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doi:10.1093/mutage/get035

Polymorphisms of CD44 gene and nasopharyngeal carcinoma susceptibility in a Chinese population

Mang Xiao, Sunhong Hu, Lei Zhang, Jun Huang1, Huifen Jiang2 and Xiujun Cai3,*

Department of Otorhinolaryngology-Head and Neck Surgery and 1Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Key Laboratory of Biotherapy of Zhejiang Province, Zhejiang University, 3 Qinchun Road, Hangzhou 310006, China, 2Department of Clinical Laboratory, Zhejiang Cancer Hospital, 38 Guangji Road, Hangzhou 310006, China and 3Department of General Surgery, Sir Run Run Shaw Hospital, Key Laboratory of Biotherapy of Zhejiang Province, Zhejiang University, 3 Qinchun Road, Hangzhou 310006, China

*To whom correspondence should be addressed. Tel: +86 571 86002167; Fax: +86 571 86002167; Email: xj2000_cai@126.com

Received on January 23, 2013; revised on May 23, 2013; accepted on June 12, 2013

As members of adhesion molecule families, CD44 transmem-brane glycoproteins have been originally thought to be essen-tial for the formation of multicellular organisms and soon recognised to be able to initiate metastatic spread of tumour cells. To investigate the association between CD44 polymor-phisms and nasopharyngeal carcinoma (NPC), we carried out a two-stage case–control study in 906 patients and 943 healthy controls in Eastern populations. Five single nucleo-tide polymorphisms of CD44 (rs10836347C>T, rs13347C>T, rs1425802A>G, rs11821102G>A and rs713330T>C) with proper frequency were selected from the HapMap data-base and genotyped with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Compared with the most common rs13347CC genotype, CT+TT geno-types significantly increased individuals’ susceptibility to NPC (odds ratio  =  2.58, 95% confidence interval  =  2.13–3.13). Furthermore, our transient transfection focusing on reporter gene expression modulated by CD44 3′UTR demonstrated that the presence of an rs13347T allele led to greater transcriptional activity than the C allele. Similarly, more CD44 expression was shown in rs13347T carriers than C carriers in our western blotting results. All these findings suggest that CD44 rs13347C>T polymorphism may affect NPC development by improving CD44 expression.

Introduction

Nasopharyngeal carcinoma (NPC), commonly known as nasopharyngeal cancer, is extremely common in southern regions of China, particularly in Guangdong Province, accounting for 18% of all cancers in China (1). It is sometimes referred to as Cantonese cancer because it occurs in ~25 cases per 100 000 people in this region, 25 times higher than the rest of the world. NPC is also quite common in Taiwan (2). This phenomenon could be due to the Southeast Asian diet, which typically includes consumption of salted vegetables, fish and meat (2). Besides, aetiology of nasopharyngeal cancer also includes Epstein–Barr virus (EBV) infection (3,4) and genetic susceptibility (5,6).

The 5-year-survival rate of NPC has been relatively low for tumour cells always have migrated at diagnosis. Cancer metasta-sis is a highly complex multistep process in the body, needing a

variety of cytokines (7,8). Studies have shown that glycoprotein molecules encoded by CD44 were involved in the specific adhe-sion process between cells, as well as cancer cells and basement membrane (9). The human CD44 gene is mapped to chromo-somal locus 11p13 and encodes a ubiquitously expressed fam-ily of cell adhesion glycoprotein comprising four regions: an N-terminal extracellular domain, a membrane proximal region, a transmembrane domain and a cytoplasmic tail (10). By cell–extracellular matrix and cell–cell adhesive interactions, CD44 plays an indispensable role in tumour pathology, involved in cell differentiation, invasion and metastasis (10–12). Also, strong association between CD44 expression and NPC risk has been reported in some studies (13,14). Correspondingly, some studies have recently indicated qualitative and quantitative changes in CD44 immunophenotype in NPC (15).

Since expression of CD44 is closely related to development of NPC and genetic variations in certain genes may change their expression level (16), we hypothesise that CD44 genetic variations that can theoretically affect its protein expression may affect individuals’ risk of NPC. Five single nucleotide pol-ymorphisms (SNPs) of CD44 gene with minor allele frequency (MAF) > 0.05 in Asian population were selected from the HapMap database to evaluate their contribution to risk of devel-oping NPC. One of them is an A/G polymorphism (rs1425802) in the promoter region, the conversion from A to G cause loss of an Nkx-2 binding site, which may theoretically affect the CD44 transcriptional activity. Another T/C (rs713330) polymorphism in the intron was linkage disequilibrium (LD) with the non-syn-onymous rs9666607 G>A polymorphism, which may change the 417 amino acid from Arg to Lys. The other three polymor-phisms (rs13347C/T, rs10836347C/T and rs11821102G/A) all locate in the 3′UTR of CD44, each of which can cause change in binding ability of certain microRNA between the two dif-ferent alleles. No published research article has investigated theses polymorphisms and NPC risk. So, we carried out a hospital-based case–control study including 906 patients with NPC and 943 cancer-free controls to investigate association between these polymorphisms and NPC risk.

Materials and methods

Study subjectsAll subjects in the two-stage case–control study were ethnically homogenous Han Chinese: 906 patients with newly diagnosed primary NPC and 943 healthy controls from Zhejiang Province in Eastern China. Patients with newly diagnosed NPC (n = 906) were consecutively recruited from 2001 to 2009 at urban hospitals including Sir Run Run Shaw Hospital of Zhejiang University (Hangzhou) and Zhejiang Cancer Hospital (Hangzhou). Cancer-free controls (n = 943) were randomly selected from a 3500 individual nutritional survey conducted in Zhejiang Province in the same period as the cases were collected (17,18). EBV capsid antigen immunoglobulin A antibodies (EB-VCA-IgA) and immunoglobulin-A antibodies to EBV early antigen were confirmed by serologic testing at the time of study enrolment. Briefly, antibodies to several antigen complexes may be measured. These antigens are the viral capsid antigen, the early antigen and the EBV nuclear antigen (EBNA). In addition, differentiation of immunoglobulin G and M subclasses to the viral capsid antigen can often be helpful for confirmation. When the ‘mono spot’ test is negative, the optimal combination of EBV serologic testing consists of the

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antibody titration of four markers: IgM and IgG to the viral capsid antigen, IgM to the early antigen and antibody to EBNA. The tumour, node, metastasis classification and tumour staging were evaluated according to the 2002 American Joint Committee on Cancer staging system. There were no age, stage and histology restriction for cases. The selection criteria for controls included no history of cancer and frequency matched to cases on age (55 years). The clinical features of the patients are summarised in Table I. Patients or controls who recently (in last 6  months) had blood transfusions were excluded. The definition of the smoking status and the family history of cancer have been described elsewhere (19). At recruitment, informed consent was obtained from each subject. All participants recruited in our study had signed a consent form before the questionnaire interview, and our study was approved by the Medical Ethics Committee of Sir Run Run Shaw Hospital, Zhejiang University.

TagSNPs selectionBioinformatics analysis with Haploview software 4.2 was performed to ana-lyse the haplotype block based on the Chinese Han Beijing population data of HapMap (HapMap Data Rel 27 Phase II + III, February 2009, on NCBI B36 assembly, dbSNP b126). Six tagSNPs were found to cover all the potential functional common SNPs (MAF > 0.05) in CD44 gene: rs8193, rs11821102, rs10836347 and rs13347 in the 3′UTR; rs1425802 in the promoter and rs9666607 in exon region. Among them, rs8193 and rs13347 were in highly LD (D′ = 1.0, r2 = 0.527), so the selection of rs13347 is enough to represent the two SNPs. Besides, due to the difficulty in genotyping rs9666607 by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) method, we choose rs713330 which is in complete LD with rs9666607 (D′ = 1.0, r2 = 1) to replace it.

Genotyping analysisGenomic DNA was isolated from the peripheral blood lymphocytes of the study subjects. MassArray (Sequenom, SanDiego, CA, USA) was used for genotyping all markers using allele-specific MALDI-TOF mass spec-trometry (20–22). Primers and multiplex reactions were designed using the RealSNP.com Website. All patients with NPC and healthy controls were genotyped for rs10836347, rs13347, rs1425802, rs11821102 and rs713330 polymorphisms.

Construction of CD44 3′UTR luciferase reporter plasmidsBased on bioinformatics analysis, CD44 rs13347 C not T is predicted to lie in a hay-mir-509-3p binding site. Therefore, we hypothesised that hsa-mir-509-3p would bind tightly to CD44 mRNA transcripts containing the C allele, nega-tively regulating CD44 expression. To test this hypothesis, the T and C allelic reporter constructs were, respectively, prepared by amplifying a 362-bp CD44 3′UTR region from subjects homozygous for the T and C allele including the artificial XhoI and NotI enzymes restriction sites with forward primer 5′-ATC GCT CGA G GG CCA TTG TCA ACG GAGA-3′ and reverse primer 5′- ATG CGC GGC CGC CAG GCT TGA AAT ATG GAT TCG-3′. The amplified fragments were then cleaved with the XhoI and NotI enzymes (New England BioLabs, Ipswich, MA, USA). The psiCHECK2 vector (Promega, Madison, WI, USA) was also cleaved with the XhoI and NotI enzymes, and the above-prepared fragment and psiCHECK2 vector were then ligated by T4 DNA ligase (New England BioLabs). The two constructs were sequenced to confirm the allele, the orientation and integrity of each insert.

Transient transfections and luciferase assaysNasopharyngeal carcinoma cell line CNE-1 was maintained in Dulbecco’s modified Eagle’s medium with high glucose (Gibco) supplemented with 10% heat-inactivated foetal bovine serum (Gibco) and 50  μg/ml streptomycin (Gibco) at a 37°C incubator supplemented with 5% CO2. Cells were seeded at 1 × 105 cells per well in 24-well plates (BD Biosciences, Bedford, MA, USA). Sixteen hours after the plating, cells were transfected by Lipofectamine 2000 according to manufacturer’s suggestion. In each well, 800 ng psiCHECK-2-CD44-3′UTR vectors were cotransfected with 50 pmol hsa-mir-509-3p mimics (Ambion, Austin, TX, USA) and 40 pmol inhibitor accordingly. There are six replicates for each group, and the experiment is repeated at least three times. Twenty-four hours after transfection, cells were harvested by the addition of 100 μl passively buffer. Renilla luciferase activities in cell lysate were meas-ured with the Dual-Luciferase Reporter assay system (Promega) in TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA, USA) and were normalised with the firefly luciferase activities.

Western blotting analysis and immunohistochemistry analysisTo analyse the correlation between rs13347 C>T polymorphism in 3′UTR of CD44 and the protein expression levels in NPC tissues, the western blotting assays and immunohistochemistry analysis were performed. The western blot-ting assay was carried out as follows: generally, 41 NPC tissues were washed with phosphate-buffered saline, and total tissue extracts were made by using a detergent lysis buffer. Sixty micrograms of total proteins were run on a sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to poly-vinylidene difluoride (Millipore, Billerica, MA, USA). The membrane was blocked with 5% milk in Tris-buffered saline with 0.05% Tween-20 for 1 h at room temperature with constant agitation. Both the polyclonal antibody against CD44 and the monoclonal antibody against β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The membranes were incubated overnight at 4°C with the primary antibody diluted 1:1000, and the proteins were detected with a Phototope-horseradish peroxidase western blot detection kit (Cell Signaling Technology, Danvers, MA, USA). The CD44 protein expression levels were normalised to that of β-actin by calculating the relative expression levels.

Statistical analysisChi square tests were used to examine the differences in the distributions of genotypes between cases and controls. The association between the CD44 poly-morphisms and risk of NPC was estimated by odds ratios (ORs) and their 95% confidence intervals, which were calculated by unconditional logistic regression models. We tested the null hypotheses of multiplicative gene–gene interactions by evaluated departures from multiplicative joint effect models by including main effect variables and their product terms in the logistic regression model (21). The correlation of genotypes and clinical parameters was analysed via the Fisher’s exact test or Chi square test as appropriate. The differences in the luciferase reporter activity and normalised expression values in cancer tissue of CD44 (western blot ratio and immunohistochemistry scores) between each allele were analysed by Kruskal–Wallis one-way analysis of variance (ANOVA). The tests were all two-sided and analysed using the SAS software (version 9.1; SAS Institute, Cary, NC, USA). P-value <0.05 was considered statistically significant.

Results

Genotypes and risk of NPCThe association between CD44 rs13347C>T, rs10836347 C>T, rs1425802A>G, rs11821102G>A and rs713330 T>C

Table I. Characteristics of selected patients with NPC and control subjects from the Chinese population used for the association study

Characteristics Patients (906), n (%) Controls (943), n (%)

Age (years) ≤55 548 (60.5) 557 (59.1) >55 358 (39.5) 386 (40.9)Sex Male 645 (71.2) 663 (70.3) Female 261 (28.8) 280 (29.7)Smoking status Ever 479 (52.9) 448 (47.5) Never 427 (47.1) 495 (52.5)Drinking status Ever 444 (49.0) 450 (47.7) Never 462 (51.0) 493 (52.3)

Body mass index ≤20 199 (22.0) 228 (24.2) 20 < body mass index ≤ 28 638 (70.4) 679 (72.0) >28 69 (7.6) 36 (3.8)

Family history of cancer Positive 96 (10.6) 93 (9.9) Negative 810 (89.4) 850 (90.1)EBV infection status Positive 685 (75.6) Negative 221 (24.4)Tumour grade Low 346 (38.2) Intermediate 461 (50.9) High 36 (4.0) Data missing 63 (6.9)Stage I 56 (11.7) II 235 (27.3) III 408 (40.4) IV 207 (20.6)

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and NPC risk was performed by our laboratories (906 cases and 943 controls). Genotypes were confirmed by direct sequencing. The observed genotype frequencies of the four polymorphisms in controls conformed to the Hardy–Weinberg equilibrium (P = 0.48 for rs13347, 0.52 for rs10836347, 0.95 for rs1425802, 0.22 for rs11821102 and 0.81 for rs713330). Genotyping results showed that only rs13347 was statistically significantly associated with NPC (Table II). The frequency of the rs13347 TT and CT genotype was significantly higher in patients with NPC (Ptrend < 10–5) compared with the healthy controls. The adjusted OR of carrying the rs13347 CT and TT genotype in cancer patient groups were 2.19 and 3.98, respectively, compared with the rs13347 CC genotype.

Stratification analysis of CD44 rs13347 genotypes and risk of NPCThe risk of NPC related to CD44 rs13347 genotypes was further examined with stratification by subgroups of age, sex, smoking, drinking, EBV infection and family history of cancer. As shown in Table III, we observed significant difference in the genotype frequency between EBV-negative patients and EBV-positive patients (P = 0.02). Compared with the CC genotype, the T allele carriers (CT+TT) had 2.82-fold increased risk for developing NPC in patients with EBV infection. As for those without EBV infection, the increased risk of CT+TT is only 1.85-fold. However, there were no differences in other subgroups.

Regulation effects of hsa-mir-509-3p on CD44 3′UTR translation efficiencyCompared with the psiCHECK-2-CD44-3′UTR-rs13347 T, the translation of Renilla luciferase of psiCHECK-2-CD44-3′UTR-rs13347 C was significantly reduced in the presence of hsa-mir-509-3p (50 pmol) in a concentration-dependent manner (P  <  0.05), which distinguished the magnitude of the effects of hsa-mir-509-3p on the transcription of different alleles (Figure 1). When psiCHECK-2-CD44-3′UTR with 50 pmol hsa-mir-509-3p and its corresponding inhibitor were cotransfected into CNE-1 cell, there appeared no significant difference in luciferase activity between the two recombinants (Figure 1). These results suggest that, indeed, hsa-mir-509-3p can bind and negatively regulate the transcription of CD44 in the presence of rs13347 C allele.

Effects of CD44 rs13347C>T variation on CD44 protein levelsAs shown in Figure 2A and B, we collected 41 tumour tissues from the untreated NPC patients with different rs13347C/T genotypes and found that the levels of CD44 protein of six cases carrying the TT genotype (0.965 ± 0.013) and 18 cases carrying the TC genotype (0.846 ± 0.247) were significantly higher than that of other 17 cases carrying the CC genotype (0.691 ± 0.0129) (ANOVA test: P < 0.001).

Table II. Genotype frequencies of the five SNPs in the CD44 gene in patients and controls and their association with NPC

Genotype Patients (906), n (%) Controls (943), n (%) Adjusted OR (95% confidence interval)a Pb

rs13347 C>T CC 386 (42.6) 606 (64.3) 1.00 (reference) CT 418 (46.1) 297 (31.5) 2.19 (1.93–2.89) TT 102 (11.3) 40 (4.2) 3.98 (2.77–6.09) <10–5

CT+TT 520 (57.4) 337 (35.7) 2.58 (2.13–3.13) C 1190 (65.7) 1509 (80.0) 1.00 (reference) T 622 (34.3) 377 (20.0) 2.17 (1.86–2.53)rs10836347 C>T CC 785 (86.7) 792 (84.0) 1.00 (reference) CT 118 (13.0) 147 (15.6) 0.80 (0.61–1.05) TT 3 (0.3) 4 (0.4) 0.78 (0.17–3.63) 0.109 CT+TT 121 (13.3) 151 (16.0) 0.80 (0.62–1.04) C 1688 (93.2) 1731 (91.8) 1.00 (reference) T 124 (6.8) 155 (8.2) 0.81 (0.63–1.05)rs1425802 A>G AA 270 (29.8) 299 (31.7) 1.00 (reference) AG 450 (49.7) 442 (46.9) 1.17 (0.94–1.45) GG 186 (20.5) 202 (21.4) 1.03 (0.80–1.35) 0.759 AG+GG 636 (70.2) 644 (68.3) 1.12 (0.92–1.38) A 990 (54.6) 1040 (55.1) 1.00 (reference) G 822 (45.4) 846 (44.9) 1.03 (0.90–1.18)rs11821102 G>A GG 796 (87.9) 805 (85.4) 1.00 (reference) AG 100 (11.0) 129 (13.7) 0.76 (0.57–1.01) AA 10 (1.1) 9 (0.9) 1.02 (0.41–2.56) 0.185 AG+AA 110 (12.1) 138 (14.6) 0.78 (0.59–1.03) A 1692 (93.4) 1739 (92.2) 1.00 (reference) G 120 (6.6) 147 (7.8) 0.81 (0.63–1.04)rs713330 T>C TT 732 (80.8) 751 (79.6) 1.00 (reference) CT 164 (18.1) 180 (19.1) 0.93 (0.74–1.19) CC 10 (1.1) 12 (1.3) 0.90 (0.38–2.15) 0.513 CT+CC 174 (19.2) 192 (20.4) 0.93 (0.74–1.18) T 1628 (89.8) 1682 (89.2) 1.00 (reference) C 184 (10.2) 204 (10.8) 0.94 (0.76–1.16)

aData were calculated by logistic regression analysis with adjusted for age, sex, body mass index, family history of cancer, smoking and drinking status.bTests for trend of odds were two sided and were based on likelihood ratio tests assuming a multiplicative model.

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Discussion

Association between CD44 polymorphisms and NPC risk has not been investigated in any population using case–control study. In our present molecular epidemiological study, we

sought to identify genetic factors that confer individual susceptibility to NPC. Our results obtained by genotyping 906 patients with NPC and 943 controls from two study centres showed that the functional variation rs13347 T in the CD44 was associated with increased risk for developing NPC, especially in individuals with EBV infection. However, there exists no significant difference in the susceptibility to NPC between different genotypes of the other four polymorphisms.

CD44 is a polymorphic family of immunologically related cell surface proteoglycans and glycoprotein, of which the multiple functions are generated by their binding with the hyaluronic acid (HA) and some other extracellular molecules (23,24). By cell–cell and cell–extracellular matrix adhesive interactions, CD44 not only participates in some fundamental biological processes including lymphocyte activation and hom-ing, cell migration, haematopoiesis, wound healing, inflam-mation, embryonal development and apoptosis but also is involved in neoplasm invasion, metastasis, clinical stage and drug resistance (10,25). It has been reported that tumour cells with relatively high concentrations of CD44 proteins were capable of forming more aggressive tumours in animal experi-ments (26,27). Correspondingly, local injection of hyaluronan oligomers, on the other hand, can inhibit tumour formation by melanoma by interfering with endogenous CD44–HA binding. In addition, many molecular biology studies have found that CD44V was over-expressioned in laryngeal cancer, thyroid

Fig. 1. Transient reporter gene expression assays modulated by hsa-mir-509-3p with constructs containing 362-bp of CD44 3′UTR region. Relative luciferase activity of the psiCHECK-2-CD44-3′UTR-C-allele and psiCHECK-2-CD44-T-allele constructs cotransfected with 50 pmol hsa-mir-509-3p and 40 pmol hsa-mir-509-3p inhibitor, respectively. Assay was performed in CNE-1 cell. Six replicates for each group and the experiment repeated at least three times. Data are mean ± SE.

Table III. Stratification analysis of the CD44 rs13347C>T genotypes by selected variables in patients with NPC and controls

Characteristics Patients (n = 906) Controls (n = 943) Adjusted OR (95% confidence interval)a

Pb

CC, n (%) CT+TT, n (%) CC, n (%) CT+TT, n (%) CT+TT versus CC

Age (years) ≤55 231 (25.5) 317 (35.0) 372 (39.5) 185 (19.6) 2.76 (2.16–3.52) >55 155 (17.1) 203 (22.4) 234 (24.8) 152 (16.1) 2.00 (1.50–2.68) 0.10Sex Male 280 (30.9) 365 (40.3) 421 (44.6) 242 (25.7) 2.27 (1.82–2.83) Female 106 (11.7) 155 (17.1) 185 (19.6) 95 (10.1) 2.85 (2.01–4.04) 0.28Smoking status Ever 203 (22.4) 276 (30.5) 276 (29.3) 172 (18.2) 2.18 (1.68–2.84) Never 183 (20.2) 244 (26.9) 330 (35.0) 165 (17.5) 2.67 (2.04–3.49) 0.29Drinking status Ever 192 (21.2) 252 (27.8) 290 (30.8) 160 (16.9) 2.38 (1.82–3.11) Never 194 (21.4) 268 (29.6) 316 (33.5) 177 (18.8) 2.47 (1.90–3.20) 0.85Body mass index ≤20 88 (9.7) 111 (12.3) 146 (15.5) 82 (8.7) 2.25 (1.52–3.31) 20 < body mass index ≤ 28 270 (29.8) 368 (40.6) 432 (45.8) 242 (25.7) 2.46 (1.97–3.07) 0.91 >28 28 (3.1) 41 (4.5) 28 (3.0) 13 (1.4) 2.59 (1.13–5.96)Family history of cancer Positive 34 (3.8) 62 (6.8) 75 (8.0) 18 (1.9) 2.17 (1.78–2.64) Negative 352 (38.9) 458 (50.5) 531 (56.3) 319 (33.8) 1.82 (0.84–3.96) 0.67EBV infection status Positive 267 (29.5) 418 (46.1) 606 (64.3) 337 (35.7) 2.82 (2.30–3.45) Negative 109 (12.0) 112 (12.4) 606 (64.3) 337 (35.7) 1.85 (1.38–2.48) 0.02Tumour grade Low 153 (16.9) 193 (21.3) 606 (64.3) 337 (35.7) 2.27 (1.77–2.91) Intermediate 194 (21.4) 267 (29.5) 606 (64.3) 337 (35.7) 2.47 (1.97–3.11) High 16 (1.8) 20 (2.2) 606 (64.3) 337 (35.7) 2.25 (1.15–4.40) Data missing 23 (2.5) 40 (4.4) 606 (64.3) 337 (35.7) 2.88 (1.72–4.83) 0.86Stage I 18 (2.0) 38 (4.2) 606 (64.3) 337 (35.7) 3.80 (2.13–6.76) II 97 (10.7) 138 (15.2) 606 (64.3) 337 (35.7) 2.56 (1.91–3.43) III 174 (19.2) 234 (25.8) 606 (64.3) 337 (35.7) 2.42 (1.91–3.07) IV 97 (10.7) 110 (12.2) 606 (64.3) 337 (35.7) 2.04 (1.51–2.76) 0.30

aORs were adjusted for age, sex, body mass index and family history of cancer in a logistic regression model.bP-value of the test for multiplicative interaction between stratum-related variables and CD44 gene (rs13347C>T CT+TT versus CC genotypes), P < 0.05 was defined as statistically significant.

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cancer, oesophageal cancer, lung cancer, gastric cancer, colo-rectal cancer, breast cancer, bladder cancer, liver cancer, cervi-cal cancer, ovarian cancer and some other tumour tissues than in normal tissues, and it is thought that CD44V expression is closely related to the occurrence, development and prognosis of malignant tumour (28).

Unexceptionally, CD44 also plays an important role in NPC development. Shi et al. (29) used RNA silencing technique to knock down CD44 gene expression human NPC CNE-2L2 cells and characterised the consequence of such inhibition on tumour cell proliferation in vitro and tumorigenicity and metas-tasis potentiality in vivo. It was found that the CD44 suppres-sion made CNE-2L2 cells display decreased cell proliferation and colony formation rate and G1 arrest in cell cycle, inhibi-tion of tumour formation and lung metastasis in nude mice. Moreover, an inhibitive effect of siCD44 therapy was also showed to be mediated by adenovirus on growth of the tumours inoculated with CNE-2L2 cells in nude mice (29).

Recent studies suggest that cancer-initiating cells (CICs) may be responsible for tumorigenesis and contribute to some indi-viduals’ resistance to cancer therapy (30). In the past 10 years, a large number of articles have studied the role of such cells in the development and maintenance of the tumours. However, only three articles (two in Chinese) about the existence and function of CIC or stem-like cancer cells in NPC cells have been reported (31). Su et al. (32) have separated stem-like can-cer cells from human NPC SUNE-1 5-8F cell line with cell sur-face marker CD44. They found that sorted CD44+ cells showed stronger proliferative capacity than unsorted cells and CD44− cells. Moreover, they detected the gene expression levels of Bmi-1 and Oct-4 which are related to stem cell self-renewal and differentiation (33) separately in CD44+ cells and CD44− cells, and it was found that CD44+ cell group had significantly higher expression of the two genes than CD44− group, suggesting a stronger self-renewal ability of CD44+ cells. Similar result was also proved in another article by Prince et al. (34). In addition,

CD44+ cell group was also found to be more resistant to chemo-therapeutic drugs and X-rays than CD44− cells. All these results suggested that CD44 was a surface marker of human NPC initi-ating cells and played an important role in the development and maintenance of the tumours NPC. Consistently, our study also found that carriers of rs13347T allele had more CD44 expres-sion and were more susceptible to NPC.

Here, we found that rs13347T allele could increase the risk of NPC, especially in EBV carriers. It has been identified that the strong association between NPC and EBV is a unique fea-ture of NPC (35). The products of EBV genome might involve in the pathogenesis of NPC, considering the EBV genome can be detected in all cases of this malignancy. In addition to EBV-encoded protein-coding genes, such as EBNA1 and LMP1, high levels of non-coding EBV RNAs, including EBER1, EBER2 and multiple microRNAs, are also detected in latently infected NPC cells and tissues (36–39). However, for EBV affecting the association between rs13347 and the risk of NPC, the molecular mechanism underlying this still needs our further study.

As we all know, microRNAs have important function in regulating multiple processes linked to human cancer, such as cell apoptosis, proliferation and migration (40), and it has been found by increasing evidence that microRNAs play an impor-tant role in the occurrence and development of many human cancers (41,42). Zhai et  al. (43) found that miR-509-3p was associated with cell invasion and migration and involved in the apoptosis of renal cell carcinoma. Jiang et al. (22) found that miR-509-3p might affect breast cancer development and prog-nosis. In this study, we provided the first evidence that miR-509-3p could involve in the development of NPC.

Although it has been found in our study that CD44 rs13347 CT and TT genotypes were associated with increased risk of NPC, the study design may still cause certain limitations. For example, selection bias and/or systematic error may occur because the cases were from hospital and the controls were from community. However, the fact that the genotype frequencies of all the five studied polymorphic sites among controls could fit the Hardy–Weinberg disequilibrium law suggested the randomness of subject selection; we have achieved a >95% study power (two-sided test, α  =  0.05) to detect an OR of 2.58 for the rs13347 CT+TT genotypes compared with the rs13347 CC genotype, suggesting that our finding is noteworthy.

In conclusion, our study indicated that compared with CC genotype, the CD44 rs13347 variant genotypes (CT+TT) can significantly elevate the risk of NPC in both southern and east-ern Chinese populations. Moreover, the phenomenon is more obvious in EBV carriers. To our best knowledge, our study first demonstrated a significant association between the CD44 rs13347 C/T polymorphism and risk of NPC. Moreover, larger, preferably population-based case–control studies, as well as well-designed mechanistic studies, are warranted to validate our findings in Chinese population or to investigate the associa-tion between this polymorphism with different tumour in dif-ferent ethnicity.

Funding

Zhejiang Provincial Natural Science Foundation of China (Y2100449 and Y2110410).

Conflict of interest statement: None declared.

Fig. 2. Association between the CD44 protein expression and the CD44 rs13347C>T polymorphism. (A) CD44 protein levels in 41 NPC tissues from individuals who carried different rs13347 genotypes. The CD44 protein expression levels were normalised to that of β-actin by calculating the relative expression levels. (B) Relative protein expression levels in 41 NPC tissues from individuals who carried different genotypes. Bars, standard deviation; P-value for all comparisons of expression difference by different genotypes.

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