This study was carried out in tissue samples of patients with either HCC undergoing surgical resection or chronic virus-related liver damage undergoing US-guided liver biopsy and, as control group, in patients undergoing cholecystectomy.
Sixty-one patients (corresponding to 90 samples) entered the study;
twenty-nine patients with HCC, 20 males and 9 females, mean age 62 (±13), entered the study and during the surgical resection both a neoplastic sample (HCC) and a sample from the non-cancerous cirrhotic tissue surrounding the resected nodules (NCCT) were obtained, for a total of 58 samples. Etiology of the disease was as follows: HBV or HCV-related 15 patients (52%), alcohol 5 patients (17%), other factors or cryptogenic 9 patients (31%);
twenty-two patients with HCV or HBV-related chronic liver damage (CH) (20 HCV, 2 HBV) 12 males, 10 females, mean age 53 (±8), also entered the study. In this case, part of the liver biopsy samples (obtained by 16/17 Gauges Menghini modified needles under US guidance) was devoted to the planned studies while the bulk (always longer than 2
cm) was used for the routine histological examination;
finally, ten patients undergoing cholecystectomy were submitted, during laparoscopy, to liver biopsy with the same modalities as above (6 females and 4 males, mean age 56 (±7) (CON).
In each patient anti-HCV antibodies were looked for using a second-generation ELISA and all positive sera were confirmed by RIBA II assay. In all patients, anti-HCV sera positivity was confirmed by positive HCV-RNA levels determination using the Amplicor HCV test (Amplicor PCR Diagnostic, Hoffman-La Roche, Basel Switzerland). A standardized genotyping assay (Inno-Lipa HCV III, Innogenetics, Gent, Belgium) was used.
The HBV group consisted of individuals who were HBsAg, anti-HBe, and HBV-DNA positive at PCR. HBV serum markers were tested by radioimmunoassay (RIA) (Abbott, Chicago, IL, USA), while HBV-DNA levels were tested using a commercially-available fluid phase hybridization assay (Abbott, Chicago, IL, USA).
After obtaining informed consent, the patients provided full information relative to their drinking and smoking habits and completed a food frequency questionnaire with particular attention to their intake of vitamins. Patients with concurrent diseases or those taking medications capable of interfering with free radical production, such as non-steroidal anti-inflammatory drugs (NSAIDs) or anti-oxidants (vitamin C), were excluded from the study.
The study that was approved by the Ethic Committee of Padua Hospital.
Genomic DNA extraction from biopsy and surgical liver sample
Genomic DNA extraction from surgical liver samples and biopsies was made on portions of tissues immediately snap-frozen in liquid nitrogen and stored at - 80°C until use. For DNA extraction we used a Wizard Genomic DNA Purification Kit (Promega Italia, Milano, Italy) according to the protocol provided. The liver tissues (approximately 10
mg) were homogenized with pestle in a solution of EDTA 0.5
M pH 8, Nuclei Lysis Solution and Proteinase K (20mg/ml).
Quantification of 8-OHdG adduct
This assay was performed with a portion of the liver material (both surgical and biotic samples), and consisted of 3 steps: (i
) genomic DNA extraction using a Wizard Genomic DNA Purification Kit (Promega Italia, Milano, Italy); (ii
) nuclease P1 and alkaline phosphatase hydrolysis of DNA; (iii
) 8-OHdG determination using HPLC-ED, which is a highly sensitive method with a detection limit of ~2 adducts per 105
deoxyguanosine (dG). Following nuclease P1 and alkaline phosphatase hydrolysis, samples were filtered through 0.22-mm nylon filters (Scientific Resources, Alfatech, Genova, Italy), and 20
μL DNA per sample was injected in the HPLC (Alliance 2695, Waters, Milano). Eight-OHdG and normal deoxynucleosides were separated in a 3-mm Supelcosil LC-18-DB analytical column (7.5
cm × 4.6
mm, Supelco, Bellefonte, PA) equipped with a 5-mm SupelguardTM LC-18-DB guard column cartridge.
The solvent system consisted of an isocratic mixture of 90% of 50
mM potassium phosphate, pH 5.5, and 10% methanol at a 1
ml/min flow rate.
The 8-OHdG was detected using an electrochemical detector (ESA Coulochem II 5200 A, Bedford, MA) equipped with a high-sensitivity analytical cell, model 5011, with the oxidation potentials of electrodes 1 and 2 adjusted to 0.15 and 0.35
V, respectively. The levels of 8-OHdG were referred to the amount of dG detected in the same sample by UV absorbency at 254
nm. The amount of DNA was determined from a calibration curve vs
. known amounts of calf thymus DNA. The 8-OHdG levels were expressed as the number of 8-OHdG adducts per 105
dG. An 8-OHdG standard (Sigma-Aldrich, St. Louis, MO, USA), prepared immediately before the assay, was injected before any set of samples. The coefficient of variation was <10%; the amount of DNA required for the assay was 100 μg. Samples with lower amounts of DNA were rejected, since the risk of methodological error is only acceptable above this cut-off.
OGG1 gene: PCR and Restriction Fragment Length Polymorphism (RFLP) Analyses
Cellular DNA isolated from tissue samples was analyzed by PCR to study the C:G transversion at nucleotide 1245 of the OGG1 gene. An aliquot of 100
ng of genomic DNA was added to a 25
μl PCR mixture and PCR was performed using the primers described by Kohno et al. [27
], 5′-AGGGGAAGGTGCTTGGGGAA--3′ as the forward primer and 5′ –ACTGTCACTAGTCTCACCAG - 3′as the reverse primer.
The thermoprofile consisted of 30 cycles of denaturation at 94°C for 15
sec, annealing at 58°C for 15
sec and extension at 72°C for 40
sec, preceded by an initial denaturation step at 94°C for 2
min followed by a terminal extension at 72°C for 5 min. The 1245 C:G transversion was identified using PCR-RFLP. Briefly, 10 μl of the 200-bp PCR product was digested with Fnu4HI. The presence of a C:G transversion creates a Fnu4HI recognition site, which leads to digestion of the 200-bp PCR product into 2 fragments of 100 bp. Heterozygous subjects exhibit 2 fragments (200 and 100 bp), and a homozygous C:G transversion results in the appearance of a single fragment of 100 bp. Fnu4HI digests of PCR amplification products were observed by electrophoresis on 3% agarose gels after ethidium bromide staining.
Protein extraction from surgical and bioptic liver sample
Liver samples were lysed in 200 μl of CHAPS buffer containing Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 4°C for 30 min. The lysate was centrifuged at 14000 rpm for 45 min at 4°C and the supernatant was collected −80°C. The protein concentration was measured using RCDC Protein Assay (Bio-Rad, Milano, Italy).
Real Time Quantitative–Telomeric Repeat Amplification Protocol (RTQ-TRAP) for telomerase activity
Each hepatic sample was assayed for telomerase activity, starting from protein extracts of the liver tissue stored at −80°C, by an ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The total volume of the reaction mixture was 25 μl in SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) contained DNA polymerase Ampli-Taq Gold, dNTPs and dUTP, 1X SYBR Green buffer, 0.2 μg of T4 gene protein, primers TS (5′-AATCCGTCGAGCAGAGTT-3′) 0.6 μM, ACX [5′-GCGCGG(CTTACC)3CTAACC-3′] 0.4 μM and 1 μg/μl of protein extract. The PCR was performed in a 96-well micro titer plate and the reaction mixture was first incubated at 25°C for 20 min to allow the telomerase in the protein extract to elongate the TS primer by adding TTAGGG repeat sequences.
PCR then started at 95°C for 10 min, followed by a 40-cycle amplification at 95°C 20 s and 60°C for 30 s. All samples were assayed in triplicate to test the reproducibility of the RTQ-TRAP assay, and in each assay also a protein extract of a cell line (PLC/PRF/5) as positive control and water as negative control were evaluated. Following amplification, a dissociation curve was performed in order to confirm the specificity of the reaction. Fluorescence signals and dissociation curves were collected and analyzed by ABI 7900 SDS 2.3 software so telomerase activity in cells line and samples was calculated based on the threshold cycle (Ct).
Telomerase activity in cells line and samples was extrapolated using a standard curve prepared with serial dilutions of Telomerase Control Oligo Standard (amol/μl) containing telomeric repeats (TAAGGG). Telomerase activity was expressed as percentage compared to that in PLC/PRF/5 cells.
Telomere length analysis by quantitative PCR
Genomic DNA extracted was used also to measure telomere length by using method developed by Cawthon [28
]. Two 96-well plates were prepared for each experiment, one of which containing telomere primers
(Tel-1:5′-GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT-3′, Tel-2: 5′- TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA-3′) and the other for 36B4, encoding acidic ribosomal phosphoprotein P0 used as the single copy gene (36B4u: 5′-CAGCAAGTGGGAAGGTGTAATCC-3′, 36b4d: 5′-CCCATTCTATCATCAACGGGTACAA-3′).
Each 25 μl PCR reaction included 40 ng of DNA, SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and primers at final concentrations 270 nM Tel-1 and 900 nM Tel-2 or 300 nM 36B4u and 500 nM 36B4d, respectively.
PCR amplification was performed in a ABI PRISM 7900 (Applied Biosystems, Foster City, CA, USA). In each plate, a standard curve was produced ranging from 4 to 80 ng of human reference DNA (Applied Biosystems, Foster City, CA, USA). The thermal cycling profile for the telomere amplification was 95°C for 10 min, followed by 30 cycles of 95°C for 5 s, 56°C for 10 s and 72°C for 60 s or 30 cycles of 95°C for 5 s, 58°C for 10 s and 72°C for 40 s. Following amplification, a dissociation curve was performed in order to confirm the specificity of the reaction.
All samples were assayed in triplicate to test the reproducibility and in each assay was evaluated a negative control. Fluorescence signals and dissociation curves were analyzed by ABI 7900 SDS 2.3 software. Telomeres/single copy gene (T/S) ratio for each sample were obtained by dividing the mean amount of telomeres by the mean amount of 36B4 gene.
miRNAs expression: Taqman microRNA assay
We started from 0.1g of frozen tissue for each sample and RNA extraction was performed through mirVana miRNA isolation kit (Ambion, Austin, TX, USA), a method which combines phenol-chloroform and fiber-glass columns to obtain total RNA enriched in miRNAs. RNA was eluted in RNAse-free water and stored at −80°C; concentration and quality were checked with Nanodrop (Thermo Scientific, Canada, USA). The expression of a selected panel of miRNAs was then evaluated by mean of RT-PCR, using TaqMan microRNA assay (Applied Biosystems, Foster City, CA, USA). 10ng of RNA were reverse translated for each sample using looped primers specific for each miRNA and the obtained cDNA was assayed with real-time PCR with miRNA specific primers and TaqMan® probes. Data obtained for each miRNA were normalized versus a control RNA (RNU6B) and the relative expression for coupled tumour and normal samples was expressed by ΔΔCT method.
Reverse transcription for Bad/Bax mRNA determination
For the synthesis of complementary DNA (cDNA), 2 μg of RNA were reverse transcribed in a final volume of 40 μl in the presence of 1X PCR buffer, 1 mM each of dNTPs (dATP, dTTP, dCTP, dGTP), 1 U RNase inhibitor, 2.5 μM random hexamers, and 2.5 U of murine leukemia virus (Perkin Elmer, Foster City, CA, USA). The reverse transcription reaction was completed at 25°C for 10 min, 42°C for 15 min and 99°C for 5 min, in a Perkin Elmer GeneAmp PCR System 2400. The cDNA was stored at −20°C.
Quantitative absolute real-time PCR
Real-time PCR was conducted in an ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using SYBR Green I. The reaction was obtained on 96-well plates, in a 25 μL final volume containing 1X TaqMan buffer, 5.5 mmol of MgCl2, 200 μmol of nucleotides with dUTP, 0.25 U of AmpliTaq Gold Polymerase (SYBR Green Master Mix), 300 nM of each primer and 200 ng of cDNA template. Nucleotide sequences for the sense and antisense primers used for real-time PCR were: 5′-CTTTTGCTTCAGGGTTTCATCC-3′, 5′-TTGAGACACTCGCTCAGCTTCT-3′ for Bax [ENST00000356483], and the length of this amplicon was 119 bp; 5′- TCTATGCAAGTTTTGCCCTTTGTA-3′, 5′-GCCAGCCTGAATGAAATGA- 3′ for BI-1 [ENST00000267115], and the length of this amplicon was 84 bp; 5′-CCTGGCACCCAGCACAA- 3′, 5′-GCCGATCCACACGGAGTACT for β-actin [ENST00000158302], and the length of this amplicon was 70 bp. After one 2-min step at 50°C and a second step at 95°C for 10 min, samples underwent 45 cycles of 45 s at 94°C and then: 45 s at 60°C for Bax and β-actin; 45 s at 65°C for Bad. A final extension step was performed at 60°C for 10 min.
The data obtained were initially examined for their distribution with the Kolmogorov-Smirnov test and then compared by either Anova One Way or Kruskal Wallis test. Correlation analysis were performed by either linear correlation analysis or Spearman Rank correlation analysis, as appropriate. The chi square test was used when indicated. A p value < 0.05 was considered as significant. The StatsDirect and PASW Statistics programs were used.
The power analysis for the data evaluated with unpaired two sample Student t tests showed the all the comparison with controls, with an 80% power and a 5% α error, were based on numbers of patients large enough to allow comparison.