Analysis of the mitochondrial 4977 bp deletion in patients with hepatocellular carcinoma

Blood and tissues of 105 patients with HCC were collected at Zhuji People’s Hospital, Shaoxing, People’s Republic of China (PRC). The lesion and the nearby normal tissue from the subjects were confirmed histologically by a pathologist. Moreover, a total of 69 peripheral blood samples from age-matched donors who had a cancer free history and had no other known diseases that could be associated with mt defects were also recruited at the physical examination center in our hospital. Informed consent from all participants in this study was obtained under protocols approved by Zhuji People’s Hospital. Tumor tissues on slides were extracted separately under a microscope, and genomic DNA was isolated as previously described [8]. Briefly, total DNA was extracted from formalin-fixed, paraffinembedded tissues using the E.Z.N.A.® FFPE DNA isolation kit (Omega BioTek Inc, Norcross, GA, USA; http://omegabiotek.com/store/product/e-z-n-a-tissue-dna-kit/), according to the manufacturer’s protocol. The isolated DNA was eluted in 100 μL Tris-EDTA buffer included in the E.Z.N.A.® FFPE DNA kit and stored at −20 °C until required. Moreover, the genomic DNA from peripheral blood leukocytes of the patients and the controls were also extracted, using the universal Genomic DNA Extraction kit version 3.0 (Takara Bio Inc, Dalian, Japan). The quality of the isolated DNA was assessed by NanoDrop spectrophotometer (NanoDrop Technologies, Thermo Fisher Scientific Inc, Waltham, MA, USA). The optical density values of all samples ranged from 1.8 to 2.0.

To screen the 4977 bp deletion in mtDNA, nested-polymerase chain reaction (nested-PCR) analysis was performed; we used the DNA samples extracted from the tissues as templates. In brief, two pairs of nested primers for detection of the 4977 bp deletion were as follows: 1F: 5’-AAC CAC AGT TTC ATG CCC ATC-3’; 1R: 5’-TGT TAG TAA GGG TGG GGA AGC-3’; 2F: 5’-ACC CTA TTG CAC CCC CTC TAC-3’; 2R: 5’-CTT GTC AGG GAG GTA GCG ATG-3’. The PCR condition was as follows: pre denaturation at 94 °C for 5 min.; then 30 cycles at 94 °C for 10 seconds, 58 °C for 45 seconds and 72 °C for 50 seconds; and a final extension at 72 °C for 10 min. The PCR products were then electrophoresed on a 2.0% agarose gel. The presence of the 4977 bp deletion was indicated by the appearance of a 358 bp band that was verified by sequencing analysis [9]. The wild-type mtDNA as the template would not yield any PCR products under such conditions because of the large flanking region (>5 kb).

The DNA extracted from blood samples was used to determine its mtDNA copy number. The mtDNA copy number was measured with a real-time-PCR using an Applied Biosystems 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). One primer pair specific for the mtDNA (ND1) and another primer pair specific for the nuclear DNA (18s) were designed for relative quantification of the mtDNA copy number. The primer sequences for the mt ND1 gene were: forward primer (ND1-F): 5’-CCC TAA AAC CCG CCA CAT CT-3’; reverse primer (ND1-R): 5’-GAG CGA TGG TGA GAG CTA AGG T-3’. The primer pair used for the amplification of the nuclear gene 18s was as follows: forward primer (18s-F): 5’-TAG AGG GAC AAG TGG CGT TC-3’; reverse primer (18s-R): 5’-CGC TGA GCC AGT CAG TGT-3’. The 14 μL PCR mixture contained 1X SYBR Green mixture, 215 nM 18s-R primer, 215 nM 18s-F primer, and 0.4 ng of genomic DNA for ND1 and 18s. The thermal cycling conditions were 95 °C for 10 min., followed by 40 cycles of 95 °C for 15 seconds, and 60 °C for 1 min. for ND1 and 62 °C for 1 min. for 18s. Standard curves made by serial dilution of a reference DNA sample was used to determine the ratio of mtDNA copy number to the amount of nuclear DNA that was proportional to the mtDNA copy number in each cell. The efficiency of all quantitative PCR ranged from 99.0 to 110.0%. The R² for all standard curves was ≥0.99. Standard deviations (SD) for the cycle of threshold (Ct) duplicates was ≤0.25. All samples were assayed in duplicate on a 96-well plate.

The blood samples were immediately placed into sterile EDTA test tubes and centrifuged at 1500 g for 20 min. at 4 °C to collect plasma. Plasma was stored at −70 °C until assayed. The concentration of ROS was analyzed by enzyme-linked immunosorbent serologic assay (ELISA) using commercial kits (Nova Biomedical, Boston, MA, USA), in accordance with the manufacturer’s instructions.

All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software version 17.0 (SPSS Inc, Chicago, IL, USA). The mtDNA relative content and common deletion level were analyzed by the independence-samples t-test. A p value of <0.05 was considered statistically significant.

Nested-PCR is a highly sensitive method for detecting large scale deletions, even at very low levels. To investigate clinical implications of the common mt deletion, we first extracted the DNA from HCC patients and the normal tissues, as well as 69 age- and sex-matched healthy subjects. We also summarized some clinical characteristics and other risk factors for HCC in these patients including gender, age, history of alcohol consumption and tumor metastasis (Table 1). Of the 105 HCC patients, 10 (9.52%) showed the 4977 bp deletion, while this deletion was absent in healthy subjects and normal tissues (Figure 1). By molecular level, the 4977 bp deletion occurs between two 13 bp direct repeats at position 13447-13459 and 8470-8482, removing all or part of the genes encoding four complex I subunits, one complex IV subunit, two complex V subunits and five transfer RNA (tRNA) genes, which are indispensable for maintaining normal mitochondrial function.

Figure 1

Table 1

Characterization of patients with hepatocellular carcinoma.

Alternations in mtDNA content have been reported in increasing numbers of cancer types [10]. To investigate the mtDNA copy number changes and to determine if there was a correlation between the 4977 bp deletion and the overall mtDNA content in HCC, we further analyzed the mtDNA copy numbers in those individuals carrying the 4977 bp deletion. For this purpose, we used real-time-PCR to determine the mtDNA content. As shown in Figure 2, the mtDNA content was significantly higher in HCC patients carrying the 4977 bp deletion when compared with the healthy subjects (p <0.05), suggesting that the 4977 bp deletion may increase the mtDNA copy number.

Figure 2

As shown in Figure 3, the concentration of ROS was significantly higher in HCC patients than the controls (p <0.05).

Figure 3