3C; Supporting Fig. 4), already described to be involved in HCC.[16] Remarkably, among the most dysregulated there was the NRF2-dependent pathway (Fig. 3C), whose role in cancer development has recently become a topic of an important controversy.[17] The finding that the majority of NRF2 target genes were up-regulated indicates PD0325901 research buy activation of the NRF2 pathway (Fig. 3D). Notably, several of these genes were among the most up-regulated in both KRT-19+ lesions and aHCCs (see Table 1). Moreover, the small musculoaponeurotic fibrosarcoma oncogene homolog (MAF) family of transcription factors, which operate as coactivators

of NRF2,[18] was found activated at all stages of HCC development (Fig. 3C; Supporting Fig. 4). To identify a possible correlation between miRNAs and genes dysregulated at the initial stage of the process, we selected conserved putative miRNA targets in rat, predicted by the TargetScanS algorithm. For each of the differentially expressed genes in KRT-19+ nodules, we extracted the

annotated regulating miRNAs and performed an intersection with the differentially expressed miRNAs in the same stage. We found Ku-0059436 in vivo that a consistent percentage of modified genes are targets of dysregulated miRNAs (Fig. 4). To study the relative expression of miRNAs and of their target genes, we evaluated the number of positive and negative correlations between the predicted miRNA-target gene pairs by means of the fold-change value reported by the Limma package. The results show that 171 out of 215 miRNA/mRNA pairs (79%) shown in Fig. 4 displayed anticorrelated expression, while 44 were positively correlated. In the transition from normal liver to KRT-19+ nodules we observed at least two relevant nodes, where two miRNA families, miR-30 and miR-200, control the expression of many modified MCE genes. While expression of the miR-30 family was mainly modified at this

initial stage, miR-200 family was up-regulated in both preneoplastic lesions and aHCCs (Supporting Fig. 5). Notably, one of the target genes of miR-200a is kelch-like ECH-associated protein 1 (KEAP1), a negative regulator of NRF2.[19] NRF2 is an integrated redox sensitive signaling system that regulates 1%-10% of human genes and is negatively controlled by KEAP1, which promotes NRF2 proteasome-mediated degradation.[19] As mentioned above, the NRF2 pathway was already activated in early preneoplastic lesions and along tumor progression (Fig. 3C,D). Accordingly, immunohistochemistry (IHC) performed on lesions at different stages revealed that all of them were strongly positive for NRF2 (Fig. 5A); most important, IHC showed NRF2 nuclear staining in several preneoplastic and neoplastic hepatocytes, clearly demonstrating translocation and suggesting activation of this transcription factor.