The Contribution of XRCC6/Ku70 to Hepatocellular Carcinoma in Taiwan

Materials and Methods

Study population. Two-hundred and ninety-eight patients diagnosed with HCC were recruited at the Departments of General Surgery at the China Medical University Hospital, Taiwan, in 2004-2010. Each patient and non-cancerous healthy person (matched by gender, age and individual habits, such as smoking and alcohol drinking, from a random sampling from the Health Examination Cohort of China Medical University Hospital) completed a self-administered questionnaire and provided their peripheral blood samples. Each patient donated 3-5 ml venous blood and their tumor and non-tumor tissues after providing a written informed consent. The study was approved by the Institutional Review Board of the China Medical University.

Genotyping protocol. The total genomic DNA of each participant was extracted from the leucocytes of peripheral blood using a QIAamp Blood Mini Kit (Qiagen, Taipei, Taiwan) and stored as previously published (30, 31). The primers used for XRCC6 promoter C-991T were: forward 5’-AACTCATGGACCCACGGTTGTGA-3’, and reverse 5’-CAACTTAAATACAGGAATGTCTTG-3’; for promoter G-57C were: forward 5’-AAACTTCAGACCACTCTCTTCT-3’, and reverse 5’-AAGCCGCTGCCGGGTGCCCGA-3’; for promoter G-31A were: forward 5’-TACAGTCCTGACGTAGAAG-3’, and reverse 5’-AAGC GACCAACTTGGACAGA-3’; for intron-3 were: forward 5’-GTATAC TTACTGCATTCTGG-3’, and reverse 5’-CATAAGTG CTCAGTA CCTAT-3’. The following cycling conditions were performed: 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 C-991T, the resultant 301-bp PCR product was mixed with 2 U DpnII. The restriction site was located at -991 with a CT polymorphism, and the C form PCR products could be further digested, while that of the T form could not. Two fragments, 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 were loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either CC homozygote (digested), TT homozygote (undigested), or CT heterozygote. For the XRCC6 promoter G-57C, the resultant 298 bp PCR products were mixed with 2 U HaeII. The restriction site was located at -57 with a C/G polymorphism, and the G form PCR products could be further digested, while that of the C form could not. Two fractions, 103 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 were loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either GG homozygote (digested), CC homozygote (undigested), or CG heterozygote. For the XRCC6 promoter A-31G, the resultant 226 bp PCR products were mixed with 2 U MnlI. The restriction site was located at -31 with an AG polymorphism, and the A form PCR products could be further digested, while that of the G form could not. Two fractions, 80 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 were loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either AA homozygote (digested), GG homozygote (undigested), or AG heterozygote. For the XRCC6 promoter intron-3, the resultant 160 bp PCR products were mixed with 2 U MscI. The restriction site was located at intron 3 with a TGGCCA polymorphism, and the CCA form PCR products could be further digested while that of the TGG form could not. Two fractions, 46 and 114 bp, were present if the product was the digestible CCA form. The reaction was incubated for 2 h at 37°C. Then 10 μl of product were loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either CCACCA homozygote (digested), TGGTGG homozygote (undigested), or CCATGG heterozygote.

XRCC6 mRNA expression pattern. To evaluate the correlation between the XRCC6 mRNA expression and XRCC6 polymorphism, 30 surgically-removed liver tissue samples adjacent to tumors with different genotypes were subjected to extraction of the total RNA using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. The total RNA was measured by real-time quantitative RT-PCR using as FTC-3000 real-time quantitative PCR instrument (Funglyn Biotech Inc., Toronto, Canada). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal quantitative control. The primers used for amplification of the XRCC6 mRNA were: forward: 5’-CGATAA TGAAGGTTCTGGAAG-3’ and reverse: 5’-CTGGAAGTGCTT GGTGAG-3’, while for GAPDH the primers were: forward: 5’-GAAATCCCATCACCATCTTCCAGG-3’ and reverse: 5’-GAGCC CCAGCCTTCTCCATG-3’. Fold changes were normalized to the level of GAPDH expression, and each assay was carried out at least in triplicate.

Immunohistochemical staining for XRCC6. For liver specimens, tissue sections (5 μm) mounted on silanized slides (Dako Japan, Kyoto, Japan) were de-paraffinized with xylene and dehydrated in a graded series of ethanol. After rehydration in absolute ethanol for 15 s, the slides were heated by microwave in 10 mM citrate buffer (pH 6.0; Zymed Lab Inc., San Francisco, CA, USA) for 8 min. After washing in ice-cold phosphate-buffered saline (PBS), sections were pre-blocked for 10 min in an autoblocker (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK). They were then incubated overnight with mouse monoclonal antibody against human XRCC6 (1:100; Transduction Lab Inc., Franklin Lakes, NJ, USA). After three washes in PBS, the sections were incubated with horseradish peroxidase (HRP)-conjugated antibody against mouse IgG (Santa Cruz, California, USA) at room temperature for one hour. Finally, 3,3’-diaminobenzidine (Sigma, St. Louis, MO, USA) was added. Counterstaining was carried out with hematoxylin (Sigma). Images were captured using an Olympus BX 50 fluorescence microscope (Olympus Optical, Tokyo, Japan) and a Delta Vision disconsolation microscopic system operated by SPOT software (Diagnostic Instruments Inc., Michigan USA).

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Figure 1.

Analysis of X-ray repair complementing defective repair in Chinese hamster cells 6 (XRCC6) mRNA expression levels. A: Quantitative real-time polymerase chain reaction (RT-PCR) for three genotypes of XRCC6 from liver tissue samples was performed and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal quantitative control. Fold changes were normalized by the level of GAPDH expression, and each assay was performed at least in triplicate. B: The groups of TC and CC in (A) were combined and compared with the TT group.

Western blotting analysis. The liver specimens were homogenized in RIPA lysis buffer (Upstate Inc., Lake Placid, NY, USA); the homogenates were then 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, then separated on a 10% gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane (Bio-Rad, CA, USA). The membrane was blocked with 5% non-fat milk and incubated overnight at 4°C with mouse monoclonal antibody against human XRCC6 (1:1000; Transduction Lab Inc.), then with the corresponding horseradish peroxidase-conjugated goat antibody to mouse IgG (Chemicon, Temecula, CA) for 1 h at room temperature. After reaction with ECL solution (Amersham, Arlington Heights, IL, USA), the 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, 0.1M mercaptoethanol) and re-probed with a monoclonal mouse antibody to β-actin (Sigma) as the loading control. The optical density of each specific band was measured using a computer-assisted imaging analysis system (Gene Tools Match software; Syngene).

Statistical analyses. To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotypic frequencies of XRCC6 single nucleotide polymorphisms in the controls from those expected under the 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 HCC risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. A value of p<0.05 was considered statistically significant, and all statistical tests were two-sided.