The association between XRCC6/Ku70 , an upstream player in the DNA double-strand break repair system, and the risk of nasopharyngeal carcinoma (NPC) was examined. In this case–control study, 176 NPC patients and 352 cancer-free controls were genotyped, and the associations of XRCC6 promoter T–991C (rs5751129), promoter G–57C (rs2267437), promoter G–31A (rs132770), and intron 3 (rs132774) polymorphisms with NPC risk were evaluated. NPC tissue samples were also assessed for their XRCC6 mRNA and protein expression by real-time quantitative reverse transcription PCR and Western blotting, respectively. With regard to the XRCC6 promoter T–991C, the TC and CC genotypes were associated with a significantly increased risk of NPC compared with wild-type TT genotype (adjusted odds ratio 2.02 and 3.42, 95% confidence interval 1.21–3.32 and 1.28–8.94, P = 0.0072 and 0.0165, respectively). The mRNA and protein expression levels for NPC tissues revealed significantly lower XRCC6 mRNA and protein expression in the NPC samples with TC/CC genotypes compared to those with the TT genotype ( P = 0.0210 and 0.0164, respectively). These findings suggest that XRCC6 may play an important role in the carcinogenesis of NPC and could serve as a chemotherapeutic target for personalized medicine and therapy.
Nasopharyngeal carcinoma (NPC) – cancer originating in the nasopharynx – occurs relatively infrequently in the West (age-standardized incidence rate (ASR) of <1/100,000), but remains a leading tumour among those in southern China (ASR 30–50/100,000), Southeast Asia (ASR 9–12/100,000), and Taiwan (ASR 8.2–8.4/100,000). The geographical pattern of incidence suggests an interaction of complicated environmental and genetic factors. Although the aetiology of NPC remains to be elucidated, those people with an Epstein–Barr virus (EBV) infection, environmental risk factor exposure, risky dietary habits, and risky genotypes with single nucleotide polymorphisms (SNPs), may have a higher susceptibility to NPC.
The integrity of the human genome is controlled by the human DNA repair system, and accumulated mutations or defects are thought to be essential for carcinogenesis. Therefore, it is reasonable to hypothesize that the loss of DNA repair capacity or a decrease in the function via genomic variation might have a significant influence on the carcinogenesis of NPC. In humans, genetic variations affecting non-homologous end-joining (NHEJ), together with those affecting the alternative homologous recombination DNA double-strand break (DSB) repair system, have been postulated to be important contributors to the aetiology of cancer.
In recent years, several proteins involved in the NHEJ pathway have been identified, including ligase IV, XRCC4, XRCC6 (Ku70), XRCC5 (Ku80), DNA-PKcs, Artemis, and XLF. Inappropriate NHEJ has been shown to lead to translocations and telomere fusion, which are hallmarks of tumour cells. As for NHEJ, some genetic polymorphisms have been reported to influence DNA repair capacity and confer a predisposition to several types of cancer, including skin, breast, bladder, lung, and oral cancers. However, there is no information regarding NPC and NHEJ gene polymorphisms.
Recent epidemiological studies have investigated the association between XRCC6 polymorphism and the risk of various types of cancer, including gastric cancer, oral cancer, breast cancer, lung cancer, and renal cell carcinoma. We hypothesized that the different XRCC6 genotypes, together with their RNA and protein expression, may also contribute to NPC susceptibility.
To test this hypothesis, the present study was designed to investigate the association of XRCC6 genotypes with the risk of NPC; this was a case–control study involving a population in central Taiwan. In addition, we also investigated the association of the XRCC6 mRNA and protein expression patterns with NPC risk by real-time polymerase chain reaction (PCR) and Western blotting, respectively, in order to assess the potential functional effect of XRCC6 genotype on NPC risk. To the best of our knowledge, this is the first study to evaluate the association between the XRCC6 genotypes and NPC susceptibility and to explore the potential function of XRCC6 in NPC at the same time.
One hundred and seventy-six patients diagnosed with NPC were recruited at the general surgery outpatient clinics of the study hospital in Taichung, Taiwan, between 2003 and 2009. All patients participated voluntarily, completed a self-administered questionnaire, and provided peripheral blood samples. The questionnaire included questions on history and frequency of alcohol consumption, betel quid chewing, and smoking habits, and ‘ever’ was defined as more than twice a week for years. Self-reported alcohol consumption, betel quid chewing, and smoking habits were evaluated and classified as categorical variables.
For each case patient, two age- and gender-matched healthy controls, who had no NPC or other type of cancer, were selected from those attending the hospital for a health examination (age matching was done within less than 5 years of the case patient’s first diagnosis). These volunteers attended the hospital for regular health assessments by multidisciplinary team approach with registered health practitioners during the years 2002–2012; most of the volunteers underwent health examinations every 5–6 months. A total of 10,358 participants aged 1–104 years were recruited into this cohort, and those who were cancer-free by the age at diagnosis of the case patient, according to the International Classification of Diseases, Ninth Revision (ICD-9) codes, were chosen. Finally, 352 participants were included for analysis in the present study. For the convenience of the gene–environment interaction analysis, we preferentially selected those with alcoholism, betel quid chewing, and smoking habits when selecting the controls for genotyping and further analysis. Thus, the control group was not a general population control, but rather an alcohol-, betel quid-, and smoking-related control group. The overall agreement rate in this study was more than 85% in collection.
The study was approved by the institutional review board of the medical university hospital and written informed consent was obtained from all participants.
The total genomic DNA of each subject was extracted from peripheral blood leucocytes using a QIAamp Blood Mini Kit (Qiagen, Taipei, Taiwan) and stored as reported previously. The primers used for XRCC6 were as follows: promoter T–991C, forward 5′-AACTCATGGACCCACGGTTGTGA-3′, and reverse 5′-CAACTTAAATACAGGAATGTCTTG-3′; promoter G–57C, forward 5′-AAACTTCAGACCACTCTCTTCT-3′, and reverse 5′-AAGCCGCTGCCGGGTGCCCGA-3′; promoter G–31A, forward 5′-TACAGTCCTGACGTAGAAG-3′, and reverse 5′-AAGCGACCAACTTGGACAGA-3′; intron 3, forward 5′-GTATACTTACTGCATTCTGG-3′, and reverse 5′-CATAAGTGCTCAGTACCTAT-3′. The following cycling conditions were used: one cycle at 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s; and a final extension at 72 °C for 10 min.
Restriction fragment length polymorphism (RFLP) conditions
For the XRCC6 promoter T–991C, the resulting 301-base pair (bp) PCR product was mixed with 2 U Dpn II. The restriction site was located at −991, with a T/C polymorphism; the C form PCR products could be digested further, while the T form could not. Two fragments of 101 bp and 200 bp were present if the product was the digestible C form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) C/C homozygote (digested), (2) T/T homozygote (undigested), or (3) C/T heterozygote.
For the XRCC6 promoter G–57C, the resulting 298-bp PCR product was mixed with 2 U Hae II. The restriction site was located at −57, with a G/C polymorphism; the G form PCR products could be digested further, while the C form could not. Two fractions of 103 bp and 195 bp were present if the product was the digestible G form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) G/G homozygote (digested), (2) C/C homozygote (undigested), or (3) C/G heterozygote.
For the XRCC6 promoter G–31A, the resulting 226-bp PCR product was mixed with 2 U Mnl I. The restriction site was located at −31, with a G/A polymorphism; the A form PCR products could be digested further, while the G form could not. Two fractions of 80 bp and 146 bp were present if the product was the digestible A form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) A/A homozygote (digested), (2) G/G homozygote (undigested), or (3) A/G heterozygote.
For the XRCC6 intron 3, the resulting 160-bp PCR product was mixed with 2 U Msc I. The restriction site was located at intron 3, with a GG/GC polymorphism; the GG form PCR products could be digested further, while the GC form could not. Two fractions of 46 bp and 114 bp were present if the product was the digestible GG form. The reaction was incubated for 2 h at 37 °C. Then, 10 μl of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as (1) GG/GG homozygote (digested), (2) GC/GC homozygote (undigested), or (3) GG/GC heterozygote.
mRNA XRCC6 expression pattern
To evaluate the correlation between XRCC6 mRNA expression and XRCC6 polymorphism, 20 surgically removed NPC tissue samples obtained from sites adjacent to tumours with different genotypes were subjected to extraction of the total RNA using Trizol Reagent (Invitrogen, Carlsbad, CA, USA); the manufacturer’s protocol was followed. Total RNA was measured by real-time quantitative RT-PCR using an FTC-3000 real-time quantitative PCR instrument (Funglyn Biotech Inc., Canada). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal quantitative control. The primers used for amplification of XRCC6 mRNA were forward 5′-CGATAATGAAGGTTCTGGAAG-3′ and reverse 5′-CTGGAAGTGCTTGGTGAG-3′, while for GAPDH the primers were forward 5′-GAAATCCCATCACCATCTTCCAGG-3′ and reverse 5′-GAGCCCCAGCCTTCTCCATG-3′. Fold changes were normalized using the levels of GAPDH expression, and each assay was done at least in triplicate.
Western blotting analysis
The NPC specimens were homogenized in radio immunoprecipitation assay (RIPA) lysis buffer (Upstate Biotechnology Inc., Lake Placid, NY, USA), the homogenates were centrifuged at 10,000 × g for 30 min at 4 °C, and the supernatants were used for Western blotting. Samples were denatured by heating at 95 °C for 10 min, were separated on a 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel, and were then transferred to a nitrocellulose membrane (BioRad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% non-fat milk and incubated overnight at 4 °C with mouse monoclonal anti-human XRCC6 antibody (1:1000; BD Transduction Laboratories; BD Biosciences, Franklin Lakes, NJ, USA), and then with the corresponding horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Chemicon, Temecula, CA, USA) for 1 h at room temperature. After reaction with enhanced chemiluminescence (ECL) solution (Amersham, Arlington Heights, IL, USA), bound antibody was visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK). Finally, the blots were incubated at 56 °C for 18 min in stripping buffer (0.0626 M Tris–HCl, pH 6.7, 2% SDS, 0.1 M mercaptoethanol) and re-probed with a monoclonal mouse anti-β-actin antibody (Sigma, St. Louis, MO, USA) as the loading control. The optical density of each specific band was measured using a computer-assisted imaging analysis system (GeneTools Match software; Syngene).
The results only for those with successful genotyping and completed questionnaires are presented and analyzed in the tables. To ensure that the control subjects were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of XRCC6 SNPs in the control subjects from those expected under Hardy–Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s Chi-square test or Fisher’s exact test (when the expected number in any cell was less than five) was used to compare the distribution of the XRCC6 genotypes between cases and controls. The associations between the XRCC6 polymorphisms and NPC risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis, with adjustment for possible confounders. P < 0.05 was considered statistically significant, and statistical analyses were carried out using SPSS version 16.0 software (SPSS Inc., Chicago, IL, USA); tests were two-sided.
Basic comparisons between the case and control groups
Selected characteristics of the control and case subjects are summarized in Table 1 . All characteristics of the patients and controls were matched and no frequency distribution was statistically different between the two groups ( P > 0.05). In this selected population, personal habits including smoking, alcohol consumption, and betel quid chewing appeared not to be direct risk factors for NPC.
|Characteristics||Patients ( n = 176)||Controls ( n = 352)||P -value a|
|n (%), or mean ± SD||n (%), or mean ± SD|
|Age, years||49.3 ± 9.4||48.7 ± 10.8||0.7138|
|Male||128 (72.7%)||256 (72.7%)|
|Female||48 (27.3%)||96 (27.3%)|
|Cigarette smoker||73 (41.4%)||150 (42.6%)||0.8519|
|Betel quid chewer||54 (30.7%)||115 (32.7%)||0.6926|
|Alcohol drinker||72 (40.9%)||124 (35.2%)||0.2150|
Association of XRCC6 genotypes and NPC risk
The genotype distributions of the XRCC6 polymorphisms in the cases and controls are presented in Table 2 . Among the polymorphic sites, the most significant findings were found for the XRCC6 promoter T–991C genotyping. The ORs after adjusting for confounding factors (age, gender, and smoking, alcohol drinking and betel quid chewing status) for those subjects carrying TC and CC genotypes were 2.02 (95% CI 1.21–3.32) and 3.42 (95% CI 1.28–8.94), respectively, compared to those carrying the TT wild-type genotype. The P -value for trend was significant ( P = 0.0014). In the dominant (TC+CC vs. TT) and recessive (CC vs. TT+TC) models, the association between XRCC6 promoter T–991C polymorphism and the risk of NPC was also statistically significant (adjusted OR 2.15 and 2.45, 95% CI 1.40–3.19 and 1.23–7.74, respectively).
|NPC cases (%)||Controls (%)||aOR a (95% CI)||P -value b|
|Promoter T–991C (rs5751129)|
|TT||131 (74.4)||305 (86.6)||1.00 (Ref.)|
|TC||35 (19.9)||40 (11.4)||2.02 c (1.21–3.32)||0.0072|
|CC||10 (5.7)||7 (2.0)||3.42 c (1.28–8.94)||0.0165|
|P for trend||0.0014|
|(TC+CC) vs. TT||2.15 c (1.40–3.19)||0.0006|
|CC vs. (TT+TC)||2.45 c (1.23–7.74)||0.0341|
|Promoter G–57C (rs2267437)|
|CC||113 (64.2)||229 (65.1)||1.00 (Ref.)|
|CG||58 (33.0)||109 (31.0)||1.08 (0.77–1.58)||0.7644|
|GG||5 (2.8)||14 (3.9)||0.77 (0.35–1.84)||0.6239|
|P for trend||0.7479|
|(CG+GG) vs. CC||1.02 (0.78–1.49)||0.8474|
|GG vs. (CC+CG)||0.88 (0.39–1.98)||0.8140|
|Promoter G–31A (rs132770)|
|GG||144 (81.8)||298 (84.7)||1.00 (Ref.)|
|GA||25 (14.2)||43 (12.2)||1.14 (0.76–1.93)||0.4921|
|AA||7 (4.0)||11 (3.1)||1.26 (0.60–2.96)||0.6122|
|P for trend||0.6965|
|(GA+AA) vs. GG||1.18 (0.83–1.87)||0.4533|
|AA vs. (GG+GA)||1.22 (0.58–3.00)||0.6170|
|Intron 3 (rs132774)|
|GG||144 (81.8)||279 (79.3)||1.00 (Ref.)|
|GC||32 (18.2)||73 (20.7)||0.85 (0.58–1.29)||0.5633|