[15] MiRNA expression analysis between the KRT-19- and KRT-19+ preneoplastic lesions revealed that no miRNA reached the BH correction for multiple testing at the selected threshold of P < 0.05; however, using

t-test for each single miRNA, we found 23 differentially expressed miRNAs (P < 0.05; Supporting Table 4) that are likely important in the progression of KRT-19+ lesions towards malignancy. Gene expression profiling was performed in BAY 80-6946 the same lesions using the Illumina microarray. A total of 1,144 out of 21,791 genes included in the array were selected as described in the Supporting Material. Hierarchical cluster analysis stratified the rat lesions into two major clusters: (1) normal liver and preneoplastic KRT-19- lesions; (2) preneoplastic KRT-19+ lesions, adenomas/eHCCs and aHCCs, forming three distinct subclusters (Fig. 2A). A major difference between transcriptome and miRNome was the ability of the latter to clearly separate preneoplastic from neoplastic

lesions, still maintaining the difference between KRT-19- and KRT-19+ nodules. Quantitative RT-PCR validation performed on randomly selected genes confirmed the microarray expression data for all the examined genes (Supporting Fig. 3). To identify the differentially expressed genes in each type of lesion towards its age-matched control we applied the Limma analysis package. As shown in selleck chemical the Venn diagram (Fig. 2B, left), although KRT-19- and KRT-19+ lesions

are histologically very similar, they exhibited a strikingly different number of modified genes. Besides the 64 dysregulated genes shared between KRT-19- and KRT-19+ likely involved in nodule formation, 602 genes were exclusively altered in KRT-19+ preneoplastic lesions, suggesting that they are relevant for nodule progression. Interestingly, 216 out of 234 altered genes in aHCC were altered in KRT-19+ nodules as well (Fig. 2B, right). Among these, 33/39 of the most up-regulated (fold change versus controls >5) and 12/15 of the most down-regulated (fold change versus controls <-5) genes in aHCC medchemexpress were the most dysregulated in KRT-19+ nodules as well (Table 1). These results suggest that the major expression changes leading to HCC occur in the very first stages of tumor progression. Ingenuity Pathway Analysis (IPA) of genes altered at the final stage of the carcinogenic process (aHCC) revealed that most of the dysregulated genes are involved in metabolic pathways. Among these, there are NRF2-mediated oxidative stress response, lipopolysaccharide (LPS) / interleukin (IL)-1-mediated inhibition of retinoic-X-receptor (RXR) function, aryl hydrocarbon receptor signaling, and xenobiotic metabolism (Fig. 3A). Strikingly, most of the altered pathways in aHCC were already modified in KRT-19+ preneoplastic lesions. Functional investigation also underlined common pathway modifications between early and late stages of hepatocarcinogenesis (Fig. 3B).