Background
Hepatocellular carcinoma (HCC) is the most frequent liver tumor and the third cause of cancer mortality worldwide [
1]. HCC etiopathogenesis is mainly related to viral infections (HBV, HCV) [
2], aflatoxin B1 and tobacco exposure [
3,
4], or chronic alcohol consumption [
5]. Deregulation at the level of several key signal transduction pathways (such as Wnt/β-catenin, MAPK, JAK-STAT, p53) have been extensively described in HCC pathogenesis [
6].
Non alcoholic fatty liver disease (NAFLD) is the most frequent liver disorder in western countries and occurs in individuals who do not abuse alcohol. NAFLD can be due to higher fat intake with diet, “de novo” lipogenesisis, or adipose tissue lipolysis increase [
7]. It is characterized by accumulation of triglycerides within hepatocytes (steatosis), attributable to an imbalance between storage and removal of lipids, and it is associated with obesity and metabolic syndrome [
8]. In a number of cases, NAFLD progresses from simple steatosis to non alcoholic steatohepatitis (NASH), a form of hepatic damage characterized by the recruitment of pro-inflammatory immune cells, and lastly toward cirrhosis and hepatocellular carcinoma [
7]. It has been calculated that a percentage variable between 4 and 22 % of HCC cases can be ascribed to NAFLD [
8]. However, molecular mechanisms responsible for NAFLD-NASH-HCC progression are not fully understood.
MicroRNAs (miRs, miRNAs) are short non-coding molecules able to regulate gene expression at the post-transcriptional level. MicroRNAs are involved in fundamental cellular processes, such as growth, proliferation and differentiation, apoptosis, metabolism, oncogenesis and metastasis [
9,
10]. Many miRNAs have been described in the initiation and progression of liver cancer [
11,
12]. Several down-regulated (i.e. miR-1, miR-7, miR-34a, miR-122, miR-125b, miR-200) or up-regulated (i.e. miR-17, miR-18, miR-19, miR-155, miR-93, miR-221/222) miRNAs have been identified as tumor suppressor or oncomirs, respectively, by targeting and regulating genes involved in cell proliferation, apoptosis, angiogenesis and metastasis [
13]. Several studies have furthermore shown expression level dysregulation and modulation of microRNAs in NAFLD, NASH, and then HCC. Among them, miR-122, miR-21, miR-155, miR-23a, miR-143, whose target genes have been characterized in both NAFLD (i.e.
PPARα, PTEN C/EBPβ, ORP8, G6PC) and HCC (i.e.
CCNG1, IGF-1R, ADAM17, PTEN, SOCS1, C/EBPβ, FNDC3B) [
14]. In addition, miRNAs have been described to be modulated even in steatosis/NASH (i.e. miR-155, miR-370, miR-34a, miR-200a/b, miR-99a/b), fibrosis (i.e. miR-200a/b, miR-221/222, miR-34a, miR-16, miR-99b), cirrhosis (i.e. miR-34a, miR-21, miR-31, miR-181b), and HCC (i.e. miR-16, miR-33, miR-21, miR-31, miR-181a/b, miR-99a, miR-200a/b) [
15]. However, miRNAs specifically involved in the progression of liver disease are not fully characterized. Therefore, to better define and identify microRNAs playing a pivotal role in this process, we analyzed in a time-dependent and dynamic manner the expression levels of miRNAs in livers from a long term high fat diet fed C57BL/6J mouse model, with the purpose to put into relation the expression levels of miRNAs with the progression of the liver’s injury.
Discussion
Nonalcoholic fatty liver disease is the most frequent chronic liver disease in western countries. It exhibits intra-hepatic fat accumulation and can progress through the more severe nonalcoholic steatohepatitis, leading, in a percentage of cases, to end-stage cirrhosis and HCC. Currently, some serum biomarkers are taken into consideration to diagnose and predict the progression of the disease, despite their limited prognostic usefulness, sensitivity, and tissue specificity. Several biomarkers, such as alpha-fetoprotein, alone or also in combination with osteopontin, glypican-3, laminin, VEGF (vascular endothelial growth factor), or hyaluronic acid, have been used to assess, without particularly significant results, HCC occurrence in NAFLD patients [
23‐
27]. Liver biopsy is still the most accurate procedure to diagnose and provide information about staging of liver disease, although studies have demonstrated that patients with initial NAFLD clinical manifestation and diagnosis do not develop HCC and that a regression may be also possible in pre-cirrhotic stages of the disease [
28]. Therefore, there is an urgent need to identify new diagnostic and prognostic markers able to follow the progression through NAFLD-NASH and HCC initiation and development.
MicroRNAs are short endogenous molecules which act in post-transcriptional gene regulation. Due to their role and structure, scientific evidences highlight the promising value of microRNAs as biomarkers at the diagnostic and prognostic level. In this study, we used a mouse model predisposed to NAFLD and obesity to analyze the progression of high-fat diet induced liver disease through NAFLD-NASH up to HCC initiation and development. Depending on the treatment’s duration, HF-fed animals showed an increase of body and liver weights, degree of steatosis, presence of inflammatory infiltrate and fibrosis, demonstrating the progression of liver disease. As described, the LF group showed pathological features similar to the HF, which, however, appeared later and with lower severity. This could be explained by the fact that the control LF diet here used, with higher caloric content than a standard diet, is formulated low in fat, but high in sucrose. Previous studies have discussed the role of high-carbohydrate diets on lipid accumulation and the effects of chronic fructose consumption on different tissues: in liver, inflammation, dyslipidemia, and steatosis have been described [
29‐
31]. This could trigger the
de novo lipogenesis process, with delayed lipid accumulation and cellular damage in livers in comparison to that observed in HF mice. A recent work, performed on 15 weeks-old high fructose or sucrose diet fed C57BL/6 mice, showed fatty infiltration of necroinflammatory areas, which are characteristic features of the transition to NASH, enhanced lipogenesis, gluconeogenesis and anti-oxidant imbalance, demonstrating an adverse effect of fructose or sucrose-rich diets on liver [
32]. Biochemical assays highlighted increasing values of plasma biomarkers in HF animals, characterizing the presence of metabolic dysfunctions and liver damage. No particular evidence of cirrhosis was detected, and a percentage of HF fed mice (2/10) developed tumors after 12 months. Fifteen miRs resulted differentially expressed in livers, by comparing HF- and LF-diet treated animals, and in tumors with respect to non tumor HF liver tissues, providing evidence of their modulation during the progression of diet-induced liver damage. As summarized in Table
2, some among them were already described in NAFLD, NASH, fibrosis or HCC, whereas others are for the first time here identified. MiR-155, whose expression increased after 12 months HF diet treatment, resulted over-expressed in tumors and in HF tissues with respect to LF. Previous studies have demonstrated that miR-155 plays an important role in hepatic lipid metabolism, has a protective role against HF diet-induced non alcoholic liver steatosis [
33], and was found to be up-regulated in NASH models of methyl-deficient diet, in HCC induced by choline-deficient and amino acid-defined diet, and in primary human HCC [
34,
35]. Moreover, it has been demonstrated that miR-155 deficiency can attenuate steatosis and fibrosis [
36]. In addition, anti-miR-155 has shown in vitro and in vivo potential therapeutic efficacy, by restoring the expression of
C/EBPβ and
FOXP3 [
37].
Table 2
Dysregulated miRNAs and their involvement in liver disease. ICC intrahepatic cholangiocarcinoma, HNSCC head and neck squamous cell carcinoma
miR-155 | Protective role against non alcoholic steatosis [ 33] | LXR-α[ 33] C/EBPβ, FOXP3 [ 35‐ 37] |
Up-regulated in NASH [ 34] |
|
MiR-155 deficiency attenuates steatosis and fibrosis [ 36] |
miR-193b | Down-regulated in HBV+ HCC [ 43] | |
|
|
miR-20a | Down-regulated in HCC [ 49] | |
miR-200a | Up-regulated in NAFLD [ 51] | |
Down-regulated in HCC [ 52] |
miR-200c | Up-regulated in NAFLD [ 51] | |
Down-regulated in HCC and ICC [ 54] |
miR-27a | Up-regulated in HBV+ HCC [ 55] | RXRα, PPARα/γ, FASN, SREBP1, SREBP2 [ 58, 59] |
miR-31 | Up-regulated in fibrosis [ 60] | |
Up-regulated in HCC [ 17, 53] |
miR-93 | Up-regulated in HCC [ 61, 62] | |
miR-99b | | mTOR (pancreatic cancer) [ 66] |
|
miR-125a-5p | Down-regulated in HCC [ 89] | |
Biomarker in liver disease [ 93] |
miR-182 | Up-regulated in NAFLD-fibrosis [ 98] | |
|
Up-regulated in HCC [ 94‐ 97] | EphrinA5 [ 96], FOXO1 [ 97] |
MiR-193b was down-regulated after 3 and 6 months of HF regimen and revealed over-expression in tumor samples. The role of this miR in carcinogenesis is quite controversial: miR-193b was described as a tumor suppressor and appeared down-regulated in several cancers, such as melanoma, breast, prostate carcinoma, and human HCC tissues, mainly HBV-positive [
38‐
43]. In vitro and in vivo experimental data demonstrated that miR-193b directly targeted
CCND1 (
cyclin D1) and the transcription factor
ETS1 [
43]. In a study on two HF diet fed mouse models, showing marked susceptibility (C57BL/6J) or resistance (Balb/c) to NAFLD and insulin resistance phenotype, significant up- or down-regulation of key genes which may be involved in homeostatic adaptation to HF regimen has been detected. Among them, are
CCND1 and
ETS1, whose up-regulation was detected in both strains or in C57BL/6J alone, respectively [
44]. This evidence could be in agreement with miR-193b down-regulation detected in our model during the first 6 months of HF diet treatment. With this regard,
ETS1/miR-193b 3′UTR alignment can be identified in
Mus musculus (microrna.org: SVR score −0.121, PhastCons 0.66). On the other hand, miR-193b over-expression was described by Braconi et al. [
45] in HCV-positive HCC tissues and cells. MiR-193b over-expression was also detected in head and neck squamous cell carcinoma [
46], where
neurofibromin1 (
NF1) was described as a target, and in glioma [
47], where this miR acted as an oncomiR by targeting
Smad3, one of the major
TGF-β signaling transducers. Beside, a study on a mouse model demonstrated that forced expression of
Smad3 may reduce liver susceptibility to chemically-induced carcinogenesis by promoting apoptosis through
Bcl-2 transcriptional repression [
48]. With this regard, two miR-193b target sites are predicted on mouse
Smad3 3′-UTR (microRNA.org: miR-SVR score −0.1684 and −0.0002; PhastCons 0.5285 and 0.5702, respectively), leaving hypothesize that miR-193b over-expression could be involved in hepatocarcinogenesis through
Smad3 down-regulation.
MiR-20a was down-regulated in liver tissues and in tumors from HF mice. MiR-20a down-regulation was described in human HCC, where
Mcl-1 (
myeloid cell leukemia sequence 1), an anti-apoptotic member of
Bcl-2 family, was identified as miR-20a target [
49]. In accordance, a miR-20a predicted target site is located on
Mus musculus Mcl-1 3′UTR (microRNA.org: mirSVR score −0.9773, PhastCons 0.710).
MiR-200a and c, members of the miR-200 family, showed different behavior: after expression level’s decrease (6 months), miR-200a increased during the progression of hepatic damage (12 months), whereas miR-200c revealed a trend of down-regulation during HF diet treatment and in tumors. It is known that miR-200 family plays a role as tumor suppressor by inhibiting epithelial-to-mesenchymal transition (EMT) and repressing cancer stem cells; in addition, its deregulation has been described in several tumor types, including hepatocarcinoma [
50]. MiR-200a was found to be up-regulated in NAFLD [
51], significantly down-regulated in human HCC samples and, along with miR-200 family members, has been described as a marker able to distinguish between cirrhotic and cancer tissues [
52,
53]. MiR-200c was also found to be up-regulated in NAFLD and down-regulated in human HCC as well as in intrahepatic cholangiocarcinoma (ICC) samples [
51,
54]. With regard to ICC, Oishi et al. [
54] found that miR-200c and miR-141, were negatively correlated with genes involved in the
TGF-β,
NF-κB and
Smad signaling pathway. In addition, these two miRs were able to induce epithelial differentiation and to suppress EMT by inhibition of
ZEB1 and
ZEB2. The same authors also described
NCAM1, a known hepatic stem cells marker strictly connected to EMT process, as a miR-200c direct target. Analogously, several miR-200c binding sites are predicted on
Mus musculus ZEB1,
ZEB2 and
NCAM1 3′UTR, indicating its putative role in the mouse model here presented.
MiR-27a showed expression decrease, starting faintly after 3 months up to 12 months of HF diet administration. Conversely, it was over-expressed in tumors. Literature data reveal that miR-27a may have an oncogenic role, being up-regulated in HBV-related HCC tissues and HCC cell lines [
55], and promoting proliferation in liver cancer cells by diminishing
TGF-β tumor suppressive activity [
56]. MiR-27a was also found in a hypomethylated status which led to its over-expression in HCC cells [
57]. MIR-27a was described to be involved in lipid metabolism, by regulating
RXRα,
PPARα/γ,
FASN,
SREBP1,
SREBP2, and was able to inhibit HCV replication in human hepatoma cells [
58]. Ji et al. [
59] demonstrated that miR-27a/b were over-expressed in primary culture activated rat hepatic stellate cells (HSCs). Normal HSCs are in the space of Disse, storing bunches of vitamin A-riching lipid droplets. On the contrary, activated HSCs lose cytoplasmic lipid droplets and trans-differentiate to proliferative, fibrogenic myofibroblasts which play an essential role in liver fibrosis initiation. In the above-mentioned study, miR-27a/b downregulation was demonstrated to be able to activate HSCs to switch to a more quiescent phenotype, with decreased cell proliferation and restored cytoplasmic lipid droplets. Seen in this context, it could be supposed that miR-27a hypoexpression (6M, weak, and 12M) in HF diet model might act as a protective mechanism in limiting the progression of liver damage during the phases of the disease, and, on the other hand, its over-expression in tumors could be associated to promotion of heavier liver injury with consequent HCC initiation.
MiR-31 was detected up-regulated in tumors with respect to livers from 12 months HF mice. MiR-31 up-regulation was also described in human HCC samples and in a similar C57BL/6J high-fat diet fed model [
17,
51]. MiR-31 up-regulation was also described in fibrosis [
60].
MiR-93 showed slight hypo-expression after 12 months HF diet and resulted down-regulated in HCC. Although, previous reports described an increase of miR-93 level during hepatic tumorigenesis [
61], and over-expression in human HCC cell lines and tissues [
62], miR-93 down-regulation significantly correlated with worse prognosis in colorectal cancer, where it was described to suppress oncogenesis by regulating
Wnt/β-catenin pathway [
63,
64].
MiR-99b was weakly down-regulated during HF diet administration and, conversely, up-regulated in tumors. MiR-99b was described to contribute to irradiation resistance in human pancreatic cancer by targeting
mTOR [
65], whose activity is also known to play a role in NAFLD-NASH [
66‐
68]. In this context, miR-99b hypoexpression in our model might contribute to induce
mTOR expression and function in the progression through NAFLD and NASH. A mir-99b/
mTOR site alignment is also predicted on mouse (mirSVR score, −1.2245; PhastCons score, 0.7484). In a very recent work [
69] miR-99b was up-regulated in HCC, where it promoted metastasis by inhibiting
claudin 1 (
CLDN1).
In silico analysis displays two predicted miR-99b sites also on mouse
CLDN1 (PhastCons 0.55 and 0.60).
No data are reported about the expression and role of miR-340-5p, miR-484, miR-574-3p, and miR-720 in NAFLD, NASH and HCC tissues. The above-mentioned miRs appear to be up- (miR-484 and miR-574-3p) or down-regulated (miR-340-5p, miR-720) in tumor tissues. Just one study showed miR-574-3p increase in sera from HCC and liver cirrhosis patients [
70]. Controversial evidences about the role of those miRNAs in oncogenesis are reported in several studies. MiR-340 has been described as a tumor suppressor in breast [
71], NSCLC [
72], and melanoma [
73]. Significant miR-484 level increase was described in sera from early breast cancer [
74] and in melanoma [
75], whereas miR-484 down-regulation was displayed in urine from prostate cancer patients [
76]. MiR-574-3p was identified over-expressed in plasma from head and neck [
77] and in prostate cancer patients [
78]. On the contrary, it was found down-regulated in colorectal [
79] and esophageal cancer [
80]. MiR-720 was described to inhibit breast tumor invasion and migration by targeting the metastasis promoter TWIST1 [
81]. Conversely, it resulted hyper-expressed in colorectal cancer [
82].
MiR-125a-5p is transcribed as a cluster with let-7 and miR-199b. Similarly to miR-99b, miR-125a-5p revealed hypo-expression during the treatment. On the other hand, it increased and showed over-expression in tumors. Interestingly, miR-125a-5p differential expression, detected on pooled RNAs, is maintained with statistical significance in mice HF fed for 3, 6, and 12 months individually analyzed, and in tumors, suggesting miR-125a-5p potential high impact at the functional level starting from the early stage of the liver disease. MiR-125a-5p seems to possess oncogenic or tumor suppressor activities. It was described as an epidermal growth factor signaling-regulated miRNA which can negatively regulate human lung cancer cell migration and invasion
in vitro, is frequently down-regulated in lung cancer [
83], and seems to play a role in enhancing
in vitro cell migration and invasion in NSCLC [
84]. Yang et al. [
85] described miR-125a-5p up-regulated in lung squamous cell carcinoma (SCC), whereas miR-125a-5p low expression levels in tissues or serum have been associated with enhanced malignant potential in gastric and breast cancer [
86,
87]. MiR-125a-5p was described over-expressed in thyroid carcinomas [
88] and hypo-expressed in human HCC [
89], where it was shown to target the 3′UTR of
SIRT7, a member of the Sirtuin family, whose activity in cancer, ER and genomic stress response, hepatosteatosis has already been investigated and is still controversial [
90]. Two predicted miR-125a-5p interaction sites are also detected on mouse
SIRT7 (microRNA.org: mirSVR score −0.0006, −0.3021; PhastCons 0.5087, 0.5259, respectively). MiR-125a-5p is also involved in lipid metabolism [
91] and its level has been found increased in hyperlipidemic and/or hyperglycemic patients’ sera [
92]. MiR-125a-5p serum levels have been also described as biomarkers in liver diseases [
93].
MiR-182 showed over-expression already after 3 months of HF diet, and this trend was markedly maintained in mice, also at the individual level, during the treatment and in tumors. Several studies demonstrated miR-182 involvement in HCC and metastasis, by controlling the expression of genes with tumor suppressor activity, such as the metastasis suppressor
MTSS1 [
94],
Cebpa [
95],
ephrinA5 [
96], and
FOXO1 [
97]. MiR-182 was also down-regulated in fibrosis related to NAFLD, where
FOXO3 was described as a target [
98]. MiR-182/
Cebpa/ephrinA5/FOXO1/FOXO3 alignments can be also predicted on
Mus musculus, putatively indicating a role of miR-182 in the regulation of those genes. Our data demonstrate early involvement of miR-182 in the transition of liver injury, which is maintained up to HCC initiation and development, indicating that early deregulation of this microRNA could be one among the factors putatively responsible for the hepatic disease here represented, and for its progression.