclooxygenase-1 genepolymorphisms in patients with different asthma phenotypesand atopyrdquo European Respiratory Journal vol 26 no 2 pp 249ndash256 2005

[27] H S de Vries R H M te Morsche M G H van Oijen I DNagtegaal W H M Peters and D J de Jong ldquoThe functionalminus765119866 rarr 119862 polymorphism of the COX-2 gene may reducethe risk of developing crohnrsquos diseaserdquo PLoS ONE vol 5 no 11Article ID e15011 2010

[28] S Sugie H Tsukino S Mukai et al ldquoCyclooxygenase 2genotypes influence prostate cancer susceptibility in JapaneseMenrdquo Tumor Biology vol 35 no 3 pp 2717ndash2721 2014

[29] H-C Wu C-H Chang H-L Ke et al ldquoAssociation ofcyclooxygenase 2 polymorphic genotypes with prostate cancerin Taiwanrdquo Anticancer Research vol 31 no 1 pp 221ndash225 2011

[30] I Cheng X Liu S J Plummer L M Krumroy G Casey andJ S Witte ldquoCOX2 genetic variation NSAIDs and advancedprostate cancer riskrdquo British Journal of Cancer vol 97 no 4 pp557ndash561 2007

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Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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of the protein products [11] and many studies have showeda relationship between inherited variations in inflammatoryresponse genes and the risk of prostate carcinogenesis [12ndash16] However the influence of several common SNPs inthe inflammatory response genes either individually or incombinations on prostate cancer risk has been understudiedin theChinese populationThis includes thePPARGPro12AlapolymorphismNFKB1 -94 insdel polymorphismNFKBIA -826CT polymorphism COX-1 (50CgtT) polymorphism andCOX-2 (-1195GgtA) polymorphism The present study aimedto investigate the association of the above polymorphismswith the risk of prostate cancer in the Chinese population

2 Materials and Methods

21 Study Subjects The study was approved by the MedicalEthics Committee of the Xuanwu Hospital Beijing ChinaAll subjects were recruited from Xuanwu Hospital BeijingChina between October 2010 and August 2014 A total of 543males who were diagnosed by qualified physicians as prostatecancer patients based on clinical pathological and laboratorytests were recruited into the study To ensure consistencyonly patients with high grade prostate cancer (Gleason scoregt 7) who had a PSA level of gt10 ngmL were included inthe study At the same period of time a total of 753 healthycontrols were enrolled into this study Eligibility criteria forcontrol included no individual history of cancer and noknown genetic syndromes associated with prostate cancerand a PSA level of lt2 ngmL Blood samples were collectedfrom the subjectswhomeet the criteriamentioned above afterobtaining written informed consent

22 SNP Genotyping Genomic DNA extraction was doneby using EasyPure Blood Genomic DNA Kit (TransGenBiotech Beijing China) after the blood samples were takenfrom peripheral blood of all subjects The DNA was usedfor genotyping of the five SNPs by using the PCR-RFLPmethod All PCRwere performed in a 25 120583L reactionmixturecontaining standard PCR buffer 15mM MgCl

2 025mM

dNTP 1 unit Taq polymerase and 04 120583Mof each primer Forall polymorphisms approximately 10 of the samples werechosen at random and sequenced to confirm the genotypesfrom the amplified PCR product The laboratory personnelperforming the genotyping were blinded to the identity thecase-control status and source of the samples

221 PPARG Pro12Ala Polymorphism The PCR primersused for amplification of the region containing the PPARGPro12Ala polymorphism were 51015840-GCC AAT TCA AGC CCAGTC-31015840 and 51015840-GAT ATG TTT GCA GAC AGT GTATCA GTG AAG GAA TCG CTT TCC-31015840 The annealingtemperature of the reaction was 58∘CThe PCR amplificationproduced a fragment of 270 bp in size which was thendigested using BstUI restriction enzyme The fragment wascleaved into a 227 bp and a 43 bp fragment for the AlaAlagenotype while the ProPro genotype was uncut Apart fromthat heterozygous genotype (ProAla) was detected by thepresence of all the above bands in agarose gel

222 NFKB1 -94 insdel Polymorphism The PCR for ampli-fication of the region containing the NFKB1 -94 insdelpolymorphism was performed by using 51015840-TGG GCA CAAGTC GTT TAT GA-31015840 and 51015840-CTG GAG CCG GTA GGGAAG-31015840 primers which were annealed to the genomic DNAat 61∘C PflMI restriction enzyme was subsequently usedto digest the amplified PCR product of 281 bp in size Forinsins genotype the absence of restriction site resulted inthe undigested fragment of 281 bp after the reaction On theother hand a 240 bp and a 45 bp fragment were obtained forthe deldel genotype while a 281 bp a 240 bp and a 45 bpfragment were found in the insdel genotype

223 NFKBIA -826CT Polymorphism The NFKBIA -826CT polymorphismwas genotyped by first amplifying theregion of interest by using the following PCR primers 51015840-GGT CCT TAA GGT CCA ATC G-31015840 and 51015840-GTT GTGGAT ACC TTG CAC TA-31015840 at an annealing temperature of595∘CThis produced an amplicon of 200 bp in size Restric-tion enzyme digestion was performed with BfaI restrictionenzyme The TT genotype was uncut by the enzyme andremained 200 bp but the CC genotype was cleaved into180 bp and 20 bp bandsHeterozygotes showed all three bandson agarose gel

224 COX-1 (50CgtT) Polymorphism ThePCRprimers usedwere 51015840-GGT GCC CGG TGG GGA ATT TTC-31015840 and 51015840-GAG GGG AAA GGA GGG GGT TG-3 The annealingtemperature used was 60∘C The PCR reaction generated aproduct of 245 bp Then the fragment was digested by usingSmuI restriction enzymeTheTT genotype was not cut by theenzyme hence remaining as a 245 bp band on agarose gelsThe CC genotyped on the other hand was cleaved into twofragments of 120 bp and 125 bp in size Heterozygotes showedthe presence of all the above bands in agarose gel

225 COX-2 (-1195GgtA) Polymorphism The PCR primersused for amplification of the region containing the COX-2 (1195GgtA) polymorphism were 51015840-CCC TGA GCA CTACCCATGAT-31015840 and 51015840-GCCTTCATAGGAGATACTGG-31015840The annealing temperature of the reaction was 62∘C ThePCR amplification gave a product of 273 bp in size whichwas then digested using PvuII restriction enzyme The AAgenotype was undigested while the PCR product was cleavedinto a 220 bp and a 53 bp fragment for theGGgenotype Apartfrom that heterozygous genotype (AG) was detected by thepresence of all the above bands on the agarose gel

23 Statistical Analysis Statistical analyses were carried outusing the SPSS software package 180 (SPSS Inc ChicagoIL) Mean age for cases and controls was evaluated by usingStudentrsquos 119905-test On the other hand chi square test (1205942 test)was used to determine the significance difference in thesmoking status and the distribution of genotypes of thecases and controls Odds ratios (ORs) and the corresponding95 confidence intervals (CI) calculated by using logisticregression analysis were used to analyze the associationbetween the polymorphic genotypes and prostate risk based







3 Results







Control(119899 = 753) 120594

2119875


NFKBIA -826CT 051 077CC 382 541CT 152 198TT 9 14


COX-1 (50CgtT) 405 013CC 452 655CT 83 92TT 8 6



















4 Discussion






















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results







Control(119899 = 753) 120594

2119875


NFKBIA -826CT 051 077CC 382 541CT 152 198TT 9 14


COX-1 (50CgtT) 405 013CC 452 655CT 83 92TT 8 6



















4 Discussion






















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























4 Discussion






















References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































References





































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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