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hep26616-sup-0001-suppfig1.tif4140KFigure S1. MiRNA profile classifies the different steps of hepatocarcinogenesis. Hierarchical clustering of miRNA expression profile in KRT-19- and KRT-19+ preneoplastic lesions, adenomas (Ade), early HCCs (eHCCs) and advanced HCCs (aHCCs). Only miRNAs whose expression was dysregulated more than 4-fold in a minimum of two samples in at least one experimental group were considered. Each row represents the expression profile of a miRNA expressed as ΔΔ ct calculated respect to age-matched controls (see Supporting Material for details) and each column represents a sample. Red and green colors represent higher or lower expression levels of the miRNA (median centered), respectively.
hep26616-sup-0002-suppfig2.tif200KFigure S2. QRT-PCR validation of randomly selected miRNAs in the stepwise development of HCC. MiRNA expression was determined in KRT-19- and KRT-19+ nodules, adenomas, eHCCs and aHCCs. MiRNA expression is reported as log fold-change relative to age-matched controls. TLDA: TaqMan Low Density Array.
hep26616-sup-0003-suppfig3.tif178KFigure S3. QRT-PCR validation of randomly selected genes in the stepwise development of HCC. Dynamic mRNA expression was determined in KRT-19- and KRT-19+ nodules, adenomas, eHCCs and aHCCs. Gene expression is reported as log fold-change relative to age-matched controls.
hep26616-sup-0004-suppfig4.tif3344KFigure S4. Network visualization of the indicated transcription factors. Data and visualization were obtained using IPA. Red: up-regulated genes; green: downregulated genes. TF → target gene.
hep26616-sup-0005-suppfig5.tif6612KFigure S5. The miR-200 and miR-30 families are dysregulated during hepatocarcinogenesis. MiR-200 (A) and miR-30 (B) family expression evaluated by TLDA in different stages. MiRNA expression is reported as log fold-change compared to age-matched controls. TLDA: TaqMan Low Density Array.
hep26616-sup-0006-suppfig6.tif6612KFigure S6. Modulation of the NRF2 pathway affects HCC cell growth. A) HepG2 cells were transiently transfected with NRF2 or KEAP1 or control (siC) siRNAs or a miR-200a mimic and seeded in 96-well plates. Cell growth was evaluated by crystal violet staining 72 hours after seeding, in either the presence (H2O2) or the absence (NT) of 25uM hydrogen peroxide. B) Expression of the NRF2 target genes NQO1, GCLC and GSTA4 was evaluated by qRT-PCR on RNA extracted from the cells transfected in (A). Values are shown as fold change difference compared to control transfected cells (siC). C) HepG2 and rat cells (FaO and RH) were transiently transfected with the indicated siRNAs or miR-200a. Total cell lysates were extracted 48 hours upon transfection and analyzed by Western blot. Vinculin or βactin were used as loading control. Quantification of KEAP1 related to the corresponding βactin is shown for miR-200a (control siC was set as 1).
hep26616-sup-0007-suppfig7.tif72KFigure S7. In vitro T3 treatment of HepG2 cells inhibits the NRF2 pathway. HepG2 cells were treated with 10-6 M triiodothyronine for 48 hours. Total RNA was then extracted and expression of NRF2 target genes (NQO1, GCLC, GSTA4) was evaluated. Data are indicated as fold changes compared to not treated cells (NT).
hep26616-sup-0008-suppfig8.tif712KFigure S8. Flowchart for the intersection with the Liverome database. We first downloaded gene signatures from the Liverome database, for a total of 6.927 human genes. Then we focused on the dataset of 234 rat genes dysregulated in our model (aHCC vs ctr). For these genes, we looked for reliable human orthologous by means of the Ensembl database (version 66). Commonly dysregulated genes were then identified according to matching gene ids. Up/Down common modulations were assessed by means of the Fold-Change provided by the Limma package for the rat case and by the internal Liverome annotation for the human case.
hep26616-sup-0009-supptable1.doc29KSupp. Table 1. Percentage of early preneoplastic nodules (10 weeks after initiation with DENA) and aHCCs (14 months after initiation) positive for KRT-19.
hep26616-sup-0010-supptable2.doc78KSupp. Table 2A and Table 2B.
hep26616-sup-0011-supptable3.doc67KSupp. Table3A, Table3B, Table3C and Table3D.
hep26616-sup-0012-supptable4.doc36KSupp. Table 4. MiRNAs differentially expressed in KRT-19+ vs. KRT-19-
hep26616-sup-0013-supptable5.doc112KSupp. Table 5. List of the 86 rat/human orthologous genes involved in HCC progression
hep26616-sup-0014-supptable6.doc52KSupp. Table 6. List of the 26 miRNAs involved in both rat and human HCC progression
hep26616-sup-0015-suppinfo.doc67KSupporting Information

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