The Institutional Review Board on Medical Ethics, Zhejiang Provincial People Hospital (China), approved the method of tissue collection. The present study was conducted in the department of surgery, Zhejiang Provincial People Hospital, on 32 patients who underwent hepatectomy for sporadic HCC without preoperative radio- or chemotherapy. All of tissue samples were immediately frozen in liquid nitrogen, and stored at -80°C until use. A total of 32 HCC samples from 15 lymph node negative and 17 lymph node positive cases were used (Table ).
Clincal data of patients with hepatocellular carcinoma
Eight μm-thick sections of the frozen tissue were cut at -20°C and stained with HE. Under microscopic observation, parts of cancer cells nests in the invasive and intraductal components were microdissected, using the LM100 laser capture microdissection system (Arcturus Engineering, Mountain View, CA, USA). A 15 μm-diameter beam was used to capture the tumor cells and the corresponding non-cancerous liver tissues. The cell nests were transferred to a LCM transfer film (CapSure TF-100S transfer film carrier, 5 mm-diameter optical-grade transparent plastic; Arcturus Engineering).
RNA preparation and T7-based RNA amplification
Total RNA was isolated from the dissected specimens using Trizol reagent (Gibco BRL) and a modified acidic guanidinium phenol-chloroform method, following the manufacturer’s recommendations. Total RNA was treated with DNaseIfor removal of genomic DNA, and the mRNA was purified using a poly(A) purification kit (Oligotex, Qiagen), according to the manufacturer’s instructions. The quality of mRNA was assessed by A260/280 ratios and the contamination of genomic DNA was checked using the PCR method. cDNA was synthesized with T7-oligo (dT) primer (Ambion) and Superscript II enzyme (Gibco BRL), as described in the instruction manual. cDNA was purified by cDNA clean-up column (DNA clearTM kit, Ambion). cRNA was generated by T7 MEGAscriptTM kit (MEGAscript in vitro Transcription Kit, Ambion, AUSTIN, Tex), per the manufacturer’s recommendations. Column purification of cRNA was performed with RNeasy kit (Qiagen), according to the manufacturer’s protocol. The concentration and quality of cRNA were analyzed by GeneQuant pro RNA/DNA Calculator (Amersharmacia biotech).
Microarray hybridization and scanning
Human Cancer Chip version 4.0 (IntelliGene, TaKaRa) was used for these studies. This array was spotted on a glass slide with 886 cDNA fragments of human genes, which are composed of 588 human identified genes related to cancer, and 298 cDNA fragments prescreened by differential display method between cancer tissue and normal tissue. Three μg of cRNA from the tumor and the matched normal tissue were labeled with Cy3-dUTP and Cy5-dUTP respectively (Amersham Pharmacia Biotech, Buckinghamshire, England), using a labeling kit (RNA Fluorescence Labeling Core kit, TaKaRa), according to the manufacturer’s instructions. The labeled probe was purified by centrifugation in a spin column (Centrisep, Princeton Separations, Adelphia, NJ). Two separate probes were combined, and 2 μL of 5 × competitor containing CotI(Gibco BRL), poly dA (Amersham Pharmaca Biotech), and tRNA (TaKaRa) were added. After addition of 50 μL of 100% ethanol and 2 μL of 3 mol/L sodium acetate (pH 5.2), the mixture was cooled at -80°C for 30 min, followed by centrifugation at 15 000 g for 10 min, and pelleted down. For final probe preparation, the pellet was washed in 500 μL of 70% ethanol twice, and eluted in 10 μL hybridization buffer (6 × SSC, 0.2% SDS, 5 × Denhardt's solution, 0.1 mg/mL salmon sperm solution). The probe were denatured by heating for 2 min at 95°C, cooled at room temperature, and centrifuged at 15 000 g for 10 min (20-26°C). Supernatants were placed on the array and covered with a 22-mm × 22-mm glass coverslip. The coverslip was sealed with a glue, and the probes were incubated overnight at 65°C for 16 h in a custom-made slide chamber with humidity maintained by underlying moist papers. After hybridization, the slides were washed in 2 × SSC with 0.1% SDS, 1 × SSC, and 0.05 × SSC, sequentially for 1 min each, and then spin dried. Hybridized arrays were scanned using a confocal laser-scanning microscope (Affymetrix 428 array scanner, Santa Clara, CA). Image analysis and quantification were performed with ImaGene 4.2 software (BioDiscovery), according to the manufacture’s instructions.
Each spot was defined by manual positioning of a grid of circles over the array image. For each fluorescent image, the average pixel intensity within each circle was determined, and a local background, outside of 3 pixel buffer range from the circle was computed for each spot. Net signal was determined by subtraction of the local background from the average intensity of each spot. Signal intensities between the two fluorescent images were normalized by the intensities of the house-keeping genes provided on the arrays. The fluorescence intensities of Cy5 (non-tumor) and Cy3 (tumor) for each target spot were adjusted so that the mean Cy3:Cy5 ratios of 32 housekeeping gene spots were equal to one. Because data derived from low signal intensities are less reliable, we first determined the cutoff values for signal intensities on each slide so that all of the filtered genes had greater S:N (signal to noise) ratios of Cy3 or Cy5 than three, and we excluded genes for further analysis when both Cy3 and Cy5 dyes gave signal intensities lower than the cutoff. To estimate the range of expression ratio within which the expression change could be considered as fluctuation in non-cancerous cells, we compared expression profiles of non-cancerous cells from 6 patients. Because 90% of expression ratios in non-cancerous cells fell within the range of 1.726 and 0.503, we categorized genes into three groups according to their expression ratios (Cy3:Cy5): up-regulated (ratio, 2.0); down-regulated (ratio 0.5); and unchanged expression (ratios, between 0.5 and 2.0); provided that signal counts of T (Cy3) and R (Cy5) were > 500. Genes with Cy3:Cy5 ratios > 2.0 or < 0.5 in more than 75% of the cases examined were defined as commonly up- or down-regulated genes, respectively.
Real-time reverse transcription PCR
LightCycler (Roche Diagnostics) technology was applied to confirm the data obtained by cDNA microarray. The primer sequences of 11 genes were obtained from the GDB Human Genome Database (http://www.gdb.org/gdb/
) (Table ). We used the same RNA from the dissected cells for the microarray analysis. First-strand cDNA was obtained by reverse transcription using a commercially available kit (first strand synthesis kit, Amersham). For each PCR, 2 μL (20 ng) first strand cDNA template, 50 pmol of each primer, 2.4 μL (3 mmol) MgCl2
, and 2 μL 10 × SYBR GreenI(Roche Laboratories)were mixed in 20 μL of PCR mixture. The running protocol was programmed as follows. In the first step, initial denaturation, reaction mixture was incubated for 10 min at 95°C. In the second step, DNA was amplified for 45 cycles at 95°C for 10 s, specific annealing temperature (the primer sequences dependent) for 0-10 s, and elongation at 72°C for some seconds [amplicon (bp)/25 s]. Finally, the temperature was raised gradually (0.2°C/s) from the annealing temperature to 95°C for the melting curve analysis. Twelve μL of PCR product were visualized by electrophoresis on 2% agarose gel stained with ethidium bromide. The amount of gene expression was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Human GAPDH kit (GmbH Heidelberg, Heidelberg, Germany). The qRT-PCR analysis was carried out in triplicate for each cDNA sample, and the median values were used for the three experiments. Up- and down-regulation were defined as the median value > 2.0 and < 0.5, respectively.
Primers used to amplify cDNA at various genes in real time RT-PCR
Statistical analysis among mean values was performed on the association of lymph node metastasis with expression levels by applying non-parametric Kruskal-Wallis and Mann-Whitney U tests. Statistical significance was defined as a P-value of < 0.05. Differential expression between the groups of HBV-infected and HCV-infected HCC was considered significant, with P < 0.05.