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. 2016 Jan 5:16:3.
doi: 10.1186/s12885-015-2007-1.

MicroRNA expression analysis in high fat diet-induced NAFLD-NASH-HCC progression: study on C57BL/6J mice

Alessandra Tessitore  1 , Germana Cicciarelli  2 , Filippo Del Vecchio  3 , Agata Gaggiano  4 , Daniela Verzella  5 , Mariafausta Fischietti  6 , Valentina Mastroiaco  7 , Antonella Vetuschi  8 , Roberta Sferra  9 , Remo Barnabei  10 , Daria Capece  11 , Francesca Zazzeroni  12 , Edoardo Alesse  13
Affiliations

MicroRNA expression analysis in high fat diet-induced NAFLD-NASH-HCC progression: study on C57BL/6J mice

Alessandra Tessitore et al. BMC Cancer. .

Abstract

Background: Hepatocellular carcinoma (HCC) is the most common malignant tumor of the liver. Non-alcoholic fatty liver disease (NAFLD) is a frequent chronic liver disorder in developed countries. NAFLD can progress through the more severe non alcoholic steatohepatitis (NASH), cirrhosis and, lastly, HCC. Genetic and epigenetic alterations of coding genes as well as deregulation of microRNAs (miRNAs) activity play a role in HCC development. In this study, the C57BL/6J mouse model was long term high-fat (HF) or low-fat (LF) diet fed, in order to analyze molecular mechanisms responsible for the hepatic damage progression.

Methods: Mice were HF or LF diet fed for different time points, then plasma and hepatic tissues were collected. Histological and clinical chemistry assays were performed to assess the progression of liver disease. MicroRNAs' differential expression was evaluated on pooled RNAs from tissues, and some miRNAs showing dysregulation were further analyzed at the individual level.

Results: Cholesterol, low and high density lipoproteins, triglycerides and alanine aminotransferase increase was detected in HF mice. Gross anatomical examination revealed hepatomegaly in HF livers, and histological analysis highlighted different degrees and levels of steatosis, inflammatory infiltrate and fibrosis in HF and LF animals, demonstrating the progression from NAFLD through NASH. Macroscopic nodules, showing typical neoplastic features, were observed in 20% of HF diet fed mice. Fifteen miRNAs differentially expressed in HF with respect to LF hepatic tissues during the progression of liver damage, and in tumors with respect to HF non tumor liver specimens were identified. Among them, miR-340-5p, miR-484, miR-574-3p, miR-720, whose expression was never described in NAFLD, NASH and HCC tissues, and miR-125a-5p and miR-182, which showed early and significant dysregulation in the sequential hepatic damage process.

Conclusions: In this study, fifteen microRNAs which were modulated in hepatic tissues and in tumors during the transition NAFLD-NASH-HCC are reported. Besides some already described, new and early dysregulated miRNAs were identified. Functional analyses are needed to validate the results here obtained, and to better define the role of these molecules in the progression of the hepatic disease.

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Figures

Fig. 1
Fig. 1
Body weight patterns. a Mice were high fat (HF) or low fat (LF) diet fed, and weighed at the indicated time points. Values are means of 10 mice ± SEM. b Representative picture of a 6 months LF (left) and HF (right) diet fed mouse. c Mean of body mass index values ± SEM
Fig. 2
Fig. 2
Livers from HF and LF diet fed mice. a Livers from 3, 6, 12 months LF (left) and HF (right) diet fed mice. b Liver weights, expressed as mean ± SEM. Statistical significance is indicated as follows: **, P < 0.08; *, P = 0.05. c Macroscopic nodules in 12 months HF diet fed mice
Fig. 3
Fig. 3
Histopathological features of hepatic tissues. A Histopathological features of hepatic tissues from 3 (a, a1), 6 (b, b1), 12 (c, c1, c2) months LF (left) and HF (right) mice (H&E staining; original magnification 10X). The microphotographs, from LF mice, show a normal liver architecture (a), scattered inflammation (b, arrow) and simple steatosis with mild inflammation (c, arrows). A wide spectrum of liver damage ranging from simple steatosis (a1) to mild steatosis (b1) and a severe steatosis with massive inflammation (c1, c2) are shown in microphotographs from HF mice. B Fibrosis is not evident in 6 months LF (a) and HF (a1) mice (Masson’s trichrome staining, original magnification, 10X). Mild fibrosis appears after 12 months in LF mice (b, arrow, original magnification, 10X), whereas 12 months HF mice show more severe fibrosis (b1, original magnification 10X), often organized in irregular thin trabeculae that border nodules with a variable number of small microscopic arteries (arrows), and a disarrangement of normal hepatic architecture with an increase of cell density and frequent steatosis (b2). Moreover, there is a certain degree of cellular atypia, rare pseudoglandular structures and steatosis (b3, H&E original magnification 40X, red box and arrows respectively). These aspects are common features of dysplastic nodules or early HCC
Fig. 4
Fig. 4
Progression of liver disease. a Percentage of HF/LF mice showing steatosis, hepatic inflammation and fibrosis. b Degree of steatosis in HF and LF diet fed animals
Fig. 5
Fig. 5
MiRNAs differentially modulated during the progression of the hepatic damage. a RQ (relative quantification) values ± SE (Y axis) obtained by comparing HF to LF pooled RNAs from hepatic tissues. b RQ values ± SE (Y axis) of pooled RNAs from tumor tissues with respect to pooled RNAs from HF hepatic non-tumor tissues. Results are from 3 replicates. MammU6 was used as endogenous control
Fig. 6
Fig. 6
Differential expression of miR-125a-5p and miR-182 in livers and tumors, at the individual level. (a) (c) RQ (relative quantification) values ± SE (Y axis) in LF and HF mice (X axis) after diet treatment for 3, 6, and 12 months. MammU6 was used as endogenous control. RQ values were calculated with respect to one reference LF mouse in each experimental group (ID#: 2LF 3 M; 11LF, 6 M; 21LF, 12 M). Statistical significance is marked as follows: *, P < 0.05; **, P < 0.004; ***, P < 0.002 (b) (d) RQ values ± SE (Y axis) obtained by comparing tumors (HFT, black) vs. paired non tumor HF livers (light gray). 26 and 29 are ID numbers of mice with tumors
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