Materials and methods
Patients
A total of 23 Tibetan patients with gastric cancer were recruited in this study. All the patients were admitted to the Affiliated Hospital of Qinghai University and diagnosed according to standard clinical and pathological criteria within the 30 months preceding the initiation of the study. The patients were diagnosed with stage II gastric cancer according to the approved international standards described by the American Joint Committee on Cancer Cancer Staging Manual (
4). A total of 40 Tibetans were randomly selected among healthy volunteers who were free of cancer and donated a blood sample. The control subjects were individually matched to the gastric cancer patients with respect to age, body mass index, educational level and
Helicobacter pylori (
H. pylori) infection.
This study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University and conformed to the tenets of the Helsinki Declaration. All the patients provided written informed consent prior to their inclusion in this study.
Mitochondrial gene sequence analysis and haplogroup classification
Mitochondrial gene sequence analysis was performed on all the participants. Total DNA was extracted from blood samples by conventional salting-out protocols. The entire mitochondrial genome was amplified by polymerase chain reaction (PCR) into 8 overlapping fragments using sets of light- and heavy-strand oligonucleotide primers. Each fragment was purified and analyzed by direct sequencing. The sequence results were then compared with the revised Cambridge reference sequence (
http://www.mitomap.org), GenBank ID:
NC_012920.1. PCR was performed in a 25-µl reaction containing 50 ng DNA, PCR buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.3 µM of each primer and 1.25 U Taq DNA polymerase. The reaction was performed with an initial denaturation step at 95°C for 5 min, followed by 30 cycles at 95°C for 30 sec, at 55°C for 30 sec and at 72°C for 45 sec, with a final elongation step at 72°C for 4 min.
The haplogroups of mtDNA were determined using phylogenetic analysis based on the mitochondrial phylogenetic tree as updated by van Oven and Kayser (
5). The classification details were previously described by Fan and Yao (
6).
mtDNA copy number assay
The mtDNA copy number assay was previously described in detail (
7). The ratio between the multi-copy mtDNA ND1 gene and the double-copy nuclear gene hemoglobin β was determined using an Applied Biosystems 7900HT Sequence Detection system (Applied Biosystems, Foster City, CA, USA). The mitochondrial ND1 and the nuclear hemoglobin β genes were individually amplified, using the ND1-forward (F)/ND1-reverse (R) and globin-F/globin-R primers, respectively. The primer pair for the mitochondrial ND1 gene was: ND1-F, 5′-ATTCGATGTTGAAGCCTGAGACT-3′; and ND1-R, 5′-TGACCCTTGGCCATAATATGATT-3′. The primer pair for the globin gene was: globin-F, 5′-ACCTCAAGGGCACCTTTGC-3′; and globin-R, 5′-AAAACATCAAGCGTCCCATAGAC-3′.
The PCR reaction mixture (15 µl) contained 0.5 ng DNA, 1X SYBR-Green master mix (Applied Biosystems) and 300 nmol of each primer. The PCR amplification consisted of a single denaturation-enzyme activation step for 8 min at 95°C, followed by 50 amplification cycles of 10 sec at 95°C, 10 sec at 60°C and 6 sec at 72°C. The data were analyzed using LightCycler software, version 3.5.3 (Roche Diagnostics, Mannheim, Germany). The ratio of mtDNA copy number to nuclear DNA was used as a measurement of mtDNA content in each specimen. All the samples were examined in triplicate and the average value was used for the ratio calculations. The R2 for each standard curve was ≥0.99. Standard deviations for the cycle of threshold (Ct) value were accepted at 0.25. The relative mtDNA copy number was calculated by a comparative Ct method, using the following equation: relative copy number (mtDNA/nDNA) = 2ΔCt (ΔCt = Ctβ-globin - CtND1). High reproducibility was observed with this assay, with a coefficient of variance of 8% (
8).