LEGENDS OF FIGURES
Figure 1. Pairwise gene expression correlation. For each gene (24), based on the average of samples in each compendium (full and partial), we measure for pairwise gene association using Pearson‘s correlation and the data was displayed using the heatmap.2 function. The analysis evidenced 3 groups of genes with similar expression patterns: 1. AFP, CK19, UHRF1, DNMT3B, HNF1A, FOXA2, HNF4A ; 2. SOX2 ;DNMT3A, TET3, TET1, DNMT1 , and 3. TET2 ; ALB, TDO2, CYP3A4, CK18 , and UGT1A1 . In the heatmap, the green color represents a reduction in gene expression, while the red color indicates an increase in expression.
Figure 2. Analysis of PCA K-means of hepatoblastomas and control liver samples based on their expression profile of 24 genes. The analysis was performed grouping HBs and non-tumoral liver samples with similar levels of gene expression which resulted in four clusters, indicated by color legends. In the figure, the triangles indicate samples of non-tumoral tissues; the squares indicate HB samples; circles indicate samples derived from cell culture (iPSC lines). The samples grouped in Set-1 are represented in black; samples grouped in Set-2 are in red; in green, samples grouped in Set-3; in blue, samples grouped in Set-4.
Figure 3. Analysis of gene expression of 13 genes in hepatoblastoma groups according to their clustering. Set-1 presents the upregulation of hepatoblast markers AFP , HNF4 A,FOX2A (A-C ), as well as the high expression level of the epigenetic machinery genes, mainly UHRF1 and TET1(D-I ). The Set-2 cluster samples revealed the upregulation of the epigenetic machinery genes, highlighting TET2 (D-I ), and low expression of markers of both the intermediary stages of differentiation (AFP ,HNF4A and FOXA2 ) (A-C ) and mature hepatocyte genes as ALB , UGT1A1 , and TDO2 (J-I; M ). Set-3 group the samples showed down-regulation of the epigenetic machinery genes (TET1, TET2, TET3, DNMT1, DNMT3A, UHRF1 ) (D-I ), associated with high expression of mature hepatocyte marker genes (ALB , UGT1A1, CYP3A4 , and TDO2 ) (J-M ).
Figure 4. Global methylation analysis of the three different clusters of hepatoblastomas and liver control samples. On the y-axis, the percentage of methylation is indicated for each sample. The boxplots referring to the overall methylation level show that the average content of this epigenetic mark differs between different HB clusters and non-tumoral liver tissues.
Figure 5. Hepatoblastoma stratification according to global content of DNA methylation and expression profile of DNA methylation enzymes and markers of hepatocyte differentiation. In the middle is depicted differentiation stages from stem cells to hepatocytes, with the respective genes used in the present study as markers of each stage. The blue bar indicates the global DNA methylation level, with the darkest color corresponding to the highest level of the epigenetic mark; similarly, the red gradient bar indicates the level of expression of DNA methylation genes (DNMT1, DNMT3A, DNMT3B, DNMT3L, TET1, TET2, TET3, and UHRF1 ), with the darkest one corresponding to the highest level. Clusters of HBs (Set-1, 2 and 3) are indicated on the top.