Research Article Differential Expression of Long Noncoding RNA...
10
Research Article Differential Expression of Long Noncoding RNA in Primary and Recurrent Nasopharyngeal Carcinoma Wei Gao, Jimmy Yu-Wai Chan, and Thian-Sze Wong Department of Surgery, LKS Faculty of Medicine, e University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, Hong Kong Correspondence should be addressed to ian-Sze Wong; [email protected] Received 11 February 2014; Accepted 20 March 2014; Published 14 April 2014 Academic Editor: ean Hock Tang Copyright © 2014 Wei Gao et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Recent studies suggested that non-protein-coding genes are implicated in the tumorigenic process of nasopharyngeal carcinoma (NPC). In the present study, we aimed to identify the differentially expressed long noncoding RNA (lncRNA) using data available in the public domain. Methods. Microarray data set GSE12452 was reannotated with ncFANs. Real-time quantitative PCR was used to quantify and validate the identified lncRNAs in NPC. Results. In primary NPC, upregulation of lnc-C22orf32-1, lnc- AL355149.1-1, and lnc-ZNF674-1 was observed. High levels of lnc-C22orf32-1 and lnc-AL355149.1-1 were significantly associated with the male patients. In addition, increased expression of lnc-C22orf32-1 and lnc-ZNF674-1 was associated with advanced tumor stages. Recurrent NPC displayed a distinctive lncRNA expression pattern. lnc-BCL2L11-3 was significantly increased in the recurrent NPC tissues. In addition, significant reduction of lnc-AL355149.1-1 and lnc-ZNF674-1 was observed in the recurrent NPC tissues. Conclusions. Our results demonstrated that it is feasible to identify the differentially expressed lncRNA in the microarray dataset by functional reannotation. e association of lncRNA with gender and tumor size implicated that lncRNA possibly plays a part in the pathogenesis of primary NPC. Further, the distinctive lncRNA identified in the recurrent NPC may reveal a distinctive development mechanism underlying tumor recurrence. 1. Introduction Undifferentiated nasopharyngeal carcinoma (NPC) is preva- lent in specific geographic localities including Southern China and East Asia, Southeast Asia, Greenland (native), Canada (Northwest Territories), and Alaska. Undifferenti- ated NPC is characterized by its close association with the virology of Epstein-Barr virus, a gamma-herpes DNA virus that infects more than 90% of world’s population and is found in nearly all undifferentiated NPC cells [1]. According to the World Health Organization classification (WHO 2005), NPC could be classified based on the cancer differentiation status into type 1 keratinising squamous cell carcinoma and type 2 nonkeratinising carcinoma (differentiated and undif- ferentiated NPC). In endemic regions, the major histological type is type 2 NPC [2]. Early diagnosis of NPC is difficult as the cancer is developed from the epithelial lining around the ostium of the Eustachian tube in the lateral wall of the nasopharynx with close proximity to the skull [3]. Later diagnosis will usually lead to unsatisfactory treatment results and high incidence of local regional recurrence [4]. lncRNAs are non-protein-coding transcripts transcribed by either RNA Pol I, RNA Pol II, or RNA Pol III. Dif- ferent from the protein-coding genes, lncRNA has distinct biological function in maintaining the cellular homeosta- sis [5]. Prensner and Chinnaiyan summarized our current knowledge on lncRNA in the pathogenesis and progression of human malignancies [6]. At present, we know that long or large intergenic ncRNAs, transcribed ultraconserved regions, pseudogenes, enhancer RNAs, antisense RNAs, and long stress-induced noncoding transcribes are involved in the development and tumorigenesis of cancers. e existence of these noncoding RNAs used to be regarded as transcriptional byproducts with no significant functions. Over the past decade, however, increasing evidence suggested that lncRNA could function as epigenetic regulators, modulators of gene expression, and protein degradation machinery as well as reg- ulators of organelle biogenesis and subcellular trafficking [7]. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 404567, 9 pages http://dx.doi.org/10.1155/2014/404567
Transcript of Research Article Differential Expression of Long Noncoding RNA...
Research ArticleDifferential Expression of Long Noncoding RNA inPrimary and Recurrent Nasopharyngeal Carcinoma
Wei Gao Jimmy Yu-Wai Chan and Thian-Sze Wong
Department of Surgery LKS Faculty of Medicine The University of Hong Kong Queen Mary Hospital102 Pokfulam Road Hong Kong Hong Kong
Correspondence should be addressed toThian-Sze Wong thianszegraduatehkuhk
Received 11 February 2014 Accepted 20 March 2014 Published 14 April 2014
Academic Editor Thean Hock Tang
Copyright copy 2014 Wei Gao et alThis 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
Background Recent studies suggested that non-protein-coding genes are implicated in the tumorigenic process of nasopharyngealcarcinoma (NPC) In the present study we aimed to identify the differentially expressed long noncoding RNA (lncRNA) using dataavailable in the public domainMethods Microarray data set GSE12452 was reannotated with ncFANs Real-time quantitative PCRwas used to quantify and validate the identified lncRNAs in NPC Results In primary NPC upregulation of lnc-C22orf32-1 lnc-AL3551491-1 and lnc-ZNF674-1 was observed High levels of lnc-C22orf32-1 and lnc-AL3551491-1 were significantly associatedwiththemale patients In addition increased expression of lnc-C22orf32-1 and lnc-ZNF674-1was associatedwith advanced tumor stagesRecurrent NPC displayed a distinctive lncRNA expression pattern lnc-BCL2L11-3 was significantly increased in the recurrentNPC tissues In addition significant reduction of lnc-AL3551491-1 and lnc-ZNF674-1 was observed in the recurrent NPC tissuesConclusions Our results demonstrated that it is feasible to identify the differentially expressed lncRNA in the microarray dataset byfunctional reannotationThe association of lncRNAwith gender and tumor size implicated that lncRNA possibly plays a part in thepathogenesis of primaryNPC Further the distinctive lncRNA identified in the recurrentNPCmay reveal a distinctive developmentmechanism underlying tumor recurrence
1 Introduction
Undifferentiated nasopharyngeal carcinoma (NPC) is preva-lent in specific geographic localities including SouthernChina and East Asia Southeast Asia Greenland (native)Canada (Northwest Territories) and Alaska Undifferenti-ated NPC is characterized by its close association with thevirology of Epstein-Barr virus a gamma-herpes DNA virusthat infectsmore than 90 of worldrsquos population and is foundin nearly all undifferentiated NPC cells [1] According tothe World Health Organization classification (WHO 2005)NPC could be classified based on the cancer differentiationstatus into type 1 keratinising squamous cell carcinoma andtype 2 nonkeratinising carcinoma (differentiated and undif-ferentiated NPC) In endemic regions the major histologicaltype is type 2 NPC [2] Early diagnosis of NPC is difficultas the cancer is developed from the epithelial lining aroundthe ostium of the Eustachian tube in the lateral wall of thenasopharynx with close proximity to the skull [3] Later
diagnosis will usually lead to unsatisfactory treatment resultsand high incidence of local regional recurrence [4]
lncRNAs are non-protein-coding transcripts transcribedby either RNA Pol I RNA Pol II or RNA Pol III Dif-ferent from the protein-coding genes lncRNA has distinctbiological function in maintaining the cellular homeosta-sis [5] Prensner and Chinnaiyan summarized our currentknowledge on lncRNA in the pathogenesis and progressionof human malignancies [6] At present we know that long orlarge intergenic ncRNAs transcribed ultraconserved regionspseudogenes enhancer RNAs antisense RNAs and longstress-induced noncoding transcribes are involved in thedevelopment and tumorigenesis of cancers The existence ofthese noncoding RNAs used to be regarded as transcriptionalbyproducts with no significant functions Over the pastdecade however increasing evidence suggested that lncRNAcould function as epigenetic regulators modulators of geneexpression and protein degradationmachinery aswell as reg-ulators of organelle biogenesis and subcellular trafficking [7]
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 404567 9 pageshttpdxdoiorg1011552014404567
2 BioMed Research International
Hence further understanding of the aberrant expressionpattern of lncRNA in cancers is essential
Study on lnRNA deregulation in undifferentiated NPCremains raw Recently with the use of candidate geneapproach it was identified that HOX antisense intergenicRNA (HOTAIR) one of the first described lncRNAs tran-scribed in the HOXC locus at 12q1313 was suggested toplay a part in NPC HOTAIR was implicated in cancerprogression and metastasis In primary NPC high HOTAIRexpression was specific to the primary NPC and was absentin the normal nasopharyngeal epithelia HOTAIR expressionlevel was linked with the tumor size nodal status anddistantmetastasis Further patients with highHOTAIR levelswill have poor clinical outcome with tumor recurrence anddistant metastasis [8 9]
High-throughput microarray was originally designed todetect and quantify the protein-coding genes However itis now noted that many of the probes on the microarrayalso match with the sequence of lncRNA By rematchingand reannotation of the existing microarray datasets theresults obtained from the microarray experiments could beused for lncRNA analysis concurrently For example withthe use of Affymetrix U133A and B microarrays it has beendemonstrated that the microarray data can be reannotatedfor lncRNA expression analysis [10] Here we explored thedifferential lncRNA expression patterns in nasopharyngealcarcinoma (NPC) based on the high-throughputNPCdatasetin the public domain using the functional reannotationservice provided by noncoding RNA Function ANnotationserver (ncFANs) [11] The identified lncRNAs were validatedin both primary and recurrent NPC samples
2 Materials and Methods
21 Dataset Microarray data set GSE12452 containing 41microarray data (httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE12452) was collected from Gene Expres-sion Omnibus (GEO) The dataset contains 31 NPC and10 normal nasopharyngeal tissue samples examined withHuman Genome U133 Plus 20 Array (HG-U133 Plus 2) fromAffymetrix [12] The tissues were confirmed histologicallybefore the microarray experiment Tumor tissue was definedby the positive stain for both cytokeratins and EBV EBERsusing immunohistochemical staining All the samples wereprocessed by laser-captured microdissection to enrich theepithelial cells before total RNA extraction
22 Patient Samples Undifferentiated NPC tissue sampleswere obtained from Department of Surgery The Univer-sity of Hong Kong Queen Mary Hospital Hong KongWritten consent of tissue donation for research purposeswas obtained from patients before tissue collection Theprotocol was approved by the IRB of the hospital (referencenumber UW 10-142) Tissue samples were stored at liquidnitrogen for transportation and stored at minus70∘C before use Intotal nasopharyngeal tissue samples were collected from 42patients with primary NPC 33 patients with recurrent NPCand 29 noncancer volunteers The 42 patients with primary
NPC included 32 male and 10 female with age ranging from12 to 75 yearsThe 33 recurrentNPCpatients included 24maleand 9 female with age ranging from 29 to 75 years The 29healthy volunteers included 20 male and 9 female with agesranging from 6 to 81 years
23 Reannotation and Selection of Differential ExpressedlncRNA The microarray data was preprocessed with thepreprocessing program and reannotated with the AffymetrixCDF file from the web server of ncFANs (noncoding RNAFunction ANnotation server) The data was normalized byMAS50 before lncRNAs annotation Transcripts with lengthlt200 were excluded [11] Differentially expressed lncRNAswere selected using Studentrsquos 119905-test using 119875 value below 005as cutoff
24 RNA Extraction and Real-Time Quantitative RT-PCRVal-idation of the Microarray Results Total RNA was extractedwith TRIZOL reagent (Invitrogen) following the manufact-urerrsquos protocol The cDNA product was generated usingcDNA conversion kit (Invitrogen) Primers were designedwith Primer3 provided by the Universal ProbeLibrary AssayDesign Center (httpwwwroche-applied-sciencecom)The LNA-labelled probe was obtained from the UniversalProbeLibrary (UPL) The lncRNA transcript levels werequantified on LightCycler 480 (Roche Applied Science)and normalized with the GAPDH levels by 2minusΔCt methodAll reactions were done in triplicate Primer and probesequences were shown in Table 1
25 Statistical Analysis Statistical analysis was performedwith SPSS V160 (SPSS Chicago IL) The difference betweenprimary NPC and controls was calculated using the Mann-Whitney 119880 test The statistical difference between recur-rent NPC and paired normal tissues was examined usingWilcoxon Signed Rank test All the tests were two-sided 119875value lt005 was considered statistically significant
3 Results
31 Functional Annotation of lncRNA with ncFANs Thedifferentially expressed lncRNAs identified were listedin Table 2 Five lncRNAs were found to be differentiallyexpressed in the nasopharyngeal carcinoma tissues in com-parison with the normal nasopharyngeal epithelial tissuesin the microarray data The lncRNAs identified were lnc-C22orf32-1 lnc-TLR4-1 lnc-BCL2L11-3 lnc-AL3551491-1and lnc-ZNF674-1
32 lncRNA Validation on Primary NPC Tissues and Nor-mal Nasopharyngeal Epithelia To validate the identifiedlncRNA we performed real-time quantitative analysis on thecandidate lncRNA in our cohort First we examined thefold changes in the primary nasopharyngeal carcinoma andnormal nasopharyngeal tissues (Figure 1) Among the 5 can-didate lncRNAs lnc-C22orf32-1 lncTLR4-1 lnc-AL3551491-1 and lnc-ZNF674-1 demonstrated significant expression
BioMed Research International 3
Table 1 Primer and probe sequences used in quantitative PCR
Vega gene ID LNCipedia gene ID Forward primer (51015840-31015840) Reverse primer (51015840-31015840) UPL numberlowast
OTTHUMG00000153695 lnc-BCL2L11-3 agcagatgctgtgcctgata cctttctcgacccagaagc 67OTTHUMG00000002490 lnc-AL3551491-1 gaaaactaggcgtctgggaac caaacaatgggagcaagtcc 25OTTHUMG00000150697 lnc-C22orf32-1 tgctcatcttctgccacagt agggcagtgatgaggaacc 38OTTHUMG00000021416 lnc-ZNF674-1 agcacttggccctaaagaga aacatactggcccaaacagc 88OTTHUMG00000020561 lnc-TLR4-1 ccacacaaatgggcaagaat gcaaaatcctgaaggttcaaa 6lowastProbe number in the universal probe library (UPL)
Primary NPC Controls
15 times 1004
10 times 1004
50 times 1003
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0001
(a)
Primary NPC Controls
4 times 1002
3 times 1002
2 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0433
(b)
Primary NPC Controls0
4 times 1004
2 times 1004
4 times 1003
3 times 1003
2 times 1003
1 times 1003Re
lativ
e exp
ress
ion
of ln
c-A
L3551491
-1
P = 0015
(c)
Primary NPC Controls
8 times 1004
4 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0034
(d)
Figure 1 Relative expression levels of lncRNAs in tissue samples frompatientswith primaryNPCandhealthy controlsThe relative expressionlevels were normalized to GAPDH by qPCR analysis The difference between primary NPC and controls was calculated using the Mann-Whitney 119880 test
4 BioMed Research International
Table2Differentia
llyexpressedlnRN
Ain
NPC
microarraydatasetidentified
byncFA
Nsreann
otation
Vega
gene
IDLN
Cipediag
eneID
Locusc
onservationlowast
Exon
number
Transcrip
tsize(bp
)Genom
elocation
Adjust119875value
OTT
HUMG00
000153695
lnc-BC
L2L11-3
No
2757
chr2112248850-112268567+
0002
OTT
HUMG00
0000
02490
lnc-AL3551491-1
No
2342
chr116
847189-16848303+
000
4OTT
HUMG00
000150697
lnc-C2
2orf32-1
No
2544
chr2242672361-42673057+
000
6OTT
HUMG00
000021416
lnc-ZN
F674-1
No
2626
chrX
46185359-46187080minus
0030
OTT
HUMG00
000020561
lnc-TL
R4-1
Mou
se6
1299
chr912
0410884-120419305+
0033
lowast
Locusc
onservationin
Mus
musculusand
Daniorerio
comparedto
Hom
osapiens
BioMed Research International 5
100806040200
Sens
itivi
ty10
08
06
04
02
00
ROC curve
Area under curve = 0734
1 minus specificity
lnc-C22orf32-1
(a)
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
Area under curve = 0670
lnc-AL3551491-1
1 minus specificity
(b)
Area under curve = 0649
lnc-ZNF674-1
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
1 minus specificity
(c)
Figure 2 Receiver operating characteristic (ROC) curve analysis of lncRNAs in tissue samples from patients with primary NPC and healthycontrols
difference between the primary NPC and normal nasopha-ryngeal tissues
Of the 4 lncRNAs the expression difference of lnc-C22orf32-1 was highly significant (119875 = 0001 Mann-Whitney119880 test) High lnc-C22orf32-1 was found in the primary NPCtissues in comparison with the normal controls (Figure 1) InROC analysis the AUC value of lnc-C22orf32-1 was 0734 indifferentiating NPC from the normal nasopharyngeal tissues(Figure 2) lnc-AL3551491-1 and lnc-ZNF674-1 were alsofound to be overexpressed in the primary NPC tissues (119875 =0015 and 0034 respectively Mann-Whitney 119880 test) The 3lncRNAs above are detectable in all the tissues examined Incontrast lnc-TLR4-1 was detectable in 21 (942) patientswith primary NPC and 66 (1929) healthy controls Inrecurrent NPC lnc-TLR4-1 was detectable in 27 (933)tumor tissues and 15 (533) paired normal tissues Low copynumber of lnc-TLR4-1 in tissue samples may account for
the low detection rate The expression pattern of lnc-TLR4-1 was not correlated with age or gender (data not shown)Since lnc-TLR4-1 was only expressed in a subset of NPC andnormal nasopharyngeal tissues it was not suitable for studyof differential expression
Next we compared the expression level of the 3 universalexpressed lncRNAs with the clinicopathological parametersof the primary NPC patients (Table 3) High levels of lnc-C22orf32-1 and lnc-AL3551491-1 were significantly associatedwith the male patients (Figure 3(b)) In addition increasedexpression of lnc-C22orf32-1 and lnc-ZNF674-1 was associ-ated with advanced tumor stages (T2ndash4) in comparison withthe early stage (T1) NPC (Figure 3(a))
33 Differential lncRNAs Expression in Recurrent NPC and thePaired Normal Epithelia We compared the lncRNA expres-sion patterns with the paired normal tissues obtained from
6 BioMed Research International
Table 3 Association of lncRNA expression levels with the clinicopathological variables in primary undifferentiated NPC patients
lnc-ZNF674-1 lnc-AL3551491-1 lnc-C22orf32-1119875 value
Gender NumberMale 32 0108 0028lowast 0033lowastFemale 10
Agelt49 19 0733 0970 0810gt49 23
T stageT1 11 0030lowast 0062 0019lowastT2ndash4 31
Nodal statusNegative 7 0895 0692 0692Positive 35
lowast
119875 value below 005 was considered statistically significant
the same recurrent NPC patients (Figure 4) lnc-BCL2L11-3was found to be upregulated in the recurrent NPC tissuesin comparison with the paired normal tissues (119875 = 0002Wilcoxon Signed Rank test) For lnc-AL3551491-1 and lnc-ZNF674-1 high expression was found in primary NPCHowever significant expression reduction was observed inthe recurrent NPC tissues (119875 = 0022 and 0002 respectivelyWilcoxon Signed Rank test)
4 Discussion
The differential expression of lnc-AL3551491-1 lnc-C22orf32-1 and lnc-ZNF674-1 observed in NPC might beattributed to epigenetic regulation including microRNADNAmethylation and histone modification As predicted bya bioinformatics tool MirTarget2 (httpmirdborgmiRDBindexhtml) lnc-AL3551491-1 is targeted by hsa-miR-939hsa-miR-4283 and hsa-miR-3191-5p lnc-C22orf32-1 is tar-geted by hsa-miR-4751 lnc-ZNF674-1 is targeted by hsa-miR-1296 hsa-miR-1292 and hsa-miR-3179 In additionDNA methylation and histone modification may alsocontribute to the aberrant expression of these lncRNAs Forexample DNA methylation H3K4 histone methylationand H3K9 histone methylation are found in lnc-AL3551491-1 gene in different human cell lines (lncRNomehttpgenomeigibresinlncRNome)
lncRNAs could exert biological functions by interactingwith RNA or protein molecules An lncRNA highly upreg-ulated in liver cancer (HULC) could bind to miR-372 thusmodulating the expression of miR-372 at posttranscriptionallevel [13] lncRNA HOTAIR interacted with polycomb chro-matin remodeling complex 2 (PRC2) by its 51015840 domain andbind to LSD1CoRESTREST complex via its 31015840 domainresulting in a specific histone methylation pattern [14] Toexplore the possible model by which these differentially
expressed lncRNAs exert their biological functions we triedto predict their binding capacity via database lncRNomeThe nucleotides 595ndash597 of lnc-ZNF674-1 are predicted to beprotein binding sites indicating that itmay play a role inNPCby interacting with specific proteins
Primary NPC is highly sensitive to radiotherapy andthe use of concurrent chemoradiotherapy has significantlyimproved the local control rates of NPC [15] Neverthelesslocal recurrence still appears in approximately 10ndash30 ofpatients after primary radiotherapy [16] Since recurrentNPC is possibly derived from those NPC cells that couldescape from radiotherapy the recurrent NPC might exhibitdistinctive mRNA miRNA and protein expression patternsin comparison with primary NPC For instance the expres-sion levels of miR-98 TR-1205722 mRNA multidrug resistance 1(MDR-1) protein and glutathione-S-transferase-120587 (GST-120587)protein were different between recurrent NPC and primaryNPC [17 18] Likewise in this study we also observed adiverse lncRNA expression pattern between recurrent NPCand primary NPC For example the aberrant expression oflnc-BCL2L11-3 was only observed in recurrent NPC but notin primaryNPC suggesting that lncRNAsmight play distinctroles in the initiation and progression of recurrent NPC andprimary NPC
According toGlobal Cancer Statistics [19] NPC is amale-dominant disease with incidence rates usually 2 to 3 timeslower in females In our cohort the ratio between malesand females is about 3 Therefore one of the limitationsof this study is the small sample size of females Anotherconcern about our patient samples is the large age rangeTo investigate whether the large age range would affect theexpression level of lncRNAs we divided our patients into twogroups according to various cutoff values of ageThere are nosignificant differences in the expression of lncRNAs betweenthe two groups of patients divided by varied cutoff values ofage
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 BioMed Research International
Hence further understanding of the aberrant expressionpattern of lncRNA in cancers is essential
Study on lnRNA deregulation in undifferentiated NPCremains raw Recently with the use of candidate geneapproach it was identified that HOX antisense intergenicRNA (HOTAIR) one of the first described lncRNAs tran-scribed in the HOXC locus at 12q1313 was suggested toplay a part in NPC HOTAIR was implicated in cancerprogression and metastasis In primary NPC high HOTAIRexpression was specific to the primary NPC and was absentin the normal nasopharyngeal epithelia HOTAIR expressionlevel was linked with the tumor size nodal status anddistantmetastasis Further patients with highHOTAIR levelswill have poor clinical outcome with tumor recurrence anddistant metastasis [8 9]
High-throughput microarray was originally designed todetect and quantify the protein-coding genes However itis now noted that many of the probes on the microarrayalso match with the sequence of lncRNA By rematchingand reannotation of the existing microarray datasets theresults obtained from the microarray experiments could beused for lncRNA analysis concurrently For example withthe use of Affymetrix U133A and B microarrays it has beendemonstrated that the microarray data can be reannotatedfor lncRNA expression analysis [10] Here we explored thedifferential lncRNA expression patterns in nasopharyngealcarcinoma (NPC) based on the high-throughputNPCdatasetin the public domain using the functional reannotationservice provided by noncoding RNA Function ANnotationserver (ncFANs) [11] The identified lncRNAs were validatedin both primary and recurrent NPC samples
2 Materials and Methods
21 Dataset Microarray data set GSE12452 containing 41microarray data (httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE12452) was collected from Gene Expres-sion Omnibus (GEO) The dataset contains 31 NPC and10 normal nasopharyngeal tissue samples examined withHuman Genome U133 Plus 20 Array (HG-U133 Plus 2) fromAffymetrix [12] The tissues were confirmed histologicallybefore the microarray experiment Tumor tissue was definedby the positive stain for both cytokeratins and EBV EBERsusing immunohistochemical staining All the samples wereprocessed by laser-captured microdissection to enrich theepithelial cells before total RNA extraction
22 Patient Samples Undifferentiated NPC tissue sampleswere obtained from Department of Surgery The Univer-sity of Hong Kong Queen Mary Hospital Hong KongWritten consent of tissue donation for research purposeswas obtained from patients before tissue collection Theprotocol was approved by the IRB of the hospital (referencenumber UW 10-142) Tissue samples were stored at liquidnitrogen for transportation and stored at minus70∘C before use Intotal nasopharyngeal tissue samples were collected from 42patients with primary NPC 33 patients with recurrent NPCand 29 noncancer volunteers The 42 patients with primary
NPC included 32 male and 10 female with age ranging from12 to 75 yearsThe 33 recurrentNPCpatients included 24maleand 9 female with age ranging from 29 to 75 years The 29healthy volunteers included 20 male and 9 female with agesranging from 6 to 81 years
23 Reannotation and Selection of Differential ExpressedlncRNA The microarray data was preprocessed with thepreprocessing program and reannotated with the AffymetrixCDF file from the web server of ncFANs (noncoding RNAFunction ANnotation server) The data was normalized byMAS50 before lncRNAs annotation Transcripts with lengthlt200 were excluded [11] Differentially expressed lncRNAswere selected using Studentrsquos 119905-test using 119875 value below 005as cutoff
24 RNA Extraction and Real-Time Quantitative RT-PCRVal-idation of the Microarray Results Total RNA was extractedwith TRIZOL reagent (Invitrogen) following the manufact-urerrsquos protocol The cDNA product was generated usingcDNA conversion kit (Invitrogen) Primers were designedwith Primer3 provided by the Universal ProbeLibrary AssayDesign Center (httpwwwroche-applied-sciencecom)The LNA-labelled probe was obtained from the UniversalProbeLibrary (UPL) The lncRNA transcript levels werequantified on LightCycler 480 (Roche Applied Science)and normalized with the GAPDH levels by 2minusΔCt methodAll reactions were done in triplicate Primer and probesequences were shown in Table 1
25 Statistical Analysis Statistical analysis was performedwith SPSS V160 (SPSS Chicago IL) The difference betweenprimary NPC and controls was calculated using the Mann-Whitney 119880 test The statistical difference between recur-rent NPC and paired normal tissues was examined usingWilcoxon Signed Rank test All the tests were two-sided 119875value lt005 was considered statistically significant
3 Results
31 Functional Annotation of lncRNA with ncFANs Thedifferentially expressed lncRNAs identified were listedin Table 2 Five lncRNAs were found to be differentiallyexpressed in the nasopharyngeal carcinoma tissues in com-parison with the normal nasopharyngeal epithelial tissuesin the microarray data The lncRNAs identified were lnc-C22orf32-1 lnc-TLR4-1 lnc-BCL2L11-3 lnc-AL3551491-1and lnc-ZNF674-1
32 lncRNA Validation on Primary NPC Tissues and Nor-mal Nasopharyngeal Epithelia To validate the identifiedlncRNA we performed real-time quantitative analysis on thecandidate lncRNA in our cohort First we examined thefold changes in the primary nasopharyngeal carcinoma andnormal nasopharyngeal tissues (Figure 1) Among the 5 can-didate lncRNAs lnc-C22orf32-1 lncTLR4-1 lnc-AL3551491-1 and lnc-ZNF674-1 demonstrated significant expression
BioMed Research International 3
Table 1 Primer and probe sequences used in quantitative PCR
Vega gene ID LNCipedia gene ID Forward primer (51015840-31015840) Reverse primer (51015840-31015840) UPL numberlowast
OTTHUMG00000153695 lnc-BCL2L11-3 agcagatgctgtgcctgata cctttctcgacccagaagc 67OTTHUMG00000002490 lnc-AL3551491-1 gaaaactaggcgtctgggaac caaacaatgggagcaagtcc 25OTTHUMG00000150697 lnc-C22orf32-1 tgctcatcttctgccacagt agggcagtgatgaggaacc 38OTTHUMG00000021416 lnc-ZNF674-1 agcacttggccctaaagaga aacatactggcccaaacagc 88OTTHUMG00000020561 lnc-TLR4-1 ccacacaaatgggcaagaat gcaaaatcctgaaggttcaaa 6lowastProbe number in the universal probe library (UPL)
Primary NPC Controls
15 times 1004
10 times 1004
50 times 1003
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0001
(a)
Primary NPC Controls
4 times 1002
3 times 1002
2 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0433
(b)
Primary NPC Controls0
4 times 1004
2 times 1004
4 times 1003
3 times 1003
2 times 1003
1 times 1003Re
lativ
e exp
ress
ion
of ln
c-A
L3551491
-1
P = 0015
(c)
Primary NPC Controls
8 times 1004
4 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0034
(d)
Figure 1 Relative expression levels of lncRNAs in tissue samples frompatientswith primaryNPCandhealthy controlsThe relative expressionlevels were normalized to GAPDH by qPCR analysis The difference between primary NPC and controls was calculated using the Mann-Whitney 119880 test
4 BioMed Research International
Table2Differentia
llyexpressedlnRN
Ain
NPC
microarraydatasetidentified
byncFA
Nsreann
otation
Vega
gene
IDLN
Cipediag
eneID
Locusc
onservationlowast
Exon
number
Transcrip
tsize(bp
)Genom
elocation
Adjust119875value
OTT
HUMG00
000153695
lnc-BC
L2L11-3
No
2757
chr2112248850-112268567+
0002
OTT
HUMG00
0000
02490
lnc-AL3551491-1
No
2342
chr116
847189-16848303+
000
4OTT
HUMG00
000150697
lnc-C2
2orf32-1
No
2544
chr2242672361-42673057+
000
6OTT
HUMG00
000021416
lnc-ZN
F674-1
No
2626
chrX
46185359-46187080minus
0030
OTT
HUMG00
000020561
lnc-TL
R4-1
Mou
se6
1299
chr912
0410884-120419305+
0033
lowast
Locusc
onservationin
Mus
musculusand
Daniorerio
comparedto
Hom
osapiens
BioMed Research International 5
100806040200
Sens
itivi
ty10
08
06
04
02
00
ROC curve
Area under curve = 0734
1 minus specificity
lnc-C22orf32-1
(a)
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
Area under curve = 0670
lnc-AL3551491-1
1 minus specificity
(b)
Area under curve = 0649
lnc-ZNF674-1
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
1 minus specificity
(c)
Figure 2 Receiver operating characteristic (ROC) curve analysis of lncRNAs in tissue samples from patients with primary NPC and healthycontrols
difference between the primary NPC and normal nasopha-ryngeal tissues
Of the 4 lncRNAs the expression difference of lnc-C22orf32-1 was highly significant (119875 = 0001 Mann-Whitney119880 test) High lnc-C22orf32-1 was found in the primary NPCtissues in comparison with the normal controls (Figure 1) InROC analysis the AUC value of lnc-C22orf32-1 was 0734 indifferentiating NPC from the normal nasopharyngeal tissues(Figure 2) lnc-AL3551491-1 and lnc-ZNF674-1 were alsofound to be overexpressed in the primary NPC tissues (119875 =0015 and 0034 respectively Mann-Whitney 119880 test) The 3lncRNAs above are detectable in all the tissues examined Incontrast lnc-TLR4-1 was detectable in 21 (942) patientswith primary NPC and 66 (1929) healthy controls Inrecurrent NPC lnc-TLR4-1 was detectable in 27 (933)tumor tissues and 15 (533) paired normal tissues Low copynumber of lnc-TLR4-1 in tissue samples may account for
the low detection rate The expression pattern of lnc-TLR4-1 was not correlated with age or gender (data not shown)Since lnc-TLR4-1 was only expressed in a subset of NPC andnormal nasopharyngeal tissues it was not suitable for studyof differential expression
Next we compared the expression level of the 3 universalexpressed lncRNAs with the clinicopathological parametersof the primary NPC patients (Table 3) High levels of lnc-C22orf32-1 and lnc-AL3551491-1 were significantly associatedwith the male patients (Figure 3(b)) In addition increasedexpression of lnc-C22orf32-1 and lnc-ZNF674-1 was associ-ated with advanced tumor stages (T2ndash4) in comparison withthe early stage (T1) NPC (Figure 3(a))
33 Differential lncRNAs Expression in Recurrent NPC and thePaired Normal Epithelia We compared the lncRNA expres-sion patterns with the paired normal tissues obtained from
6 BioMed Research International
Table 3 Association of lncRNA expression levels with the clinicopathological variables in primary undifferentiated NPC patients
lnc-ZNF674-1 lnc-AL3551491-1 lnc-C22orf32-1119875 value
Gender NumberMale 32 0108 0028lowast 0033lowastFemale 10
Agelt49 19 0733 0970 0810gt49 23
T stageT1 11 0030lowast 0062 0019lowastT2ndash4 31
Nodal statusNegative 7 0895 0692 0692Positive 35
lowast
119875 value below 005 was considered statistically significant
the same recurrent NPC patients (Figure 4) lnc-BCL2L11-3was found to be upregulated in the recurrent NPC tissuesin comparison with the paired normal tissues (119875 = 0002Wilcoxon Signed Rank test) For lnc-AL3551491-1 and lnc-ZNF674-1 high expression was found in primary NPCHowever significant expression reduction was observed inthe recurrent NPC tissues (119875 = 0022 and 0002 respectivelyWilcoxon Signed Rank test)
4 Discussion
The differential expression of lnc-AL3551491-1 lnc-C22orf32-1 and lnc-ZNF674-1 observed in NPC might beattributed to epigenetic regulation including microRNADNAmethylation and histone modification As predicted bya bioinformatics tool MirTarget2 (httpmirdborgmiRDBindexhtml) lnc-AL3551491-1 is targeted by hsa-miR-939hsa-miR-4283 and hsa-miR-3191-5p lnc-C22orf32-1 is tar-geted by hsa-miR-4751 lnc-ZNF674-1 is targeted by hsa-miR-1296 hsa-miR-1292 and hsa-miR-3179 In additionDNA methylation and histone modification may alsocontribute to the aberrant expression of these lncRNAs Forexample DNA methylation H3K4 histone methylationand H3K9 histone methylation are found in lnc-AL3551491-1 gene in different human cell lines (lncRNomehttpgenomeigibresinlncRNome)
lncRNAs could exert biological functions by interactingwith RNA or protein molecules An lncRNA highly upreg-ulated in liver cancer (HULC) could bind to miR-372 thusmodulating the expression of miR-372 at posttranscriptionallevel [13] lncRNA HOTAIR interacted with polycomb chro-matin remodeling complex 2 (PRC2) by its 51015840 domain andbind to LSD1CoRESTREST complex via its 31015840 domainresulting in a specific histone methylation pattern [14] Toexplore the possible model by which these differentially
expressed lncRNAs exert their biological functions we triedto predict their binding capacity via database lncRNomeThe nucleotides 595ndash597 of lnc-ZNF674-1 are predicted to beprotein binding sites indicating that itmay play a role inNPCby interacting with specific proteins
Primary NPC is highly sensitive to radiotherapy andthe use of concurrent chemoradiotherapy has significantlyimproved the local control rates of NPC [15] Neverthelesslocal recurrence still appears in approximately 10ndash30 ofpatients after primary radiotherapy [16] Since recurrentNPC is possibly derived from those NPC cells that couldescape from radiotherapy the recurrent NPC might exhibitdistinctive mRNA miRNA and protein expression patternsin comparison with primary NPC For instance the expres-sion levels of miR-98 TR-1205722 mRNA multidrug resistance 1(MDR-1) protein and glutathione-S-transferase-120587 (GST-120587)protein were different between recurrent NPC and primaryNPC [17 18] Likewise in this study we also observed adiverse lncRNA expression pattern between recurrent NPCand primary NPC For example the aberrant expression oflnc-BCL2L11-3 was only observed in recurrent NPC but notin primaryNPC suggesting that lncRNAsmight play distinctroles in the initiation and progression of recurrent NPC andprimary NPC
According toGlobal Cancer Statistics [19] NPC is amale-dominant disease with incidence rates usually 2 to 3 timeslower in females In our cohort the ratio between malesand females is about 3 Therefore one of the limitationsof this study is the small sample size of females Anotherconcern about our patient samples is the large age rangeTo investigate whether the large age range would affect theexpression level of lncRNAs we divided our patients into twogroups according to various cutoff values of ageThere are nosignificant differences in the expression of lncRNAs betweenthe two groups of patients divided by varied cutoff values ofage
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 3
Table 1 Primer and probe sequences used in quantitative PCR
Vega gene ID LNCipedia gene ID Forward primer (51015840-31015840) Reverse primer (51015840-31015840) UPL numberlowast
OTTHUMG00000153695 lnc-BCL2L11-3 agcagatgctgtgcctgata cctttctcgacccagaagc 67OTTHUMG00000002490 lnc-AL3551491-1 gaaaactaggcgtctgggaac caaacaatgggagcaagtcc 25OTTHUMG00000150697 lnc-C22orf32-1 tgctcatcttctgccacagt agggcagtgatgaggaacc 38OTTHUMG00000021416 lnc-ZNF674-1 agcacttggccctaaagaga aacatactggcccaaacagc 88OTTHUMG00000020561 lnc-TLR4-1 ccacacaaatgggcaagaat gcaaaatcctgaaggttcaaa 6lowastProbe number in the universal probe library (UPL)
Primary NPC Controls
15 times 1004
10 times 1004
50 times 1003
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0001
(a)
Primary NPC Controls
4 times 1002
3 times 1002
2 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0433
(b)
Primary NPC Controls0
4 times 1004
2 times 1004
4 times 1003
3 times 1003
2 times 1003
1 times 1003Re
lativ
e exp
ress
ion
of ln
c-A
L3551491
-1
P = 0015
(c)
Primary NPC Controls
8 times 1004
4 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0034
(d)
Figure 1 Relative expression levels of lncRNAs in tissue samples frompatientswith primaryNPCandhealthy controlsThe relative expressionlevels were normalized to GAPDH by qPCR analysis The difference between primary NPC and controls was calculated using the Mann-Whitney 119880 test
4 BioMed Research International
Table2Differentia
llyexpressedlnRN
Ain
NPC
microarraydatasetidentified
byncFA
Nsreann
otation
Vega
gene
IDLN
Cipediag
eneID
Locusc
onservationlowast
Exon
number
Transcrip
tsize(bp
)Genom
elocation
Adjust119875value
OTT
HUMG00
000153695
lnc-BC
L2L11-3
No
2757
chr2112248850-112268567+
0002
OTT
HUMG00
0000
02490
lnc-AL3551491-1
No
2342
chr116
847189-16848303+
000
4OTT
HUMG00
000150697
lnc-C2
2orf32-1
No
2544
chr2242672361-42673057+
000
6OTT
HUMG00
000021416
lnc-ZN
F674-1
No
2626
chrX
46185359-46187080minus
0030
OTT
HUMG00
000020561
lnc-TL
R4-1
Mou
se6
1299
chr912
0410884-120419305+
0033
lowast
Locusc
onservationin
Mus
musculusand
Daniorerio
comparedto
Hom
osapiens
BioMed Research International 5
100806040200
Sens
itivi
ty10
08
06
04
02
00
ROC curve
Area under curve = 0734
1 minus specificity
lnc-C22orf32-1
(a)
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
Area under curve = 0670
lnc-AL3551491-1
1 minus specificity
(b)
Area under curve = 0649
lnc-ZNF674-1
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
1 minus specificity
(c)
Figure 2 Receiver operating characteristic (ROC) curve analysis of lncRNAs in tissue samples from patients with primary NPC and healthycontrols
difference between the primary NPC and normal nasopha-ryngeal tissues
Of the 4 lncRNAs the expression difference of lnc-C22orf32-1 was highly significant (119875 = 0001 Mann-Whitney119880 test) High lnc-C22orf32-1 was found in the primary NPCtissues in comparison with the normal controls (Figure 1) InROC analysis the AUC value of lnc-C22orf32-1 was 0734 indifferentiating NPC from the normal nasopharyngeal tissues(Figure 2) lnc-AL3551491-1 and lnc-ZNF674-1 were alsofound to be overexpressed in the primary NPC tissues (119875 =0015 and 0034 respectively Mann-Whitney 119880 test) The 3lncRNAs above are detectable in all the tissues examined Incontrast lnc-TLR4-1 was detectable in 21 (942) patientswith primary NPC and 66 (1929) healthy controls Inrecurrent NPC lnc-TLR4-1 was detectable in 27 (933)tumor tissues and 15 (533) paired normal tissues Low copynumber of lnc-TLR4-1 in tissue samples may account for
the low detection rate The expression pattern of lnc-TLR4-1 was not correlated with age or gender (data not shown)Since lnc-TLR4-1 was only expressed in a subset of NPC andnormal nasopharyngeal tissues it was not suitable for studyof differential expression
Next we compared the expression level of the 3 universalexpressed lncRNAs with the clinicopathological parametersof the primary NPC patients (Table 3) High levels of lnc-C22orf32-1 and lnc-AL3551491-1 were significantly associatedwith the male patients (Figure 3(b)) In addition increasedexpression of lnc-C22orf32-1 and lnc-ZNF674-1 was associ-ated with advanced tumor stages (T2ndash4) in comparison withthe early stage (T1) NPC (Figure 3(a))
33 Differential lncRNAs Expression in Recurrent NPC and thePaired Normal Epithelia We compared the lncRNA expres-sion patterns with the paired normal tissues obtained from
6 BioMed Research International
Table 3 Association of lncRNA expression levels with the clinicopathological variables in primary undifferentiated NPC patients
lnc-ZNF674-1 lnc-AL3551491-1 lnc-C22orf32-1119875 value
Gender NumberMale 32 0108 0028lowast 0033lowastFemale 10
Agelt49 19 0733 0970 0810gt49 23
T stageT1 11 0030lowast 0062 0019lowastT2ndash4 31
Nodal statusNegative 7 0895 0692 0692Positive 35
lowast
119875 value below 005 was considered statistically significant
the same recurrent NPC patients (Figure 4) lnc-BCL2L11-3was found to be upregulated in the recurrent NPC tissuesin comparison with the paired normal tissues (119875 = 0002Wilcoxon Signed Rank test) For lnc-AL3551491-1 and lnc-ZNF674-1 high expression was found in primary NPCHowever significant expression reduction was observed inthe recurrent NPC tissues (119875 = 0022 and 0002 respectivelyWilcoxon Signed Rank test)
4 Discussion
The differential expression of lnc-AL3551491-1 lnc-C22orf32-1 and lnc-ZNF674-1 observed in NPC might beattributed to epigenetic regulation including microRNADNAmethylation and histone modification As predicted bya bioinformatics tool MirTarget2 (httpmirdborgmiRDBindexhtml) lnc-AL3551491-1 is targeted by hsa-miR-939hsa-miR-4283 and hsa-miR-3191-5p lnc-C22orf32-1 is tar-geted by hsa-miR-4751 lnc-ZNF674-1 is targeted by hsa-miR-1296 hsa-miR-1292 and hsa-miR-3179 In additionDNA methylation and histone modification may alsocontribute to the aberrant expression of these lncRNAs Forexample DNA methylation H3K4 histone methylationand H3K9 histone methylation are found in lnc-AL3551491-1 gene in different human cell lines (lncRNomehttpgenomeigibresinlncRNome)
lncRNAs could exert biological functions by interactingwith RNA or protein molecules An lncRNA highly upreg-ulated in liver cancer (HULC) could bind to miR-372 thusmodulating the expression of miR-372 at posttranscriptionallevel [13] lncRNA HOTAIR interacted with polycomb chro-matin remodeling complex 2 (PRC2) by its 51015840 domain andbind to LSD1CoRESTREST complex via its 31015840 domainresulting in a specific histone methylation pattern [14] Toexplore the possible model by which these differentially
expressed lncRNAs exert their biological functions we triedto predict their binding capacity via database lncRNomeThe nucleotides 595ndash597 of lnc-ZNF674-1 are predicted to beprotein binding sites indicating that itmay play a role inNPCby interacting with specific proteins
Primary NPC is highly sensitive to radiotherapy andthe use of concurrent chemoradiotherapy has significantlyimproved the local control rates of NPC [15] Neverthelesslocal recurrence still appears in approximately 10ndash30 ofpatients after primary radiotherapy [16] Since recurrentNPC is possibly derived from those NPC cells that couldescape from radiotherapy the recurrent NPC might exhibitdistinctive mRNA miRNA and protein expression patternsin comparison with primary NPC For instance the expres-sion levels of miR-98 TR-1205722 mRNA multidrug resistance 1(MDR-1) protein and glutathione-S-transferase-120587 (GST-120587)protein were different between recurrent NPC and primaryNPC [17 18] Likewise in this study we also observed adiverse lncRNA expression pattern between recurrent NPCand primary NPC For example the aberrant expression oflnc-BCL2L11-3 was only observed in recurrent NPC but notin primaryNPC suggesting that lncRNAsmight play distinctroles in the initiation and progression of recurrent NPC andprimary NPC
According toGlobal Cancer Statistics [19] NPC is amale-dominant disease with incidence rates usually 2 to 3 timeslower in females In our cohort the ratio between malesand females is about 3 Therefore one of the limitationsof this study is the small sample size of females Anotherconcern about our patient samples is the large age rangeTo investigate whether the large age range would affect theexpression level of lncRNAs we divided our patients into twogroups according to various cutoff values of ageThere are nosignificant differences in the expression of lncRNAs betweenthe two groups of patients divided by varied cutoff values ofage
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 BioMed Research International
Table2Differentia
llyexpressedlnRN
Ain
NPC
microarraydatasetidentified
byncFA
Nsreann
otation
Vega
gene
IDLN
Cipediag
eneID
Locusc
onservationlowast
Exon
number
Transcrip
tsize(bp
)Genom
elocation
Adjust119875value
OTT
HUMG00
000153695
lnc-BC
L2L11-3
No
2757
chr2112248850-112268567+
0002
OTT
HUMG00
0000
02490
lnc-AL3551491-1
No
2342
chr116
847189-16848303+
000
4OTT
HUMG00
000150697
lnc-C2
2orf32-1
No
2544
chr2242672361-42673057+
000
6OTT
HUMG00
000021416
lnc-ZN
F674-1
No
2626
chrX
46185359-46187080minus
0030
OTT
HUMG00
000020561
lnc-TL
R4-1
Mou
se6
1299
chr912
0410884-120419305+
0033
lowast
Locusc
onservationin
Mus
musculusand
Daniorerio
comparedto
Hom
osapiens
BioMed Research International 5
100806040200
Sens
itivi
ty10
08
06
04
02
00
ROC curve
Area under curve = 0734
1 minus specificity
lnc-C22orf32-1
(a)
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
Area under curve = 0670
lnc-AL3551491-1
1 minus specificity
(b)
Area under curve = 0649
lnc-ZNF674-1
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
1 minus specificity
(c)
Figure 2 Receiver operating characteristic (ROC) curve analysis of lncRNAs in tissue samples from patients with primary NPC and healthycontrols
difference between the primary NPC and normal nasopha-ryngeal tissues
Of the 4 lncRNAs the expression difference of lnc-C22orf32-1 was highly significant (119875 = 0001 Mann-Whitney119880 test) High lnc-C22orf32-1 was found in the primary NPCtissues in comparison with the normal controls (Figure 1) InROC analysis the AUC value of lnc-C22orf32-1 was 0734 indifferentiating NPC from the normal nasopharyngeal tissues(Figure 2) lnc-AL3551491-1 and lnc-ZNF674-1 were alsofound to be overexpressed in the primary NPC tissues (119875 =0015 and 0034 respectively Mann-Whitney 119880 test) The 3lncRNAs above are detectable in all the tissues examined Incontrast lnc-TLR4-1 was detectable in 21 (942) patientswith primary NPC and 66 (1929) healthy controls Inrecurrent NPC lnc-TLR4-1 was detectable in 27 (933)tumor tissues and 15 (533) paired normal tissues Low copynumber of lnc-TLR4-1 in tissue samples may account for
the low detection rate The expression pattern of lnc-TLR4-1 was not correlated with age or gender (data not shown)Since lnc-TLR4-1 was only expressed in a subset of NPC andnormal nasopharyngeal tissues it was not suitable for studyof differential expression
Next we compared the expression level of the 3 universalexpressed lncRNAs with the clinicopathological parametersof the primary NPC patients (Table 3) High levels of lnc-C22orf32-1 and lnc-AL3551491-1 were significantly associatedwith the male patients (Figure 3(b)) In addition increasedexpression of lnc-C22orf32-1 and lnc-ZNF674-1 was associ-ated with advanced tumor stages (T2ndash4) in comparison withthe early stage (T1) NPC (Figure 3(a))
33 Differential lncRNAs Expression in Recurrent NPC and thePaired Normal Epithelia We compared the lncRNA expres-sion patterns with the paired normal tissues obtained from
6 BioMed Research International
Table 3 Association of lncRNA expression levels with the clinicopathological variables in primary undifferentiated NPC patients
lnc-ZNF674-1 lnc-AL3551491-1 lnc-C22orf32-1119875 value
Gender NumberMale 32 0108 0028lowast 0033lowastFemale 10
Agelt49 19 0733 0970 0810gt49 23
T stageT1 11 0030lowast 0062 0019lowastT2ndash4 31
Nodal statusNegative 7 0895 0692 0692Positive 35
lowast
119875 value below 005 was considered statistically significant
the same recurrent NPC patients (Figure 4) lnc-BCL2L11-3was found to be upregulated in the recurrent NPC tissuesin comparison with the paired normal tissues (119875 = 0002Wilcoxon Signed Rank test) For lnc-AL3551491-1 and lnc-ZNF674-1 high expression was found in primary NPCHowever significant expression reduction was observed inthe recurrent NPC tissues (119875 = 0022 and 0002 respectivelyWilcoxon Signed Rank test)
4 Discussion
The differential expression of lnc-AL3551491-1 lnc-C22orf32-1 and lnc-ZNF674-1 observed in NPC might beattributed to epigenetic regulation including microRNADNAmethylation and histone modification As predicted bya bioinformatics tool MirTarget2 (httpmirdborgmiRDBindexhtml) lnc-AL3551491-1 is targeted by hsa-miR-939hsa-miR-4283 and hsa-miR-3191-5p lnc-C22orf32-1 is tar-geted by hsa-miR-4751 lnc-ZNF674-1 is targeted by hsa-miR-1296 hsa-miR-1292 and hsa-miR-3179 In additionDNA methylation and histone modification may alsocontribute to the aberrant expression of these lncRNAs Forexample DNA methylation H3K4 histone methylationand H3K9 histone methylation are found in lnc-AL3551491-1 gene in different human cell lines (lncRNomehttpgenomeigibresinlncRNome)
lncRNAs could exert biological functions by interactingwith RNA or protein molecules An lncRNA highly upreg-ulated in liver cancer (HULC) could bind to miR-372 thusmodulating the expression of miR-372 at posttranscriptionallevel [13] lncRNA HOTAIR interacted with polycomb chro-matin remodeling complex 2 (PRC2) by its 51015840 domain andbind to LSD1CoRESTREST complex via its 31015840 domainresulting in a specific histone methylation pattern [14] Toexplore the possible model by which these differentially
expressed lncRNAs exert their biological functions we triedto predict their binding capacity via database lncRNomeThe nucleotides 595ndash597 of lnc-ZNF674-1 are predicted to beprotein binding sites indicating that itmay play a role inNPCby interacting with specific proteins
Primary NPC is highly sensitive to radiotherapy andthe use of concurrent chemoradiotherapy has significantlyimproved the local control rates of NPC [15] Neverthelesslocal recurrence still appears in approximately 10ndash30 ofpatients after primary radiotherapy [16] Since recurrentNPC is possibly derived from those NPC cells that couldescape from radiotherapy the recurrent NPC might exhibitdistinctive mRNA miRNA and protein expression patternsin comparison with primary NPC For instance the expres-sion levels of miR-98 TR-1205722 mRNA multidrug resistance 1(MDR-1) protein and glutathione-S-transferase-120587 (GST-120587)protein were different between recurrent NPC and primaryNPC [17 18] Likewise in this study we also observed adiverse lncRNA expression pattern between recurrent NPCand primary NPC For example the aberrant expression oflnc-BCL2L11-3 was only observed in recurrent NPC but notin primaryNPC suggesting that lncRNAsmight play distinctroles in the initiation and progression of recurrent NPC andprimary NPC
According toGlobal Cancer Statistics [19] NPC is amale-dominant disease with incidence rates usually 2 to 3 timeslower in females In our cohort the ratio between malesand females is about 3 Therefore one of the limitationsof this study is the small sample size of females Anotherconcern about our patient samples is the large age rangeTo investigate whether the large age range would affect theexpression level of lncRNAs we divided our patients into twogroups according to various cutoff values of ageThere are nosignificant differences in the expression of lncRNAs betweenthe two groups of patients divided by varied cutoff values ofage
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 5
100806040200
Sens
itivi
ty10
08
06
04
02
00
ROC curve
Area under curve = 0734
1 minus specificity
lnc-C22orf32-1
(a)
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
Area under curve = 0670
lnc-AL3551491-1
1 minus specificity
(b)
Area under curve = 0649
lnc-ZNF674-1
100806040200
Sens
itivi
ty
10
08
06
04
02
00
ROC curve
1 minus specificity
(c)
Figure 2 Receiver operating characteristic (ROC) curve analysis of lncRNAs in tissue samples from patients with primary NPC and healthycontrols
difference between the primary NPC and normal nasopha-ryngeal tissues
Of the 4 lncRNAs the expression difference of lnc-C22orf32-1 was highly significant (119875 = 0001 Mann-Whitney119880 test) High lnc-C22orf32-1 was found in the primary NPCtissues in comparison with the normal controls (Figure 1) InROC analysis the AUC value of lnc-C22orf32-1 was 0734 indifferentiating NPC from the normal nasopharyngeal tissues(Figure 2) lnc-AL3551491-1 and lnc-ZNF674-1 were alsofound to be overexpressed in the primary NPC tissues (119875 =0015 and 0034 respectively Mann-Whitney 119880 test) The 3lncRNAs above are detectable in all the tissues examined Incontrast lnc-TLR4-1 was detectable in 21 (942) patientswith primary NPC and 66 (1929) healthy controls Inrecurrent NPC lnc-TLR4-1 was detectable in 27 (933)tumor tissues and 15 (533) paired normal tissues Low copynumber of lnc-TLR4-1 in tissue samples may account for
the low detection rate The expression pattern of lnc-TLR4-1 was not correlated with age or gender (data not shown)Since lnc-TLR4-1 was only expressed in a subset of NPC andnormal nasopharyngeal tissues it was not suitable for studyof differential expression
Next we compared the expression level of the 3 universalexpressed lncRNAs with the clinicopathological parametersof the primary NPC patients (Table 3) High levels of lnc-C22orf32-1 and lnc-AL3551491-1 were significantly associatedwith the male patients (Figure 3(b)) In addition increasedexpression of lnc-C22orf32-1 and lnc-ZNF674-1 was associ-ated with advanced tumor stages (T2ndash4) in comparison withthe early stage (T1) NPC (Figure 3(a))
33 Differential lncRNAs Expression in Recurrent NPC and thePaired Normal Epithelia We compared the lncRNA expres-sion patterns with the paired normal tissues obtained from
6 BioMed Research International
Table 3 Association of lncRNA expression levels with the clinicopathological variables in primary undifferentiated NPC patients
lnc-ZNF674-1 lnc-AL3551491-1 lnc-C22orf32-1119875 value
Gender NumberMale 32 0108 0028lowast 0033lowastFemale 10
Agelt49 19 0733 0970 0810gt49 23
T stageT1 11 0030lowast 0062 0019lowastT2ndash4 31
Nodal statusNegative 7 0895 0692 0692Positive 35
lowast
119875 value below 005 was considered statistically significant
the same recurrent NPC patients (Figure 4) lnc-BCL2L11-3was found to be upregulated in the recurrent NPC tissuesin comparison with the paired normal tissues (119875 = 0002Wilcoxon Signed Rank test) For lnc-AL3551491-1 and lnc-ZNF674-1 high expression was found in primary NPCHowever significant expression reduction was observed inthe recurrent NPC tissues (119875 = 0022 and 0002 respectivelyWilcoxon Signed Rank test)
4 Discussion
The differential expression of lnc-AL3551491-1 lnc-C22orf32-1 and lnc-ZNF674-1 observed in NPC might beattributed to epigenetic regulation including microRNADNAmethylation and histone modification As predicted bya bioinformatics tool MirTarget2 (httpmirdborgmiRDBindexhtml) lnc-AL3551491-1 is targeted by hsa-miR-939hsa-miR-4283 and hsa-miR-3191-5p lnc-C22orf32-1 is tar-geted by hsa-miR-4751 lnc-ZNF674-1 is targeted by hsa-miR-1296 hsa-miR-1292 and hsa-miR-3179 In additionDNA methylation and histone modification may alsocontribute to the aberrant expression of these lncRNAs Forexample DNA methylation H3K4 histone methylationand H3K9 histone methylation are found in lnc-AL3551491-1 gene in different human cell lines (lncRNomehttpgenomeigibresinlncRNome)
lncRNAs could exert biological functions by interactingwith RNA or protein molecules An lncRNA highly upreg-ulated in liver cancer (HULC) could bind to miR-372 thusmodulating the expression of miR-372 at posttranscriptionallevel [13] lncRNA HOTAIR interacted with polycomb chro-matin remodeling complex 2 (PRC2) by its 51015840 domain andbind to LSD1CoRESTREST complex via its 31015840 domainresulting in a specific histone methylation pattern [14] Toexplore the possible model by which these differentially
expressed lncRNAs exert their biological functions we triedto predict their binding capacity via database lncRNomeThe nucleotides 595ndash597 of lnc-ZNF674-1 are predicted to beprotein binding sites indicating that itmay play a role inNPCby interacting with specific proteins
Primary NPC is highly sensitive to radiotherapy andthe use of concurrent chemoradiotherapy has significantlyimproved the local control rates of NPC [15] Neverthelesslocal recurrence still appears in approximately 10ndash30 ofpatients after primary radiotherapy [16] Since recurrentNPC is possibly derived from those NPC cells that couldescape from radiotherapy the recurrent NPC might exhibitdistinctive mRNA miRNA and protein expression patternsin comparison with primary NPC For instance the expres-sion levels of miR-98 TR-1205722 mRNA multidrug resistance 1(MDR-1) protein and glutathione-S-transferase-120587 (GST-120587)protein were different between recurrent NPC and primaryNPC [17 18] Likewise in this study we also observed adiverse lncRNA expression pattern between recurrent NPCand primary NPC For example the aberrant expression oflnc-BCL2L11-3 was only observed in recurrent NPC but notin primaryNPC suggesting that lncRNAsmight play distinctroles in the initiation and progression of recurrent NPC andprimary NPC
According toGlobal Cancer Statistics [19] NPC is amale-dominant disease with incidence rates usually 2 to 3 timeslower in females In our cohort the ratio between malesand females is about 3 Therefore one of the limitationsof this study is the small sample size of females Anotherconcern about our patient samples is the large age rangeTo investigate whether the large age range would affect theexpression level of lncRNAs we divided our patients into twogroups according to various cutoff values of ageThere are nosignificant differences in the expression of lncRNAs betweenthe two groups of patients divided by varied cutoff values ofage
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 BioMed Research International
Table 3 Association of lncRNA expression levels with the clinicopathological variables in primary undifferentiated NPC patients
lnc-ZNF674-1 lnc-AL3551491-1 lnc-C22orf32-1119875 value
Gender NumberMale 32 0108 0028lowast 0033lowastFemale 10
Agelt49 19 0733 0970 0810gt49 23
T stageT1 11 0030lowast 0062 0019lowastT2ndash4 31
Nodal statusNegative 7 0895 0692 0692Positive 35
lowast
119875 value below 005 was considered statistically significant
the same recurrent NPC patients (Figure 4) lnc-BCL2L11-3was found to be upregulated in the recurrent NPC tissuesin comparison with the paired normal tissues (119875 = 0002Wilcoxon Signed Rank test) For lnc-AL3551491-1 and lnc-ZNF674-1 high expression was found in primary NPCHowever significant expression reduction was observed inthe recurrent NPC tissues (119875 = 0022 and 0002 respectivelyWilcoxon Signed Rank test)
4 Discussion
The differential expression of lnc-AL3551491-1 lnc-C22orf32-1 and lnc-ZNF674-1 observed in NPC might beattributed to epigenetic regulation including microRNADNAmethylation and histone modification As predicted bya bioinformatics tool MirTarget2 (httpmirdborgmiRDBindexhtml) lnc-AL3551491-1 is targeted by hsa-miR-939hsa-miR-4283 and hsa-miR-3191-5p lnc-C22orf32-1 is tar-geted by hsa-miR-4751 lnc-ZNF674-1 is targeted by hsa-miR-1296 hsa-miR-1292 and hsa-miR-3179 In additionDNA methylation and histone modification may alsocontribute to the aberrant expression of these lncRNAs Forexample DNA methylation H3K4 histone methylationand H3K9 histone methylation are found in lnc-AL3551491-1 gene in different human cell lines (lncRNomehttpgenomeigibresinlncRNome)
lncRNAs could exert biological functions by interactingwith RNA or protein molecules An lncRNA highly upreg-ulated in liver cancer (HULC) could bind to miR-372 thusmodulating the expression of miR-372 at posttranscriptionallevel [13] lncRNA HOTAIR interacted with polycomb chro-matin remodeling complex 2 (PRC2) by its 51015840 domain andbind to LSD1CoRESTREST complex via its 31015840 domainresulting in a specific histone methylation pattern [14] Toexplore the possible model by which these differentially
expressed lncRNAs exert their biological functions we triedto predict their binding capacity via database lncRNomeThe nucleotides 595ndash597 of lnc-ZNF674-1 are predicted to beprotein binding sites indicating that itmay play a role inNPCby interacting with specific proteins
Primary NPC is highly sensitive to radiotherapy andthe use of concurrent chemoradiotherapy has significantlyimproved the local control rates of NPC [15] Neverthelesslocal recurrence still appears in approximately 10ndash30 ofpatients after primary radiotherapy [16] Since recurrentNPC is possibly derived from those NPC cells that couldescape from radiotherapy the recurrent NPC might exhibitdistinctive mRNA miRNA and protein expression patternsin comparison with primary NPC For instance the expres-sion levels of miR-98 TR-1205722 mRNA multidrug resistance 1(MDR-1) protein and glutathione-S-transferase-120587 (GST-120587)protein were different between recurrent NPC and primaryNPC [17 18] Likewise in this study we also observed adiverse lncRNA expression pattern between recurrent NPCand primary NPC For example the aberrant expression oflnc-BCL2L11-3 was only observed in recurrent NPC but notin primaryNPC suggesting that lncRNAsmight play distinctroles in the initiation and progression of recurrent NPC andprimary NPC
According toGlobal Cancer Statistics [19] NPC is amale-dominant disease with incidence rates usually 2 to 3 timeslower in females In our cohort the ratio between malesand females is about 3 Therefore one of the limitationsof this study is the small sample size of females Anotherconcern about our patient samples is the large age rangeTo investigate whether the large age range would affect theexpression level of lncRNAs we divided our patients into twogroups according to various cutoff values of ageThere are nosignificant differences in the expression of lncRNAs betweenthe two groups of patients divided by varied cutoff values ofage
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 7
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
8 times 1004
4 times 1004
20 times 1003
15 times 1003
10 times 1003
50 times 1002
0 00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0019 P = 0030
T1 T2-4T1 T2-4
(a)
Male Female
15 times 1004
10 times 1004
50 times 1003
1 times 1003
8 times 1002
6 times 1002
4 times 1002
2 times 1002
0
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0033
Male Female
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1P = 0028
(b)
Figure 3 Relative expression levels of lncRNAs in primary NPC patients with different T stage (a) and gender (b) The relative expressionlevels were normalized to GAPDH by qPCR analysis The difference was calculated using the Mann-Whitney 119880 test
The differential lncRNA expression patterns could berepresented by the microarray probsets which were orig-inally intended to map the somatic gene expression levelThis approach has proven successful in identifying the alteredlncRNA expression in the nucleus accumbens of heroinabusers Michelhaugh SK reanalyzed the existing Affymetrixmicroarray dataset and identified the 5 lncRNAs dysregulatedin the heroin abusers [10] They curated existing RNAdatabases including RNAdb and H-Invitational to generatea list of lncRNA and later matched them with the probeseton the Affymetrix U133A and U133B chips [10] Using similarapproaches we demonstrated that the differential lncRNA
expression data could possibly be retrieved from the existingmicroarray dataset for use to identify candidate lncRNAwhich is implicated in the pathogenesis of NPC
5 Conclusions
Our results demonstrated that it is feasible to identify thedifferentially expressed lncRNA in the microarray datasetby functional reannotation The association of lncRNA withgender and tumor size implicated that lncRNA possiblyplays a part in the pathogenesis of primary NPC Further
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 BioMed Research International
8 times 1002
4 times 1002
15 times 1002
10 times 1002
50 times 1001
00
Relat
ive e
xpre
ssio
n of
lnc-
C22
orf32
-1
P = 0837
Recurrent NPCn = 33n = 33
Paired normal
(a)
25 times 1002
20 times 1002
15 times 1002
1 times 1002
5 times 1001
0
Relat
ive e
xpre
ssio
n of
lnc-
BCL2
L11
-3
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(b)
6 times 1004
4 times 1004
2 times 1004
5 times 1003
4 times 1003
3 times 1003
2 times 1003
1 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
AL3551491
-1
P = 0022
Recurrent NPCn = 33n = 33
Paired normal
(c)
2 times 1005
1 times 1005
1 times 1004
8 times 1003
6 times 1003
4 times 1003
2 times 1003
0
Relat
ive e
xpre
ssio
n of
lnc-
ZNF6
74
-1
P = 0002
Recurrent NPCn = 33n = 33
Paired normal
(d)
Figure 4 Relative expression levels of lncRNAs in paired tumor and normal tissue samples from patients with recurrent NPC The relativeexpression levels were normalized toGAPDH by qPCR analysisThe difference between tumor and normal was calculated using theWilcoxonSigned Rank test
the distinctive lncRNA identified in the recurrent NPCmay reveal a distinctive development mechanism underlyingtumor recurrence Further studies are warranted to explorethe pathological significance of lncRNA in the developmentof NPC
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This study was supported by Seed Funding of Basic ResearchThe University of Hong Kong and Hong Kong Area ofExcellent Scheme Hong Kong UGC
References
[1] E T Chang and H-O Adami ldquoThe enigmatic epidemiology ofnasopharyngeal carcinomardquo Cancer Epidemiology Biomarkersand Prevention vol 15 no 10 pp 1765ndash1777 2006
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
BioMed Research International 9
[2] J E Marks J L Phillips and H R Menck ldquoThe national database report on the relationship of race and national origin tothe histology of nasopharyngeal carcinomardquoCancer vol 83 pp582ndash588 1998
[3] W I Wei and J S T Sham ldquoNasopharyngeal carcinomardquo TheLancet vol 365 no 9476 pp 2041ndash2054 2005
[4] W I Wei J Y W Chan R W M Ng and W K HoldquoSurgical salvage of persistent or recurrent nasopharyngealcarcinoma with maxillary swing approachmdashcritical appraisalafter 2 decadesrdquoHead and Neck vol 33 no 7 pp 969ndash975 2011
[5] M Huarte and J L Rinn ldquoLarge non-coding RNAs missinglinks in cancerrdquo Human Molecular Genetics vol 19 no 2 ppR152ndashR161 2010
[6] J R Prensner and A M Chinnaiyan ldquoThe emergence oflncRNAs in cancer biologyrdquo Cancer Discovery vol 1 no 5 pp391ndash407 2011
[7] R J Taft K C Pang T R Mercer M Dinger and J S MattickldquoNon-coding RNAs regulators of diseaserdquo Journal of Pathologyvol 220 no 2 pp 126ndash139 2010
[8] R A Gupta N Shah K C Wang et al ldquoLong non-codingRNA HOTAIR reprograms chromatin state to promote cancermetastasisrdquo Nature vol 464 no 7291 pp 1071ndash1076 2010
[9] Y Nie X Liu S Qu E Song H Zou and C Gong ldquoLong non-coding RNAHOTAIR is an independent prognostic marker fornasopharyngeal carcinoma progression and survivalrdquo CancerScience vol 104 no 4 pp 458ndash464 2013
[10] S KMichelhaugh L Lipovich J Blythe H Jia G Kapatos andM J Bannon ldquoMining affymetrixmicroarray data for long non-coding RNAs altered expression in the nucleus accumbens ofheroin abusersrdquo Journal of Neurochemistry vol 116 no 3 pp459ndash466 2011
[11] Q Liao H Xiao D Bu et al ldquoNcFANs a web server forfunctional annotation of long non-coding RNAsrdquoNucleic AcidsResearch vol 39 no 2 pp W118ndashW124 2011
[12] L E Dodd S Sengupta I-H Chen et al ldquoGenes involved inDNA repair and nitrosami ne metabolism and those locatedon chromosome 14q32 are dysregulated in nasopharyngealcarcinomardquo Cancer Epidemiology Biomarkers and Preventionvol 15 no 11 pp 2216ndash2225 2006
[13] J Wang X Liu H Wu et al ldquoCREB up-regulates longnon-coding RNA HULC expression through interaction withmicroRNA-372 in liver cancerrdquo Nucleic Acids Research vol 38no 16 pp 5366ndash5383 2010
[14] M-C Tsai O Manor Y Wan et al ldquoLong noncoding RNA asmodular scaffold of histone modification complexesrdquo Sciencevol 329 no 5992 pp 689ndash693 2010
[15] S-P Hao and N-M Tsang ldquoSurgical management of recurrentnasopharyngeal carcinomardquo Chang Gung Medical Journal vol33 no 4 pp 361ndash369 2010
[16] D T T Chua J S T Sham D L W Kwong W I WeiG K H Au and D Choy ldquoLocally recurrent nasopharyn-geal carcinoma treatment results for patients with computedtomography assessmentrdquo International Journal of RadiationOncology Biology Physics vol 41 no 2 pp 379ndash386 1998
[17] N M Alajez W Shi A B Y Hui et al ldquoEnhancer of Zestehomolog 2 (EZH2) is overexpressed in recurrent nasopharyn-geal carcinoma and is regulated by miR-26a miR-101 and miR-98rdquo Cell Death and Disease vol 1 no 10 article e85 2010
[18] C-L Chen T-S Sheen I-U Lou and A-C Huang ldquoExpres-sion of multidrug resistance 1 and glutathione-S-transferase-120587protein in nasopharyngeal carcinomardquo Human Pathology vol32 no 11 pp 1240ndash1244 2001
[19] A Jemal F Bray M M Center J Ferlay E Ward andD Forman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
