On ratios of these lesions compared to correspondent normal livers in each strain,, supporting the validity of interstrain comparative analysis. 3.4 Cell proliferation and cell survival genes Author Manuscript Author Manuscript Author Manuscript Author Manuscript Since a striking difference between DN and HCC developing in susceptible and resistant rats concerns their capacity to progress we selected, on the basis of a public database search, genes differently expressed in nodules and HCCs, more specifically related to cell proliferation and cell survival. 70% of cell cycle- and cell proliferation-related genes, and all genes related to cell differentiation, oxidative stress, and ubiquitination were significantly more expressed in DN of F344 than BN rats. In contrast, tumor growth inhibitors, including Gnmt, Csmd1, Dmbt1, and Dusp1, were more expressed in DN of BN than F344 rats. Remarkably, in BN HCC highest expression of numerous cell cycle- and cell proliferation-related genes, was associated with a highest expression of tumor growth inhibitors Bhmt, Dmbt1, Dusp1, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13. 3.5 Validation of BioPQQ chemical information microarray MedChemExpress GLYX 13 analysis Expression of 9 randomly chosen genes, involved in cell proliferation and cell survival, was made by qPCR on 6 normal livers, 15 DNs and 14 HCCs, including the lesions used for microarray analysis, from each strain. These samples consisted of low-grade and high-grade DNs of F344 and BN rats, respectively, 3 poorly-differentiated and 11 moderately-differentiated HCCs of F344 rats, and 3 well-differentiated and 11 moderately-differentiated BN HCC. qPCR analysis showed relatively low variance of liver lesions from both strains, supporting a low heterogeneity of rat liver lesions, and roughly confirmed the results of microarray analysis. Upregulation of Anxa5, c-Myc, Igfbp3, Ctgf, and Igfbp1 
occurred in DN and HCC from both strains, with highest values in F344 rat lesions. Bhmt downregulation occurred in DN and HCC from both strains, with significantly lower values in HCC of F344 than BN liver. A progressive decrease in Gnmt, Dusp1, and Dmbt1 expression from normal liver to DN and HCC of F344 rats, contrasted with significantly lower decrease in Gnmt, and sharp increase in Dusp1 and Dmbt1 in BN rat lesions. The validity of microarray analysis was further supported by a close correlation of expression data of qPCR and microarray analyses of cMyc and Dusp1 expression. Western blot analysis of c-Myc, Gnmt, Bhmt, Dusp1, and Pp2A proteins, performed on 5 HCCs of both strains, reproduced the results of qPCR and/or microarray analyses. Cell Oncol. Author manuscript; available in PMC 2015 July 28. Frau et al. Page 6 3.6 Comparison of gene expression pattern of subclasses of rat and human liver lesions Author Manuscript Author Manuscript Author Manuscript 
Author Manuscript We next examined the possible value of the rat model of hepatocarcinogenesis to assess the significance of a susceptible/resistant phenotype for human disease. We thus performed a comparative functional genomic approach by integrated analysis of 28, 25, and 35 human non-tumor surrounding liver, HCCB and HCCA, respectively, and the rat liver lesions. This approach is based on the hypothesis that due to the maintenance of regulatory elements in evolutionarily related species, gene expression traits related to similar phenotypes could be conserved in different species. In favor of this hypothesis, numerous studies demonstrated that cross-compa.On ratios of these lesions compared to correspondent normal livers in each strain,, supporting the validity of interstrain comparative analysis. 3.4 Cell proliferation and cell survival genes Author Manuscript Author Manuscript Author Manuscript Author Manuscript Since a striking difference between DN and HCC developing in susceptible and resistant rats concerns their capacity to progress we selected, on the basis of a public database search, genes differently expressed in nodules and HCCs, more specifically related to cell proliferation and cell survival. 70% of cell cycle- and cell proliferation-related genes, and all genes related to cell differentiation, oxidative stress, and ubiquitination were significantly more expressed in DN of F344 than BN rats. In contrast, tumor growth inhibitors, including Gnmt, Csmd1, Dmbt1, and Dusp1, were more expressed in DN of BN than F344 rats. Remarkably, in BN HCC highest expression of numerous cell cycle- and cell proliferation-related genes, was associated with a highest expression of tumor growth inhibitors Bhmt, Dmbt1, Dusp1, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19847069 Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13. 3.5 Validation of microarray analysis Expression of 9 randomly chosen genes, involved in cell proliferation and cell survival, was made by qPCR on 6 normal livers, 15 DNs and 14 HCCs, including the lesions used for microarray analysis, from each strain. These samples consisted of low-grade and high-grade DNs of F344 and BN rats, respectively, 3 poorly-differentiated and 11 moderately-differentiated HCCs of F344 rats, and 3 well-differentiated and 11 moderately-differentiated BN HCC. qPCR analysis showed relatively low variance of liver lesions from both strains, supporting a low heterogeneity of rat liver lesions, and roughly confirmed the results of microarray analysis. Upregulation of Anxa5, c-Myc, Igfbp3, Ctgf, and Igfbp1 occurred in DN and HCC from both strains, with highest values in F344 rat lesions. Bhmt downregulation occurred in DN and HCC from both strains, with significantly lower values in HCC of F344 than BN liver. A progressive decrease in Gnmt, Dusp1, and Dmbt1 expression from normal liver to DN and HCC of F344 rats, contrasted with significantly lower decrease in Gnmt, and sharp increase in Dusp1 and Dmbt1 in BN rat lesions. The validity of microarray analysis was further supported by a close correlation of expression data of qPCR and microarray analyses of cMyc and Dusp1 expression. Western blot analysis of c-Myc, Gnmt, Bhmt, Dusp1, and Pp2A proteins, performed on 5 HCCs of both strains, reproduced the results of qPCR and/or microarray analyses. Cell Oncol. Author manuscript; available in PMC 2015 July 28. Frau et al. Page 6 3.6 Comparison of gene expression pattern of subclasses of rat and human liver lesions Author Manuscript Author Manuscript Author Manuscript Author Manuscript We next examined the possible value of the rat model of hepatocarcinogenesis to assess the significance of a susceptible/resistant phenotype for human disease. We thus performed a comparative functional genomic approach by integrated analysis of 28, 25, and 35 human non-tumor surrounding liver, HCCB and HCCA, respectively, and the rat liver lesions. This approach is based on the hypothesis that due to the maintenance of regulatory elements in evolutionarily related species, gene expression traits related to similar phenotypes could be conserved in different species. In favor of this hypothesis, numerous studies demonstrated that cross-compa.