Differential reactivation of fetal/neonatal genes in mouse liver tumors induced in cirrhotic and non-cirrhotic conditions

Abstract

Hepatocellular carcinoma develops in either chronically injured or seemingly intact livers. To explore the tumorigenic mechanisms underlying these different conditions, we compared the mRNA expression profiles of mouse hepatocellular tumors induced by the repeated injection of CCl4 or a single diethylnitrosamine (DEN) injection using a cDNA microarray. We identified tumor-associated genes that were expressed differentially in the cirrhotic CCl4 model (H19, Igf2, Cbr3, and Krt20) and the non-cirrhotic DEN model (Tff3, Akr1c18, Gpc3, Afp, and Abcd2) as well as genes that were expressed comparably in both models (Ly6d, Slpi, Spink3, Scd2, and Cpe). The levels and patterns of mRNA expression of these genes were validated by quantitative RT-PCR analyses. Most of these genes were highly expressed in mouse livers during the fetal/neonatal periods. We also examined the mRNA expression of these genes in mouse tumors induced by thioacetamide, another cirrhotic inducer, and those that developed spontaneously in non-cirrhotic livers and found that they shared a similar expression profile as that observed in CCl4 -induced and DEN-induced tumors, respectively. There was a close relationship between the expression levels of Igf2 and H19 mRNA, which were activated in the cirrhotic models. Our results show that mouse liver tumors reactivate fetal/neonatal genes, some of which are specific to cirrhotic or non-cirrhotic modes of pathogenesis.

Keywords: Hepatocellular carcinoma; insulin-like growth factor 2; liver cirrhosis; mRNA expression; trefoil factor 3.

Figure 1

Identification of differentially expressed genes in mouse liver tumors induced in cirrhotic and non-cirrhotic models. (a) Gross appearance of livers from control (olive oil-treated, 32-week-old), CCl₄-treated (32-week-old), and diethylnitrosamine (DEN)-treated (46-week-old) mice. (b) Histology of the liver tissues from control, CCl₄-treated, and DEN-treated mice; HE and Sirius Red staining. NT, non-tumor; T, tumor. Arrowheads indicate the boundary of a DEN-induced tumor. Scale bar = 50 μm. (c) cDNA microarray analysis showing genetic clusters (A–D) of differentially expressed genes in control liver tissues (olive oil-treated), CCl₄-induced cirrhotic tissues (CCl₄-NT), CCl₄-induced tumors (CCl₄-T), and DEN-induced tumors (DEN-T).

Figure 2

Differential expression of mRNA and their products in CCl₄-induced and diethylnitrosamine (DEN)-induced liver tumors. (a) Quantitative RT-PCR analyses of mRNA expression of 15 tumor-associated genes. The genes preferentially expressed in CCl₄-induced tumors and DEN-induced tumors are designated “CCl₄-associated” and “DEN-associated,” respectively, and those comparatively expressed in CCl₄-induced and DEN-induced tumors are designated “Common.” Each value is expressed as the mean ± SEM. The ages of the mice at analyses were 32–34 weeks and 46 weeks in the CCl₄-induced and DEN-induced models, respectively. The number of samples in each group was 5, 12, 15, 6, and 13 for CCl₄ control (olive oil, C), CCl₄-induced cirrhosis (NT), CCl₄-induced tumors (T), DEN control (NT), and DEN-induced tumors (T), respectively. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; one-way factorial anova. (b) In situ detection of insulin-like growth factor 2 (IGF2), H19 mRNA, trefoil factor 3 (TFF3), and α-fetoprotein (AFP) in CCl₄-induced and DEN-induced tumors. Immunohistochemistry for IGF2, TFF3, and AFP and in situ hybridization for H19 mRNA. NT, non-tumor; T, tumor. Scale bar = 20 μm.

Figure 3

Relationship between mRNA expression of tumor-associated genes and tumor cell proliferation. Scatter plots of mRNA expression levels of selected tumor-associated genes and Ki-67 labeling index (%) in CCl₄-induced tumors (n = 16) and diethylnitrosamine (DEN)-induced tumors (n = 15). Spearman correlation coefficients were used to test the association of mRNA expression and tumor cell proliferation.

Figure 4

Fetal/neonatal activation of tumor-associated genes and their products. (a) Quantitative RT-PCR analyses of mRNA expression of tumor-associated genes during fetal/neonatal periods. Each value is expressed as the mean ± SEM. The number of samples in each group was 3, 5, 7, 10, 4, 4, 4, and 7 for E13.5, E16.5, P0 (immediately after birth), P1 (1 day after birth), P3 (3 days after birth), P6 (6 days after birth), 1 m (1 month old), and 5 m (5 months old), respectively. *P < 0.05, **P < 0.01, ***P < 0.001 versus control (5 m); one-way factorial anova. (b) In situ detection of insulin-like growth factor 2 (IGF2), H19 mRNA, trefoil factor 3 (TFF3), and α-fetoprotein (AFP) in developing livers. Immunohistochemistry for IGF2, TFF3, and AFP and in situ hybridization for H19 mRNA in fetal (E16.5) and neonatal (P0) livers. Scale bar = 20 μm.

Figure 5

Differential gene expression in thioacetamide (TAA)-induced liver tumors and spontaneously developed liver tumors in mice. (a) Histology of liver tissues from TAA-induced and spontaneous tumors. The ages of mice at analyses were 38–40 weeks and 13–15 months in the TAA-induced model and spontaneous model, respectively. HE staining. NT, non-tumor; T, tumor. Scale bar = 50 μm. (b) Quantitative RT-PCR analyses of mRNA expression of 15 tumor-associated genes in TAA-induced and spontaneous (Spont.) tumors. Each value is expressed as the mean ± SEM. The number of samples in each group was 7, 6, 9, 4, and 3 for the TAA control (C), TAA-induced cirrhosis (NT), TAA-induced tumors (T), control C3H mouse liver (NT), and spontaneous tumors (T) in C3H mice, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 versus control; one-way factorial anova.

Figure 6

Heatmap of 2-D hierarchical clustering of mRNA expression of 15 tumor-associated genes in four different liver tumor models: CCl₄-induced, diethylnitrosamine (DEN)-induced, thioacetamide (TAA)-induced, and spontaneous.

Figure 7

Association between mRNA expression of Igf2 and its related genes in CCl₄- and thioacetamide (TAA)-induced tumors. (a) Scatter plot showing a positive correlation between the mRNA expression of Igf2 and H19 in CCl₄-induced tumors (n = 27) and TAA-induced tumors (n = 9). Spearman’s correlation coefficient was used to test the correlation between the expression of the transcripts. (b) Igf2bp3 mRNA expression in tumors with substantial Igf2 mRNA expression (Igf2^high, Igf2/Gapdh ≥0.01; CCl₄-induced tumors, n = 12; TAA-induced tumors, n = 3) and very low or no Igf2 mRNA expression (Igf2^low, Igf2/Gapdh <0.01; CCl₄-induced tumors, n = 19; TAA-induced tumors, n = 6). **P < 0.01; unpaired two-tailed t-test.

Figure 8

Expression of insulin-like growth factor 2 (IGF2), trefoil factor 3 (TFF3), and α-fetoprotein (AFP) in human hepatocellular carcinoma (HCC) with or without liver fibrosis. (a) Immunohistochemistry for IGF2, TFF3, and AFP in human HCC and the surrounding cirrhotic tissues. Scale bar = 20 μm. (b) Expression of IGF2, TFF3, and AFP in HCC developed in non-cirrhotic and cirrhotic livers. Tumors containing clusters of cells with unequivocal cytoplasmic staining were regarded as positive (+).