Specific genomic and transcriptomic aberrations in tumors induced by partial hepatectomy of a chronically inflamed murine liver.

Resection of hepatocellular carcinoma (HCC) tumors by partial hepatectomy (PHx) is associated with promoting hepatocarcinogenesis. We have previously reported that PHx promotes hepatocarcinogenesis in the Mdr2-knockout (Mdr2-KO) mouse, a model for inflammation-mediated HCC. Now, to explore the molecular mechanisms underlying the tumor-promoting effect of PHx, we compared genomic and transcriptomic profiles of HCC tumors developing in the Mdr2-KO mice either spontaneously or following PHx. PHx accelerated HCC development in these mice by four months. PHx-induced tumors had major chromosomal aberrations: all were amplifications affecting multiple chromosomes. Most of these amplifications were located near the acrocentric centromeres of murine chromosomes. Four different chromosomal regions were amplified each in at least three tumors. The human orthologs of these common amplified regions are known to be amplified in HCC. All tumors of untreated mice had chromosomal aberrations, including both deletions and amplifications. Amplifications in spontaneous tumors affected fewer chromosomes and were not located preferentially at the chromosomal edges. Comparison of gene expression profiles revealed a significantly enriched expression of oncogenes, chromosomal instability markers and E2F1 targets in the post-PHx compared to spontaneous tumors. Both tumor groups shared the same frequent amplification at chromosome 18. Here, we revealed that one of the regulatory genes encoded by this amplified region, Crem, was over-expressed in the nuclei of murine and human HCC cells in vivo, and that it stimulated proliferation of human HCC cells in vitro. Our results demonstrate that PHx of a chronically inflamed liver directed tumor development to a discrete pathway characterized by amplification of specific chromosomal regions and expression of specific tumor-promoting genes. Crem is a new candidate HCC oncogene frequently amplified in this model and frequently over-expressed in human HCC.

Technologies). The corresponding Cy3-and Cy5-labeled DNA were combined and hybridized at 65°C 24 hours to an Agilent Mouse Genome CGH Microarray 4x44K, using Agilent's recommended hybridization chamber and oven. Subsequently, the microarrays were washed once with Agilent Oligo aCGH Wash Buffer 1 at room temperature for 5 minutes followed by a second wash with Agilent Oligo aCGH Wash Buffer 2 at 37 °C for one minute. Finally, the microarray was washed once with acetonitril for one minute at room temperature and slowly removed from the acetonitril solution to let it dry at room temperature.

RNA purification and cDNA synthesis
Total RNA was isolated from frozen liver tissues with Trizol reagent (Invitrogen, Carlabad, CA, USA) followed by twice Phenol-Chlorophorm extraction and isopropanol precipitation. The cDNA was obtained from 1 µg total liver RNA. Twelve units of the RNasine RNase inhibitor and 1/25ul random hexamer primers (Promega, WI, USA) in DEPC-treated water were preheated at 90 o C for 1 min to denature secondary structures. The mixture was then cooled gradually (0.2 o C/S to 45 o C and then 5 μl 5X RT Buffer, and 0.5 mM dNTPs (Deoxynucleotide triphosphate set, Roche, Indianapolis, IN, USA)) with total volume of 25 μl. The RT mixture was incubated at 42 o C for 60 min. then stopped by heating at 95 o C for 5 minutes. The cDNA stock was stored at -20 o C.
For each gene, the cDNA concentration and the number of PCR cycles were established in the linear amplification range. Primers were used at a concentration of 0.5 µM. PCR was performed using the

Bioinformatic analysis of the gene expression profiling data
Genome-scale gene expression profiling data were analyzed by the GeneChip robust multiarray analysis (GCRMA) and preprocessing algorithm following median polish normalization using the Partek software (ProSoftware, Partek, St. Louis, MO). All signals below 5.0 (in log2 scale) were rounded to 5.0.
Enrichment analysis (Fisher test) for gene signatures was performed using "EDanalysis" of the "R" Package for Gene Enrichment Disequilibrium Analysis: http://cran.r-project.org/web/packages/EDanalysis/EDanalysis.pdf Histological sections were reviewed and characterized by the pathologist (O.P.) for inflammation, mitotic incidence, tissue damage, and cell homogeneity. Liver tumors were characterized, and only those assigned as "well differentiated HCCs" were used for further experiments.

Bradford protein quantification assay
The protein content of each lysate sample was quantified by the Bradford assay. A standard curve of OD against increasing protein concentration was established for each protein sample using serial dilutions of BSA standard protein (1 mg/ml) and a Coomassie blue based dye reagent (Bio-RAD). 1 / 2.5 / 5 / 10 / 20 / 40 μl of standard lysate samples were incubated with 200 μl of Coomassie reagent (diluted 1:5 with DDW) and OD at 595nm was immediately measured by spectrophotometer.

Protein immunoblotting
Lysates were obtained from the whole liver; tissues were prepared by manual homogenization of liver

Assessment of Crem protein levels by immunohistochemistry and immunoblotting.
To assess Crem protein levels in hepatocytes, paraffin-embedded slides were stained for Crem (1:100)

Assessment of proliferative response, DNA damage and apoptosis
To assess the proliferative response of hepatocytes, mice were injected intraperitoneally (i.p.) with BrdU Mitotic events were counted in slides stained for hematoxylin only. Assessment of a proliferative response was performed by counting positively stained hepatocytes cells within at least 20 fields per slide, magnification x200.
To assess the apoptosis, TUNEL staining was performed using the in situ Cell Death Detection Kit (Life Technologies) as described by the manufacturer.

Cell cultures
The human HCC cell lines Huh7, Hep3b, HepaRG, and HepG2 and the murine cell lines Bnl, Bwtg, and

Cell proliferation assay
Cell proliferation rate was measured by proliferation assay using a xCELLigence system for real-time and label-free monitoring of cell viability (4)

Statistical analysis and calculation software.
In this study, the in vitro experiments were performed in triplicates, excluding the cell proliferation rate assay using a xCELLigence system, which was performed in duplicates; in the in vivo experiments, at least 3 mice per group were used. All parameters were evaluated with the two-tailed t-test. Statistical evaluation of differential expression between the experimental groups was performed using the ANOVA   Murine