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Prevention and Epidemiology
A Sequence Polymorphism in miR-608 Predicts Recurrenceafter
Radiotherapy for Nasopharyngeal Carcinoma
Jian Zheng1, Jieqiong Deng1, Mang Xiao4, Lei Yang5, Liyuan
Zhang2, Yonghe You1, Min Hu3, Na Li1,Hongchun Wu1, Wei Li1, Jiachun
Lu5, and Yifeng Zhou1
AbstractNasopharyngeal carcinoma is treated with radiotherapy
and other modalities, but there is little infor-
mation on individual genetic factors to help predict and improve
patient outcomes. Single-nucleotidepolymorphisms (SNP) in mature
microRNA (miRNA) sequences have the potential to exert broad impact
asmiRNAs target many mRNAs. The aim of this study was to evaluate
the effects of SNPs in mature miRNAsequences on clinical outcome in
patients with nasopharyngeal carcinoma receiving radiotherapy.
Inparticular, we analyzed associations between seven SNPs and
nasopharyngeal carcinoma locoregionalrecurrence (LRR) in 837
patients from eastern China, validating the findings in an
additional 828 patientsfrom southern China. We found that miR-608
rs4919510C>G exhibited a consistent association with LRR inthe
discovery set [HR, 2.05; 95% confidence interval (CI), 1.35–3.21],
the validation set (HR, 2.24; 95% CI, 1.45–3.38), and the combined
dataset (HR, 2.08; 95% CI, 1.41–3.26). Biochemical investigations
showed thatrs4919510C>G affects expression of miR-608 target
genes along with nasopharyngeal carcinoma cell growthafter
irradiation in vivo and in vitro. Notably, X-ray radiation induced
more chromatid breaks in lymphocytecells from rs4919510CC carriers
than in those from subjects with other genotypes (P ¼ 0.0024). Our
findingsreveal rs4919510C>G in miR-608 as a simple marker to
predict LRR in patients with radiotherapy-treatednasopharyngeal
carcinoma. Cancer Res; 73(16); 5151–62. �2013 AACR.
IntroductionNasopharyngeal carcinoma is one of the most
common
head and neck malignancies in South China and SouthAsia, where
the incidence is as high as 50 of 100,000, but itis rare in the
Western world (1 of 100,000; refs. 1–3).Although significant
progress has been made in the diag-nosis and treatment of
nasopharyngeal carcinoma in recentdecades, it remains a highly
frequent cause of cancer-related death in China. The worldwide
5-year overall sur-vival (OS) rate ranges from 32% to 62% among a
series ofstudies involving more than 9,500 patients in all stages
ofnasopharyngeal carcinoma (4). The main cause of deathamong
patients with nasopharyngeal carcinoma is recur-
rence, and 80% of all recurrences occur during the first 3years
after pathogenesis (4). Currently, radiotherapy is themain
treatment modality for this malignancy; however,differences in
individual sensitivity to radiotherapy havea great impact on the
recurrence rate of nasopharyngealcarcinoma (5).
microRNAs (miRNA) comprise a group of
endogenous,single-stranded, small, noncoding RNAs that have emerged
askey regulators of fundamental biologic processes via theircontrol
over the expression of more than 30% of human genes(6, 7). mRNAs
are initially transcribed as primary miRNAs (pri-miRNA) with
several hundred nucleotides that are furtherprocessed into
hairpin-structured precursor miRNAs (pre-miRNA) and then
intomaturemiRNAs (8–10). MaturemiRNAsconsist of approximately 22 to
25 nucleotides. To date, morethan 1,000 miRNAs have been detected
in humans (11, 12).Physiologically, miRNAs act as negative gene
regulators thatfine-tune translational output through targeted mRNA
bind-ing. A variety of pathologic associations have been attributed
toaltered miRNA networks, particularly in cancer, because miR-NAs
can function as both oncogenic and tumor suppressorfactors
(13–15).
Genome-wide association studies (GWAS) have identifiedseveral
single-nucleotide polymorphisms (SNP) related tonasopharyngeal
carcinoma susceptibility (1, 16). To date,candidate gene approaches
remain the primary strategy usedin association studies of clinical
outcomes. Genetic variants,such as SNPs in miRNAs, can affect their
biogenesis, proces-sing, and target site binding in a variety of
ways (10). A SNP in
Authors' Affiliations: 1Laboratory of Cancer Molecular Genetics,
MedicalCollege of SoochowUniversity; Departments of 2Radiotherapy
&Oncologyand 3Obstetrics and Gynecology, The Second Affiliated
Hospital of Soo-chow University, Suzhou; 4Department of
Otorhinolaryngology-Head andNeck Surgery, Sir Run Run Shaw
Hospital, Zhejiang University, Hangzhou;and 5The Institute for
Chemical Carcinogenesis, The State Key Lab ofRespiratory Disease,
Guangzhou Medical University, Guangzhou, China
Note: Supplementary data for this article are available at
Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
J. Zheng and J. Deng contributed equally to this work.
Corresponding Author: Yifeng Zhou, Medical College of Soochow
Uni-versity, No.199 Ren-ai Rd., Suzhou 215123, China. Phone:
86-512-65884720; Fax: 86-512-65884720; E-mail:
[email protected]
doi: 10.1158/0008-5472.CAN-13-0395
�2013 American Association for Cancer Research.
CancerResearch
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themature sequence can alter target site interactions by
eitherstrengthening or weakening hybridization kinetics, and
SNPscan significantly transform the target library of the
miRNAitself. Numerous studies have linked genetic variation
inmature miRNA sequences to cancer risk and prognosis (17–19).
Nonetheless, to the best of our knowledge, the importanceof mature
miRNA sequence SNPs in the locoregional recur-rence (LRR; ref. 20)
of nasopharyngeal carcinoma after radio-therapy remains unknown. In
this study, we evaluated thefrequencies of mature miRNA sequence
SNPs in patients withnasopharyngeal carcinoma and assessed their
impact on LRRafter radiotherapy.
Materials and MethodsStudy population
A total of 1,665 patients with histologically confirmed
naso-pharyngeal carcinoma [International Classification of
Disease(ICD) 9:147, ICD10:C11] were recruited from Jiangsu
Province
in eastern China [The First Affiliated Hospital of
SoochowUniversity (Suzhou, China), The Second Affiliated Hospital
ofSoochow University (Suzhou, China), The Third Hospital
Affil-iated to Nantong University (Wuxi, China), and Huaian
No.1Hospital (Huaian, China)] as well as Guangzhou City in
south-ern China (The Tumor Hospitals affiliated to GuangzhouMedical
College, Guangzhou, China) between 2000 and 2009and were followed
up until 2012 (Table 1). All patients weretreated with definitive
radiotherapy at urban hospitals, and nopatients underwent surgery.
In our eastern Chinese popula-tion, 837 newly diagnosed patients
were analyzed as a discov-ery set in this study. In the southern
Chinese population, 828newly diagnosed patients were used as a
validation set (21, 22).A self-administered questionnaire was used
for all patients tocollect epidemiologic data including
demographical charac-teristics, tobacco and alcohol use, family
history of cancer,and medical history. All LRRs were diagnosed by
endoscopyand biopsy and/or computed tomography (CT) scan of the
Table 1. Baseline demographic and clinical characteristics of
study populations
Discovery set (eastern Chinese, N ¼ 837) Validation set
(southern Chinese, N ¼ 828)
VariablesRecurrence(N ¼ 176)
No recurrence(N ¼ 661) P
Recurrence(N ¼ 218)
No recurrence(N ¼ 610) P
Age, mean (SEM) 53.91 (0.879) 52.85 (0.411) 0.856 50.81 (0.821)
51.54 (0.409) 0.413Gender, N (%) 0.239 0.476Male 116 (65.91) 466
(70.50) 162 (74.32) 438 (71.81)Female 60 (34.09) 195 (29.50) 56
(25.68) 172 (28.19)
Family history of cancer, N (%) 0.859 0.466No 156 (88.64) 589
(89.11) 197 (90.37) 561 (91.96)Yes 20 (11.36) 72 (10.89) 21 (9.63)
49 (8.04)
Smoking status, N (%) 0.542 0.438Never 82 (46.59) 291 (44.02)
106 (48.62) 278 (45.57)Ever 94 (53.41) 370 (55.98) 112 (51.38) 332
(54.43)
Drinking status, N (%) 0.367 0.415Never 87 (49.43) 352 (53.25)
107 (49.08) 319 (52.30)Ever 89 (50.57) 309 (46.75) 111 (50.92) 291
(47.70)
BMI, N (%) 0.911 0.151�20 28 (15.91) 116 (17.55) 62 (28.44) 205
(33.61)20–28 133 (75.57) 481 (72.77) 148 (67.89) 387 (63.44)�28 15
(8.52) 64 (9.68) 8 (3.67) 18 (2.95)
EBV infection status, N (%) 0.024 0.007Positive 159 (90.34) 552
(83.51) 202 (92.66) 522 (85.58)Negative 17 (9.66) 109 (16.49) 16
(7.34) 88 (14.42)
Stage, N (%) 0.285 0.194I 8 (4.55) 28 (4.23) 9 (4.13) 26
(4.26)II 42 (23.86) 155 (23.45) 47 (21.55) 163 (26.72)III 68
(38.64) 316 (47.81) 92 (42.21) 245 (40.16)IV 58 (32.95) 162 (24.51)
70 (32.11) 176 (28.86)
Histologic grade, N (%) 0.119 0.434Undifferentiated 139 (78.97)
484 (73.22) 166 (76.15) 448 (73.44)Differentiated 37 (21.02) 177
(26.78) 52 (23.85) 162 (26.56)
Chemotherapy, N (%) 0.003 0.011Yes 143 (81.25) 593 (89.71) 175
(80.28) 533 (87.38)No 33 (18.75) 68 (10.29) 43 (19.72) 77
(12.62)
Zheng et al.
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nasopharynx and the skull base showing progressive boneerosion
and/or soft tissue swelling (23). Diagnosis of a secondprimary
tumor was based on a modification of the criteria ofWarren andGates
(24), andwe did notfind any second primarytumor cases in our
present study. Each patient saw his or herdoctor during the
follow-up period for assessment of recur-rence once a month in the
first year, every 2 months in years 2and 3, and every 6 months
thereafter. We reviewed patients'medical records during the
follow-up period to collect clinicalinformation including the date
of diagnosis, recurrence status,Epstein–Barr virus (EBV) infection
status, histologic grade,pathologic stage, and treatment.
Immunoglobulin-A antibo-dies to EBV capsid antigen (EBV/IgA/VCA)
and immunoglob-ulin-A antibodies to EBV early antigen were
confirmed byserologic testing at the time of study enrollment.
Thetumor, node, metastasis classification and tumor stagingwere
evaluated according to the 2002 American Joint Com-mittee on Cancer
staging system. At recruitment, informedconsent was obtained from
each patient, and the study wasapproved by the Medical Ethics
Committee of Soochow Uni-versity (SZUM2009061002) and the
Institutional Review Boardof Guangzhou Medical College
(GZMC2009060426).
Radiotherapy techniqueRadiotherapy was conducted largely using
standard proce-
dures (5). For details, see Supplementary Materials and
Meth-ods. The patients received about a month of radiotherapy,
andtreatment was delivered once daily (5 fractions/week). The
lagtime between date of diagnosis and date of first treatment
iswithin 2 weeks.
Follow-up and endpoint selectionAll patients were evaluated
weekly during the treatment
period, and after the completion of treatment, they werefollowed
up every 3 months by telephone for the first 3 years,every 6 months
in years 4 and 5, and annually thereafter.Overall, we recruited
2,073 patients with nasopharyngeal car-cinoma (1,075 from the
eastern Chinese and 998 from thesouthern Chinese). A total of 1,665
patients (837 from theeastern Chinese and 828 from the southern
Chinese) hadcomplete follow-ups and clinical information. Among
theremaining 408 patients (238 from the eastern Chinese and170 from
the southern Chinese) with incomplete follow-up orclinical
information or both, 125 cases (6.03%) lacked stageand/or histology
information, 132 cases (6.37%) had incorrecttelephone numbers, 49
cases (2.36%) refused to participate, 66cases (3.18%) had ambiguous
death date and/or indirect deathbecause of nasopharyngeal
carcinoma, and 36 cases (1.74%)moved or were unavailable for
unknown reasons. However,there was no significant difference in the
distributions ofdemographic characters (e.g., age and gender),
smoking status,drinking status, body mass index (BMI), EBV
infection status,stage, histologic grade, and chemotherapy between
thepatients with nasopharyngeal carcinoma with and
withoutfollow-up/clinical information (P ¼ 0.642 for age; P ¼
0.365for gender; P¼ 0.516 for smoking status; P¼ 0.287 for
drinkingstatus;P¼ 0.258 for BMI;P¼ 0.539 for EBV infection
status;P¼0.225 for stage; P¼ 0.169 for histologic grade; and P¼
0.258 for
chemotherapy). The endpoints of the current study includedthe
time to recurrence (TTR) and OS. The TTR was calculatedas the time
from the date of diagnosis of nasopharyngealcarcinoma to the date
of the first observation of LRR, or untilthe last follow-up if the
patient was recurrence-free at thattime. TTR was censored at the
time of death or at the lastfollow-up if the patient remained
recurrence-free at that time.The OS was defined as the time from
pathologic diagnosis todeath fromany cause, or the last contact if
the patientwas alive.
Tissue samplesTo determine the expression levels of selected
genes, we
collected 35 nasopharyngeal carcinoma tissues from patientswho
had undergone resection from The Second AffiliatedHospital of
Soochow University. All cases were histopatholog-ically diagnosed
as nasopharyngeal carcinoma by biopsy andwithout radio- or
chemotherapy.
SNP selection and genotypingAccording to the bioinformatics
analysis, SNPs located
in mature miRNA sequences with allelic frequencies inChinese
populations were selected. First, based on themiRBase database
(http://mirbase.org; up to January 1,2012), 205 polymorphic loci
located in mature miRNAsequences (Supplementary Table S1) were
screened. Second,based on the HapMap public database [HapMap Data
Rel28 phase IIþIII, August 10, on National Center for
Bio-technology Information (NCBI) B36 assembly, dbSNPb126; up to
January 1, 2012], we found that there were onlyseven SNPs in 205
polymorphic loci that had allelic fre-quencies in Chinese
populations (Supplementary Table S1).Finally, we selected these
seven SNPs located in maturemiRNA sequences with the allelic
frequencies in theChinese population for genotyping:miR-499
(rs3746444C>T),miR-608 (rs4919510C>G), miR-3152
(rs13299349A>G), miR-4513 (rs2168518C>T), miR-4520a
(rs8078913C>T), miR-4741(rs7227168C>T), and miR-4762
(rs41524547C>G).
Genomic DNA was isolated from the peripheral bloodlymphocytes of
all the study subjects. All subjects were geno-typed for the seven
SNPs by using allele-specific matrix-assisted laser
desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry
(Sequenom; refs. 25, 26). The DNAisolation and genotyping were
conducted in Suzhou Center(Suzhou, China; for the eastern Chinese
population) andGuangzhou Center (Guangzhou, China; for the southern
Chi-nese population), respectively. The cross-trained
laboratorypersonnel conducting the genotyping were blinded to
patientinformation. Approximately, 10% of the samples were
alsorandomly selected for a blinded repeat of the genotypingwithout
prior knowledge of the previous genotyping resultor the patient
information, and the results were in 100%agreement.
Cell cultureHuman nasopharyngeal carcinoma cell lines (CNE-1
and
CNE-2) and 293T cells were purchased from the Cell Bankof Type
Culture Collection of the Chinese Academy ofSciences, Shanghai
Institute of Biochemistry and Cell Biology
Polymorphism in microRNA and Recurrence of Nasopharyngeal
Carcinoma
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(Shanghai, China) and passaged for fewer than 6 months. Thecell
lines were characterized by DNA fingerprinting analysisusing
short-tandem repeat (STR) markers. Cells were main-tained according
to the Cell Bank's protocols.
Plasmids, lentiviral production, and transductionLentiviral
expression plasmids construction, lentiviral pro-
duction, and transduction were conducted by following
theestablished and previously published procedures (27, 28).
Fordetails, see Supplementary Materials and Methods.
Finally,CNE-1-empty vector, CNE-1-miR-608-C,
CNE-1-miR-608-G,CNE-2-empty vector, CNE-2-miR-608-C, and
CNE-2-miR-608-G cells were stably selected with G418 at 500 mg/mL
(Gibco),and the drug-resistant cell populations were used for
subse-quent studies.
RNA extraction and microarray analysisTotal RNA was extracted
from the cultured cells in both
experimental (CNE-1-miR-608-C and CNE-1-miR-608-G) andcontrol
(CNE-1-empty vector) groups using RNeasy Mini Kits(Qiagen)
according to the manufacturer's instructions. Geneexpression
profiling was conducted using the Human OneAr-ray microarray
(Phalanx Biotech; refs. 29, 30). Details are givenin Supplementary
Materials andMethods. The array data havebeen deposited in Gene
Expression Omnibus (GEO; accessionnumber: GSE46372).
Quantitative real-time PCR analysisTotal RNA was isolated from
35 nasopharyngeal carcinoma
tissue samples with TRIzol reagent (Molecular Research Cen-ter,
Inc.). The relative gene expression for the selected geneswas
quantified using the ABI Prism 7000 sequence detectionsystem
(Applied Biosystems) based on the SYBR Green meth-od. The primers
used for PCR amplification of the candidategenes are listed in
Supplementary Table S2. The expression ofmiR-608 in nasopharyngeal
carcinoma cells was calculatedrelative to theU6 small nuclear RNA
(SupplementaryMaterialsand Methods).
Construction of FBXO32 30-UTR luciferase reporterplasmid
The reporter vector psiCHECK-2 (Promega)was prepared
byamplifying a 683-bp FBXO32 30-untranslated region (30-UTR)region
from a human genomic DNA sample, including theartificial XhoI and
NotI enzyme restriction sites with theforward primer
50-TGTATTATGCTCGAGCCATAGTTCTC-30
and reverse primer 50-CGCTCTAAGTCTAAAGCGGCCGC-TAG-30.
Construction of the FBXO32 30-UTR luciferase reporterplasmid was
conducted according to a previously describedmethod (21). The
resulting construct (psiCHECK-2-FBXO32-30-UTR) was verified by
sequencing.
Transient transfections and luciferase assaysThe CNE-1 and CNE-2
cells were seeded at 1 � 105 cells per
well in 24-well plates (BD Biosciences). Sixteen hours
afterplating, the cells were transfected with Lipofectamine
2000(Invitrogen) according to the manufacturer's
instructions(Supplementary Materials and Methods).
Mutagen sensitivity assayTo further explore the differences in
DNA repair ability
among individuals with different miR-608 rs4919510C>G
gen-otypes, we evaluated X-ray radiation sensitivities in 135
addi-tional control subjects according to published
protocols(Supplementary Materials and Methods; refs. 31, 32).
Thevalues of chromatid breaks per cell (b/c) were used to
indicatethe DNA repair capacity of the individual (31, 33).
Cell growth analysis in response to irradiationCNE-1-empty
vector, CNE-1-miR-608-C, CNE-1-miR-608-G,
CNE-2-empty vector, CNE-2-miR-608-C, and CNE-2-miR-608-Gcells
were plated in a 24-well culture plates (2.5 � 104/well).After
incubation for 24 hours, the cells received 2 Gy ofirradiation.
Cell growth was monitored by counting cell num-bers at various time
intervals. Three independent experimentswere carried out in
triplicate.
Animal modelThe animal experiments were carried out in
accordance
with National guidelines and approved by the LaboratoryAnimal
Center of Soochow University. CNE-1-empty vector,CNE-1-miR-608-C,
CNE-1-miR-608-G, CNE-2-empty vector,CNE-2-miR-608-C, and
CNE-2-miR-608-G cells were diluted toa concentration of 5� 107/mL
in physiologic saline. Nudemicewere injected subcutaneously with
0.1 mL of the suspensioninto the back flank (6 mice/group). For
details, see Supple-mentary Materials and Methods.
Radiation deliveryLocal irradiation of the implanted tumor was
conducted
using a customized mouse jig with other parts of the
bodyshielded with lead. Each mouse was confined to a
customizedmouse jig with a circular window, through which the
tumorbed was exposed to the radiation, and was irradiated
locally.Mice were exposed to X-rays with 5-mm thick lead
shieldswhen the tumor bed was gently extended into the
radiationfield. Tumors were locally irradiated at a dose of 2, 10,
or 20 Gyusing a RS2000 X-ray Biological Irradiator (Rad Source
Tech-nologies) at a dose rate of 1 Gy/min through a 0.2-mm
copperfilter beginning 1 week after transplantation.
Statistical analysisThe x2 test or Fisher exact test was applied
separately to
compare the distribution of selected demographic and
clinicalvariables according to recurrence status. The Cox
proportionalhazards model was used to estimate HRs and their
95%confidence intervals (CIs) for multivariate analyses in
thediscovery set, validation set, and combined dataset. The
anal-yses were adjusted for age, gender, BMI, smoking
status,drinking status, family history of cancer, EBV infection
status,stage, histologic grade, and chemotherapy, as
appropriate.Associations between genotypes and TTR and OS were
esti-mated using the Kaplan–Meier method, and statistical
signif-icance was determined using the log-rank test (34). For
thefunctional analyses, data were presented by using mean �SEM; the
comparison of mean between two groups was con-ducted by using
Student t test; statistical comparisons of more
Zheng et al.
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than two groups were conducted using the one-way ANOVA(35), and
then least-significant difference (LSD) for multiplecomparisons.
The statistical analyses were conducted usingSTATA software
(version 10; STATA Corporation). All P valueswere two-sided and P
< 0.05 was considered statisticallysignificant.
ResultsPatient characteristicsThe clinical information and
demographic characteristics
of the 1,665 patients with nasopharyngeal carcinoma includ-ed in
this study are shown in Table 1. For the easternChinese population,
the median patient age was 54 years(range, 21–82 years) and the
median follow-up time was 3.1years (range, 0.4–10.8 years); 176
patients (21.03%) showedtumor LRR after radiotherapy, resulting in
a 3-year recur-rence probability of 0.21 � 0.04, and the median TTR
was 1.9years (95% CI, 1.6–2.3 years); 180 patients (21.51%) have
died(including 150 LRRs, 21 distant metastases, and 9
otherreasons), but the median OS had not been reached duringthe
follow-up time. No significant differences were notedwith regard to
sex, age, family history of cancer, smokingstatus, drinking status,
BMI, stage, or histologic gradeaccording to recurrence status;
however, significant differ-ences were found in EBV infection
status and the number ofpatients who received chemotherapy (Table
1).For the southern Chinese population, the median patient
agewas 52 years (range, 19–87 years) and themedian
follow-uptimewas 2.9 years (range, 0.5–10.2 years); 218 patients
(26.33%)exhibited tumor recurrence after radiotherapy, resulting in
a 3-year recurrence probability of 0.28� 0.04, and themedian TTRwas
2.2 years (95%CI, 2.1–2.5 years); 223 patients (26.93%) havedied
(including 182 LRRs, 29 distant metastases, and 12 otherreasons),
but median OS had not been reached during thefollow-up time.
Associations between SNPs and recurrence riskGenotype
distributions of all SNPs were found to be con-
sistent with Hardy–Weinberg equilibrium (HWE), with the
exception of rs7227168C>T and rs41524547C>G. No
significantassociations were noted in the SNPs and baseline
demo-graphic, clinical, or pathologic characteristics according
torecurrence status. The selected seven candidate SNPs
weregenotyped in a discovery set consisting of 837 patients
withnasopharyngeal carcinoma from the eastern Chinese popula-tion.
In the univariate analysis, patients carrying the
miR-608rs4910510GG genotype had a median TTR of 6.2 years,
com-pared with a median TTR of 8.9 years for patients
withrs4910510CC genotype (HR, 2.02; 95% CI, 1.29–3.16; P ¼0.0008;
Fig. 1A; Table 2). However, the other tested SNPs didnot show any
statistically significant associations with naso-pharyngeal
carcinoma LRR in the univariate analyses. In themultivariate
analysis, a Cox proportional hazards model wasadjusted for age,
gender, BMI, smoking status, drinking status,family history of
cancer, EBV infection status, stage, histologicgrade, and
chemotherapy, and the miR-608 rs4910510GGgenotype remained
significantly associated with nasopharyn-geal carcinoma LRR (HR,
2.05; 95% CI, 1.35–3.21; P ¼0.0012; Table 2). These results were
also confirmed in thesouthern Chinese population, in which
rs4910510C>G dis-played a consistent association with recurrence
in the valida-tion (HR, 2.24; 95% CI, 1.45–3.38; P ¼ 0.0006; Fig.
1B; Table 2)and combined (HR, 2.08; 95% CI, 1.41–3.26; P <
0.00001)datasets (Fig. 1C; Table 3).
About 85% of patients for both populations received
thechemotherapy during the treatment; however, there were
nosignificant differences in the associations with
miR-608rs4919510C>G polymorphism by chemotherapy (P > 0.05),
asshown in Supplementary Fig. S1.
Associations between miR-608 rs4919510C>G genotypesand OS
As shown in Supplementary Fig. S2A–S2C, we found thatmiR-608
rs4919510C>G exhibited a consistent association withthe OS in
the discovery set (HR, 2.13; 95% CI, 1.39–3.28; P ¼0.0006), the
validation set (HR, 1.89; 95% CI, 1.23–3.07; P ¼0.0023), and the
combined dataset (HR, 1.95; 95% CI, 1.31–3.15;P < 0.0001).
However, we also analyzed the time from
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Log-rank test: P = 0.0012
rs4919510C>GCC (36/201) MTTR=8.9GC (78/420) MTTR=7.9GG
(62/216) MTTR=6.2
CC (42/209) MTTR=9.0GC (110/439) MTTR=8.1GG (66/180)
MTTR=5.2
CC (78/410) MTTR=8.9GC (188/859) MTTR=8.1GG (128/396)
MTTR=5.3
rs4919510C>G rs4919510C>G
Log-rank test: P = 0.0006 Log-rank test: P < 0.00001
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Years since diagnosis of NPC Years since diagnosis of NPC6 8 10
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Figure 1. Kaplan–Meier TTRcurves of patientswith nasopharyngeal
carcinoma receiving radiotherapybygenotypesofmiR-608
rs4919510C>G.Discovery set(A), validation set (B), and combined
dataset (C). MTTR, median time to recurrence.
Polymorphism in microRNA and Recurrence of Nasopharyngeal
Carcinoma
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-
Tab
le2.
Univa
riate
andmultiv
ariate
analysis
ofmaturemiRNAsse
que
ncepolym
orphism
san
dna
sopha
ryng
ealc
arcino
marecu
rren
cein
disco
very
setan
dva
lidationse
t
Disco
very
set(eas
tern
Chine
se,N
¼83
7)Validationse
t(southe
rnChine
se,N
¼82
8)
Univa
riatean
alys
isMultiva
riatean
alys
isUniva
riatean
alys
isMultiva
riatean
alys
is
Nd
MAFe
Probab
ility
aHR
(95%
CI)
Pb
HR
(95%
CI)
Pc
Nd
MAFe
Probab
ility
aHR
(95%
CI)
Pb
HR
(95%
CI)
Pc
miR-608
rs49
1951
00.50
90.00
080.00
120.48
20.00
010.00
06CC
201
0.17
�0.03
1.00
(Referen
ce)
1.00
(Referen
ce)
209
0.21
�0.04
1.00
(Referen
ce)
1.00
(Referen
ce)
GC
420
0.19
�0.03
1.41
(0.89–
2.04
)1.45
(0.92–
2.18
)43
90.27
�0.03
1.55
(1.03–
2.15
)1.62
(1.11–
2.19
)GG
216
0.26
�0.05
2.02
(1.29–
3.16
)2.05
(1.35–
3.21
)18
00.37
�0.04
2.21
(1.49–
3.24
)2.24
(1.45–
3.38
)GCþG
G63
60.22
�0.03
1.61
(1.13–
2.29
)1.68
(1.18–
2.61
)61
90.29
�0.03
1.65
(1.17–
2.39
)1.71
(1.23–
2.31
)miR-451
3rs21
6851
80.19
40.12
00.17
80.18
20.49
80.40
1CC
552
0.19
�0.02
1.00
(Referen
ce)
1.00
(Referen
ce)
560
0.26
�0.03
1.00
(Referen
ce)
1.00
(Referen
ce)
TC24
60.23
�0.03
1.18
(0.85–
1.62
)1.21
(0.87–
1.67
)23
40.28
�0.04
1.11
(0.71–
1.53
)1.15
(0.84–
1.58
)TT
3934
TCþT
T28
50.22
�0.05
1.14
(0.82–
1.68
)1.18
(0.84–
1.71
)26
80.29
�0.03
1.17
(0.78–
1.61
)1.23
(0.82–
1.65
)miR-452
0ars80
7891
30.34
70.73
70.70
50.33
50.68
30.78
2TT
368
0.18
�0.02
1.00
(Referen
ce)
1.00
(Referen
ce)
378
0.28
�0.03
1.00
(Referen
ce)
1.00
(Referen
ce)
CT
357
0.21
�0.03
1.14
(0.83–
1.57
)1.13
(0.82–
1.56
)34
60.27
�0.03
1.08
(0.87–
1.18
)1.04
(0.87–
1.21
)CC
112
0.22
�0.04
1.02
(0.64–
1.64
)0.91
(0.54–
1.51
)10
40.25
�0.05
0.92
(0.73–
1.14
)1.01
(0.78–
1.24
)CTþ
CC
469
0.22
�0.04
1.11
(0.82–
1.59
)1.09
(0.81–
1.52
)45
00.26
�0.04
0.96
(0.78–
1.17
)1.02
(0.85–
1.23
)miR-499
rs37
4644
40.14
80.64
70.52
30.15
00.46
90.21
3TT
611
0.19
�0.02
1.00
(Referen
ce)
1.00
(Referen
ce)
605
0.28
�0.02
1.00
(Referen
ce)
1.00
(Referen
ce)
TC20
40.20
�0.05
1.16
(0.82–
1.64
)1.13
(0.79–
1.60
)19
90.25
�0.03
0.87
(0.47–
1.64
)0.93
(0.45–
1.72
)CC
2224
TCþC
C22
60.21
�0.03
1.05
(0.72–
1.53
)1.06
(0.73–
1.54
)22
30.27
�0.02
0.85
(0.57–
1.17
)0.86
(0.53–
1.19
)miR-315
2rs13
2993
490.12
00.33
90.25
60.12
20.67
50.53
4GG
653
0.18
�0.02
1.00
(Referen
ce)
1.00
(Referen
ce)
639
0.27
�0.03
1.00
(Referen
ce)
1.00
(Referen
ce)
AG
167
0.21
�0.05
1.11
(0.65–
1.64
)1.13
(0.70–
1.67
)17
70.29
�0.05
1.15
(0.80–
1.61
)1.09
(0.77–
1.56
)AA
1712
AGþA
A18
40.24
�0.04
1.18
(0.75–
1.89
)1.19
(0.78–
1.93
)18
90.30
�0.04
1.07
(0.57–
1.69
)1.06
(0.76–
1.65
)
aProbab
ility
�SEof
3-ye
arrecu
rren
ce.
bOnthebas
isof
log-rank
test.
cOnthebas
isof
Waldtest
with
inCox
proportio
nalhaz
ardsmod
elwith
adjusted
fora
ge,g
ender,B
MI,sm
okingstatus
,drin
king
status
,fam
ilyhistoryof
canc
er,E
BVinfectionstatus
,stag
e,histolog
icgrad
e,an
dch
emothe
rapy.
dNum
ber
ofpatientswith
thegive
nge
notype.
eMinor
allele
freq
uenc
y.
Zheng et al.
Cancer Res; 73(16) August 15, 2013 Cancer Research5156
on April 6, 2021. © 2013 American Association for Cancer
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http://cancerres.aacrjournals.org/
-
recurrence to death among patients with nasopharyngealcarcinoma
(Supplementary Fig. S2D) and found that there wasno significant
difference among patients carrying the differentmiR-608
rs4919510C>G genotypes (ANOVA test, P > 0.05).
Effects of the rs4919510C>G genotypes on the miR-608target
genes expressionThe nasopharyngeal carcinoma cell line CNE-1 was
infected
withmiR-608-C-allele lentivirus,miR-608-G-allele lentivirus,
orcontrol lentivirus, and the infection efficiency exceeded 90%
forall lentiviruses, as shown in Supplementary Fig. S3. Next,
theexpression levels of miR-608 target genes were detected
bymicroarray analysis (Fig. 2A). We compared RNA
transcriptionlevels between the CNE-1-miR-608-C and
CNE-1-miR-608-Ggroups. Overall, 2,242 genes were differentially
expressed witha P< 0.01. Of these genes, 801were upregulated and
1,441 geneswere downregulated (GEO; accession number: GSE46372);
thegenes with altered expression after infection induced immu-nity
and defense genes, DNA repair genes, cell growth–relatedgenes,
tumor invasion and metastasis-related genes, cancerstem
cell–related genes, and cell death–related genes. Wealso compared
gene expression levels between the CNE-1-miR-608-C and
CNE-1-miR-608-G groups and the controlgroup (CNE-1-empty vector).
We identified 108 genes thatwere downregulated with a P < 0.01
(GEO; accession number:GSE46372). Interestingly, four
differentially expressedgenes were present in both comparisons:
FBXO32, RHOD,TRIM31, and TSC22D3 (Fig. 2A). Differentially
expressedgenes from the microarray experiments were analyzed
usingSTRING, a database of known and predicted
protein–proteininteractions. Figure 2B summarizes the network of
predictedassociations for differentially expressed gene-encoded
pro-teins. The results indicated that FBXO32 was the key gene
ofthis protein interaction net. This gene was linked to TNF
andKITLG, and these genes were linked to many downstreamgenes. All
of these genes were interrelated, thereby forming a
large network. However, the other 3 genes (RHOD, TRIM31,and
TSC22D3) were not linked to other genes (outside thenetwork).
On the basis of the microarray array results, the expres-sion
levels of 20 selected genes (9 upregulated and 11downregulated)
were evaluated using the quantitative PCR(qPCR) analysis, and all
these selected genes with alteredexpression after infection induced
immunity and defensegenes, DNA repair genes, cell growth–related
genes, tumorinvasion and metastasis-related genes, cancer stem
cell–related genes, and cell death–related genes. The results
forthe 20 selected genes had shown that the direction ofexpression
changes were consistent with those found bymicroarray analysis
(Fig. 3A).
The rs4919510C>G genotypes affect FBXO32 expressionby
inhibiting the binding of miR-608 in vitro
According to a bioinformatics analysis software program(MIRanda
Java Interface v1.0), the FBXO32 30-UTR waspredicted to bear a
miR-608–binding site. CNE-1 cells weretransiently cotransfected
with twomiR-608mimics (contain-ing different rs4919510C>G
alleles) and the reporter con-structs and then assessed for
luciferase activity. Comparedwith the rs4919510C allele, the
rs4919510G allele was asso-ciated with significantly reduced
luciferase activity in aconcentration-dependent manner (Fig. 3B).
The sameexperiments were repeated using CNE-2 cells with
similarresults (Fig. 3C).
Effects of themiR-608 rs4919510C>G genotypes on
X-rayradiation-induced chromatid breaks in lymphocytes
We investigated the phenotype of X-ray
radiation-inducedchromatid breaks in lymphocyte cells from 125
controlsubjects and the rs4919510C>G genotype–phenotype
asso-ciation in these individuals. The exact b/c value was
definedas the b/c value of the treatment group minus the
Table 3. Univariate and multivariate analysis of mature miRNAs
sequence polymorphisms andnasopharyngeal carcinoma recurrence in
combined dataset
Combined dataset (eastern and southern Chinese, N ¼ 1,665)
Univariate analysis Multivariate analysis
Nd MAFe Probabilitya HR (95% CI) Pb HR (95% CI) Pc
miR-608 rs4919510 0.496
-
spontaneous b/c value of the untreated group. As shownin Fig.
3D, we found that the mean� SEM b/c value in the 28rs4919510CC
carriers was 0.225 � 0.008, which was signifi-cantly higher than
those of 39 individuals with rs4919510GGgenotype (0.182 � 0.007)
and 58 individuals with thers4919510GC genotype (0.204 � 0.007;
ANOVA test, P ¼0.0024).
Effects of the miR-608 rs4919510C>G genotypes on thecell
growth in response to irradiation
CNE-1-empty vector, CNE-1-miR-608-C, and CNE-1-miR-608-G cells
were subjected to 2 Gy of radiation to examinethe effect on cell
growth. As shown in Fig. 4A, the cell growthdelay after irradiation
was shorter for CNE-1-miR-608-G cellsthan for CNE-1-miR-608-C
cells. The same experiments were
A Lentivirus
Lentivirus
Lentivirus
miRNA-empty vector
Infect target cells
CNE-1-miR-608-C
miR-608-Cvs.
miR-608-G
miR-608-C+
miR-608-Gvs.
empty vector
108 genesdownregulated
FBXO32RHOD
TRIM31TSC22D3
801 genesupregulated1,441 genes
downregulated
CNE-1-miR-608-G
CNE-1-empty vector
miR-608-C
miR-608-G
B
Figure 2. A, the CNE-1 cells were infected with miR-608-C-allele
lentivirus, miR-608-G-allele lentivirus, or control lentivirus.
Next, the expression levelsofmiR-608 target genes were detected by
microarray analysis. B, differentially expressed genes between
CNE-1-miR-608-C group and CNE-1-miR-608-Ggroup were analyzed using
the STRING database. The network nodes represent the proteins
encoded by the differentially expressed genes. Differentline colors
represent the types of evidence for the association.
Zheng et al.
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-
repeated using CNE-2-empty vector, CNE-2-miR-608-C,
andCNE-2-miR-608-G cells with similar results (Fig. 4A).
Effects of themiR-608 rs4919510C>Ggenotypes on tumorgrowthWe
evaluated the effects of radiation on tumor growth in
different animal models. During a 1-week period, all micewere
injected with CNE-1-miR-608-C, CNE-1-miR-608-G, orCNE-1-empty
vector cells developed tumors, after whichmice were assigned to
receive 2, 10, or 20 Gy of localradiation. As shown in Fig. 4B, no
significant tumor growthinhibition was observed in CNE-1-empty
vector, CNE-1-miR-608-C, or CNE-1-miR-608-G xenografts that were
locallyirradiated with 2Gy of radiation (P ¼ 0.625). In contrast,
itwas showed that the CNE-1-miR-608-G xenografts grewfaster than
the CNE-1-miR-608-C xenografts after 10-Gyirradiation (average
volumes �SEM; 593 � 59.49 mm3 vs.891 � 93.44 mm3; P ¼ 0.009).
Unfortunately, approximately70% of the mice died after being
locally irradiated at a doseof 20 Gy (Fig. 4B). The same
experiments were repeatedusing the CNE-2-empty vector,
CNE-2-miR-608-C, and CNE-2-miR-608-G cell xenografts with similar
results (Fig. 4B).
DiscussionIn the present study, we investigated the effects of
SNPs in
mature miRNA sequences on LRR in patients with naso-pharyngeal
carcinoma after radiotherapy. We found thatcarrying at least one G
allele (GC, GG) of the miR-608rs4919510C>G SNP significantly
increased the risk of LRRcompared with that in patients carrying a
homozygous
C allele. Importantly, these results remained significantafter
adjustment for other potential predictors of patientoutcome in this
patient cohort study, and our functionalresults were also
consistent with these findings. This studyrepresents the first
finding that the miR-608 rs4919510C>GSNP may serve as a
predictive marker for the LRR ofnasopharyngeal carcinoma in
patients who were treatedwith radiotherapy.
Nasopharyngeal carcinoma is highly sensitive to radiationthat
alone for early nasopharyngeal carcinoma can achieve arelatively
high cure rate; however, its efficacy is disappointingfor locally
advanced disease. It is well known that recurrence inthe
nasopharynx is one of the important causes of treatmentfailure (36,
37); therefore, assessments of recurrence are crucialfor the
selection of appropriate treatment. Previous reportsindicated that
miRNAs were aberrantly expressed in nasopha-ryngeal carcinoma
compared with that in normal epithelialtissue, and this aberrant
expression promoted an aggressivetumor phenotype by changing the
expression of mRNA targets(38–40). Therefore, miRNA-related SNPs
may be used individ-ually and jointly to predict the risk of
recurrence of early-stagehead and neck cancer (41). Liu and
colleagues suggested thatsome important miRNAs had a significant
value for determin-ing the survival prognosis in addition to
nasopharyngealcarcinoma development and progression (42). As the
impor-tant roles of miRNAs in cancer are gradually being
revealed,their potential applications as predictive markers and
treat-ment targets have generated great interest for cancer
diagno-sis, classification, prognosis, risk factor evaluation, and
therapystrategies.
Figure 3. A, the 20 selected genes mRNA expression levels in
tissue samples from patients with nasopharyngeal carcinoma as
function of miR-608rs4919510C>Ggenotypes (7 rs4919510CC, 17
rs4919510CG, and 11 rs4919510GG); data aremean�SEM, normalized
tob-actin. Relative luciferase activityof the
psiCHECK-2-FBXO32-30-UTR construct cotransfected with miR-608
containing different rs4919510C>G alleles in CNE-1 (B) and CNE-2
cells (C).Renilla luciferase activitywasmeasured andnormalized to
firefly luciferase. Six replicateswere carried out for eachgroup,
and the experimentwas repeated atleast three times. Data are mean�
SEM. D, effect of themiR-608 rs4919510C>GSNP on X-ray–induced
chromatid b/c in peripheral blood lymphocytes from125 control
subjects. The levels of chromatid breaks in controls with different
genotypes of rs4919510C>G were analyzed with ANOVA test.
Polymorphism in microRNA and Recurrence of Nasopharyngeal
Carcinoma
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-
A series of epidemiologic studies revealed that the
miR-608rs4919510C>G SNP has a key role in cancer progression
(17),and evidence of its influence on prognosis has also
accumu-lated recently (18, 34, 43). However, the importance of
themiR-608 rs4919510C>G SNP in the LRR of nasopharyngeal
carci-noma after radiotherapy remains unclear, and the
biologicfunctions of this SNP have not yet been elucidated.
Thers4919510C>G SNP is located within the mature sequence
ofmiR-608 and at the joint of the stemwith the canonical
hairpinloop (18). Because this rigid secondary structure is a
requisitefor recognition, and thus processing, of pre-miRNA by
theRNase Drosha, structural disruptions at this critical point
mayaffect recognition or subsequent processing. Each miRNA
hashundreds of targets, and thus, a singular change in a mature
miRNA sequence could have an exponentially large effect
onprotein output, perhaps an effect sufficient to skew,
evenslightly, the clinical outcome of cancer. Our results
indicatedthat the rs4919510C>G SNP might influence the
expression ofmiR-608 target genes, which include immunity and
defensegenes, DNA repair genes, cell growth–related genes,
tumorinvasion and metastasis-related genes, cancer stem
cell–relat-ed genes, and cell death–related genes. These genes
could haveconsequences directly related to cancer cell survival and
tumorgrowth, thereby influencing the LRR of nasopharyngeal
carci-noma after radiotherapy. Moreover, the X-ray radiationinduced
more chromatid breaks in lymphocyte cells fromrs4919510CC carriers
than in those from subjects with othergenotypes. As we know,
medical radiation can cause DNA
A
0 1 2 3 4 5 6
Day
Subcutaneous injection Radiation
2 Gy 2 Gy
10 Gy 10 Gy
20 Gy 20 Gy
0 3 7 11 15 19
0 1 2 3
Time (d)
Time (d)
Time (d)
Tum
or
volu
me
(mm
3 )
Tum
or
volu
me
(mm
3 )
Tum
or
volu
me
(mm
3 )
Tum
or
volu
me
(mm
3 )
Tum
or
volu
me
(mm
3 )
Tum
or
volu
me
(mm
3 )
0
2,000
1,500
1,000
500
0
2,000
1,500
1,000
500
0
2,000
1,500
1,000
500
0
2,000
1,500
1,000
500
0
600
400
200
0
800
600
400
200
0
1 3 5 7 9 11 13 15 17 19
0 1 3 5 7 9 11 13 15 17 19
0 1 3 5 7 9 11 13 15 17 19 0 1 3 5 7 9 11 13 15 17 19
0 1 3 5 7 9 11 13 15 17 19
0 1 3 5 7 9 11 13 15 17 19Time (d)
Time (d) Time (d)
Time (d) Time (d)
Cel
l nu
mb
er (
10E
4)C
ell n
um
ber
(10
E4)
50
40
30
20
10
0
50
40
30
20
10
04 5 6
B
CNE-1-miR-608-C
CNE-1-miR-608-G
CNE-1-empty vector
CNE-1-miR-608-CCNE-1-miR-608-GCNE-1-empty vector
CNE-2-miR-608-CCNE-2-miR-608-GCNE-2-empty vector
CNE-2-miR-608-C
CNE-2-miR-608-G
CNE-2-empty vector
Figure 4. A, CNE-1-empty vector, CNE-1-miR-608-C,
CNE-1-miR-608-G, CNE-2-empty vector, CNE-2-miR-608-C, and
CNE-2-miR-608-G cells weresubjected to 2 Gy of radiation to examine
the effect on cell growth. Cells were seeded in 24-well culture
plate 24 hours before irradiation. Cellnumbers were counted at
different times after irradiation. Cell multiplication of
CNE-1-empty vector and CNE-2-empty vector cells were used
ascontrols. Three experiments were carried out; points, mean; bars,
SEM. B, subcutaneously implanted CNE-1-empty vector,
CNE-1-miR-608-C, CNE-1-miR-608-G, CNE-2-empty vector,
CNE-2-miR-608-C, and CNE-2-miR-608-G cells xenografted tumors were
established. During a 1-week period, all micedeveloped tumors,
after which mice were assigned to receive 2, 10, or 20 Gy of local
radiation. Each point represents the mean tumor volume. Bars,SEM.
Mean tumor volume from 6 nude mice of each group are shown.
Zheng et al.
Cancer Res; 73(16) August 15, 2013 Cancer Research5160
on April 6, 2021. © 2013 American Association for Cancer
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-
double-strand breaks in cancer cells and suppress cancer
cellgrowth (44), which is consistent with the rs4919510CCvariant
genotype having low DNA repair genes expression anddeficient DNA
repair capacity, and the association betweenrs4919510CC variant
genotype and medical radiation ondecreasing LRR risk of
nasopharyngeal carcinoma is bio-logically plausible. Our results
also indicated that miR-608rs4919510C>G exhibited an association
with the OS;however, there was no significant difference in time
fromrecurrence to death among patients carrying different miR-608
rs4919510C>G genotypes. All these results suggested thatmiR-608
rs4919510C>G was associated with higher risk of LRRand then the
TTR in advance, which finally may lead to thehigher risk of
all-cause mortality. Therefore, our present studydetected the
importance of mature miRNA sequence SNPs inthe LRR of
nasopharyngeal carcinoma after radiotherapy.FBXO32 (also known as
atrogin-1) is a member of the F-box
protein family and constitutes 1 of 4 subunits of the
ubiquitinprotein ligase complex (45, 46). FBXO32 has been reported
toplay a role in muscle atrophy (47), and recent findings
havesuggested that FBXO32 is a novel apoptosis regulator that
isnegatively regulated by a prosurvival signal (48, 49).
Interest-ingly, Tan and colleagues also showed that FBXO32
wastranscriptionally silenced by epigenetic mechanisms in
cancercells (48). Furthermore, Chou and colleagues found thatFBXO32
might be a novel tumor suppressor gene that wasassociated with poor
prognosis in human ovarian cancer (50).Our reporter gene assays
suggested that the rs4919510C alleleaffected FBXO32 expression by
inhibiting the binding ofmiR-608 in nasopharyngeal carcinoma cell
lines, and theseresults are consistent with previous findings on
the contribu-tions of FBXO32 to tumor suppression.In summary, our
preliminary study provides the first
evidence that miR-608 rs4919510C>G may be a predictivemarker
to identify patients with a high risk of nasopharyn-geal carcinoma
recurrence, and these data may help topredict response in a
subgroup of patients treated withradiotherapy. In addition, this
may help to select subgroups
of patients with nasopharyngeal carcinoma who may benefitfrom
newly developed gene-therapy strategies. Nevertheless,a median
follow-up time of approximately 3 years for bothpopulations
included in the present studies may not besufficient to identify
all of the recurrences that would occur,and further validation of
our hypothesis-generating findingsin prospective biomarker-embedded
clinical trials is needed.Therefore, large patient cohort studies
are warranted tofurther confirm our results.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Authors' ContributionsConception and design: J. Zheng, J. Lu, Y.
ZhouDevelopment of methodology: J. Zheng, L. Zhang, M. Hu, H. Wu,
W. Li, J. Lu,Y. ZhouAcquisition of data (provided animals, acquired
and managed patients,provided facilities, etc.): J. Zheng, J. Deng,
M. Xiao, L. Yang, L. Zhang, Y. You,W. Li, J. Lu, Y. ZhouAnalysis
and interpretation of data (e.g., statistical analysis,
biostatistics,computational analysis): J. Zheng, J. Deng, Y. You,
N. Li, J. Lu, Y. ZhouWriting, review, and/or revision of the
manuscript: J. Zheng, J. Deng,H. Wu, J. Lu, Y. ZhouAdministrative,
technical, or material support (i.e., reporting or orga-nizing
data, constructing databases): J. Zheng, J. Deng, L. Zhang, M.
Hu,N. Li, J. Lu, Y. ZhouStudy supervision: J. Lu, Y. Zhou
Grant SupportThis study was supported by the National Scientific
Foundation of China
grants 81001278, 81171895, and 81072366; a project funded by the
PriorityAcademic Program Development of Jiangsu Higher Education
Institutions,Jiangsu Provincial Natural Science Foundation (no.
BK2011297); Jiangsu Prov-ince Science and Technology Support
Program (no. BE2012648); the ScientificResearch Foundation for the
Returned Overseas Chinese Scholars, State Edu-cation Ministry (no.
20101561), Suzhou Key Laboratory for Molecular CancerGenetics (no.
SZS201209); and Zhejiang Provincial Natural Science Foundationof
China (Y2100449 and Y2110410).
The costs of publication of this article were defrayed in part
by thepayment of page charges. This article must therefore be
hereby markedadvertisement in accordance with 18 U.S.C. Section
1734 solely to indicate thisfact.
Received February 10, 2013; revised May 22, 2013; accepted June
2, 2013;published OnlineFirst June 24, 2013.
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