Gene profiling, biomarkers and pathways characterizing HCV-related hepatocellular carcinoma

Liver biopsies from fourteen HCV-positive HCC patients and seven HCV-negative non-liver cancer control patients (during laparoscopic cholecystectomy) were obtained with informed consent at the liver unit of the INT "Pascale" in Naples. In particular, from each of the HCV-positive HCC patients, a pair of liver biopsies from HCC nodule and non-adjacent non-HCC counterpart were surgically excised. All liver biopsies were stored in RNA Later at -80°C (Ambion, Austin, TX). Confirmation of the histopathological nature of the biopsies was performed by the Pathology lab at INT before the processing for RNA extraction. The non-HCC tissue from HCV-positive patient were an heterogeneous sample representing the prevalent liver condition of each subject (ranging from persistent HCV-infection to cirrhotic lesions). Furthermore, laboratory analysis confirmed that the 7 controls were seronegative for hepatitis C virus antibodies (HCV Ab).

Samples were homogenized in disposable tissue grinders (Kendall, Precision). Total RNA was extracted by TRIzol solution (Life Technologies, Rockville, MD), and purity of the RNA preparation was verified by the 260:280 nm ratio (range, 1.8-2.0) at spectrophotometric reading with NanoDrop (Thermo Fisher Scientific, Waltham, MA). Integrity of extracted RNA was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), analyzing the presence of 28S and 18S ribosomal RNA bands as well as the 28S/18S rRNA intensity ratio equal or close to 1.5. In addition, phenol contamination was checked and a 260:230 nm ratio (range, 2.0-2.2) was considered acceptable.

Double-stranded cDNA was prepared from 3 μg of total RNA (T-RNA) in 9 μl DEPC -treated H₂O using the Super script II Kit (Invitrogen) with a T7-(dT15) oligonucleotide primer. cDNA synthesis was completed at 42°C for 1 h. Full-length dsDNA was synthesized incubating the produced cDNA with 2 U of RNase-H (Promega) and 3 μl of Advantage cDNA Polymerase Mix (Clontech), in Advantage PCR buffer (Clontech), in presence of 10 mM dNTP and DNase-free water. dsDNA was extracted with phenol-chloroform-isoamyl, precipitated with ethanol in the presence of 1 μl linear acrylamide (0.1 μg/μl, Ambion, Austin, TX) and aRNA (amplified-RNA) was synthesized using Ambion's T7 MegaScript in Vitro Transcription Kit (Ambion, Austin, TX). aRNA recovery and removal of template dsDNA was achieved by TRIzol purification. For the second round of amplification, aliquots of 1 μg of the aRNA were reverse transcribed into cDNA using 1 μl of random hexamer under the conditions used in the first round. Second-strand cDNA synthesis was initiated by 1 μg oligo-dT-T7 primer and the resulting dsDNA was used as template for in vitro transcription of aRNA in the same experimental conditions as for the first round [20]. 6 μg of this aRNA was used for probe preparation, in particular test samples were labeled with USL-Cy5 (Kreatech) and pooled with the same amount of reference sample (control donor peripheral blood mononuclear cells, PBMC, seronegative for hepatitis C virus antibodies (HCV Ab)) labeled with USL-Cy3 (Kreatech). The two labeled aRNA probes were separated from unincorporated nucleotides by filtration, fragmented, mixed and co-hybridized to a custom-made 36 K oligoarrays at 42°C for 24 h. The oligo-chips were printed at the Immunogenetics Section Department of Transfusion Medicine, Clinical Center, National Institutes of Health (Bethesda, MD). After hybridization the slides were washed with 2 × SSC/0.1%SDS for 1 min, 1 × SSC for 1 min, 0.2 × SSC for 1 min, 0.05 × SSC for 10 sec., and dried by centrifugation at 800 g for 3 minutes at RT.

Hybridized arrays were scanned at 10-μm resolution with a GenePix 4000 scanner (Axon Instruments) at variable photomultiplier tube (PMT) voltage to obtain maximal signal intensities with less than 1% probe saturation. Image and data files were deposited at microarray data base (mAdb) at http://​nciarray.​nci.​nih.​gov and retrieved after median centered, filtering of intensity (>200) and spot elimination (bad and no signal). Data were further analyzed using Cluster and TreeView software (Stanford University, Stanford, CA).

The quality of extracted total RNA was verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), showing discrete 28S and 18S rRNA bands (Figure 1A) as well as a 28S/18S rRNA intensity ratio equal or close to 1.5 which is considered appropriate for total RNA extracted from liver tissue biopsies ("Assessing RNA Quality", http://​www.​ambion.​com/​techlib/​tn/​111/​8.​html). Based on this parameter, all extracted total RNA samples met the quality control criteria (Figure 1B).

The gene expression profiles of tissue samples from the three groups of analyzed samples (the HCV-related HCC, their non-HCC counterpart, as well as samples from control patients) were compared by an unsupervised analysis. No clear separation of the 3 different groups was observed, although control samples clustered mainly with samples from HCV-related non-HCC paired tissue, which includes dysplastic lesion in cirrhotic liver, representing a pre-neoplastic step (Figure 2A).

In order to identify genes differentially modulated in HCV-related lesions compared to normal liver tissue samples, an unsupervised analysis was then performed including only paired samples from HCV-related HCC and normal control samples or from the HCV-related non-HCC counterpart and control samples (Figures 2B and 2C). According to filtering described in Material and Methods, HCV-related HCC and normal control samples showed 5'473 genes differentially expressed, with a perfect clustering according to histological characteristics (Figure 2B). Similarly, HCV-related non-HCC tissue and normal control samples showed 6'069 genes differentially expressed with a perfect clustering according to histological characteristics also in this case (Figure 2C). The only exception to this pattern is represented by the normal control sample (CTR#80) which did not fall in the control cluster (CTR).

The supervised analysis was performed comparing pairs of gene sets using an unpaired Student's t-test with a cut-off set at p < 0.01.

The analysis comparing gene sets in liver tissues from HCV-related HCC and normal controls identified 825 genes differentially expressed. Among them, 465 were shown to be up-regulated and 360 down-regulated in HCV-related HCC liver tissues (Figure 3A). The first 40 genes showing the highest fold of up-regulation are listed in Table 1.

The analysis comparing gene sets in liver tissues from HCV-related non-HCC tissue and controls identified 151 genes differentially expressed. Among them, 127 were shown to be up-regulated and 24 down-regulated in HCV-related non-HCC liver tissues (Figure 3B). The first 40 genes showing the highest fold of up-regulation are listed in Table 2.

The analysis comparing gene sets in liver tissues from HCV-related HCC and HCV-related non-HCC counterpartidentified 383 genes differentially expressed. Among them, 83 were shown to be up-regulated and 300 down-regulated in HCV-related HCC liver tissues (Figure 3C). The first 40 genes showing the highest fold of up-regulation are listed in Table 3.

The pathway analysis was performed including the genes found up-regulated in the supervised comparisons, using the gene set expression comparison kit implemented in BRB-Array- Tools. The human pathway lists determined by "Ingenuity System Database" was selected. Significance threshold of t-test was set at 0.001. Samples from HCV-related non-HCC liver tissue showed strong up-regulation of genes involved in Antigen Presentation, Protein Ubiquitination, Interferon signaling, IL-4 signaling, Bacteria and Viruses cell cycle and chemokine signaling pathways. Samples from HCV-related HCC showed strong up-regulation of genes involved in Metabolism, Aryl Hydrocarbon receptor signaling, 14-3-3 mediated signaling and protein Ubiquitination pathways. Significant pathways were listed respectively in Figures 4, 5, 6 and 7.