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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

MicroRNAs (miRs) are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of messenger RNAs (mRNAs) at the posttranscriptional level. Aberrant expression of certain microRNAs plays a causal role in tumorigenesis. Here, we report identification of hepatic microRNAs that are dysregulated at early stages of feeding C57BL/6 mice choline-deficient and amino acid–defined (CDAA) diet that is known to promote nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 84 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P ≤ 0.01) altered in mice fed CDAA diet for 6, 18, 32, and 65 weeks compared with those fed choline-sufficient and amino acid–defined (CSAA) diet. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated up-regulation of oncogenic miR-155, miR-221/222, and miR-21 and down-regulation of the most abundant liver-specific miR-122 at early stages of hepatocarcinogenesis. Western blot analysis showed reduced expression of hepatic phosphatase and tensin homolog (PTEN) and CCAAT/enhancer binding protein beta (C/EBPβ), respective targets of miR-21 and miR-155, in these mice at early stages. DNA binding activity of nuclear factor kappa B (NF-κB) that transactivates miR-155 gene was significantly (P = 0.002) elevated in the liver nuclear extract of mice fed CDAA diet. Furthermore, the expression of miR-155, as measured by in situ hybridization and real-time RT-PCR, correlated with diet-induced histopathological changes in the liver. Ectopic expression of miR-155 promoted growth of hepatocellular carcinoma (HCC) cells, whereas its depletion inhibited cell growth. Notably, miR-155 was significantly (P = 0.0004) up-regulated in primary human HCCs with a concomitant decrease (P = 0.02) in C/EBPβ level compared with matching liver tissues. Conclusion: Temporal changes in microRNA profile occur at early stages of CDAA diet-induced hepatocarcinogenesis. Reciprocal regulation of specific oncomirs and their tumor suppressor targets implicate their role in NASH-induced hepatocarcinogenesis and suggest their use in the diagnosis, prognosis, and therapy of liver cancer. (HEPATOLOGY 2009.)

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer in the world, with an annual death rate exceeding 500,000.1, 2 The low survival rate is probably attributable to the late-stage diagnosis of this cancer. Primary HCC, the most common primary malignant tumor of the liver, accounts for more than 90% of all primary liver cancers. The development of HCC is a complex, multistep process that is generally characterized by steatosis, hepatocyte degeneration, fibrosis, inflammatory infiltrates, and Mallory's hyaline.3 The incidence of nonalcoholic fatty liver disease is increasing dramatically, particularly in the Western world, which can lead to an increase in the prevalence of nonalcoholic steatohepatitis (NASH) and associated complications such as cirrhosis and HCC.4 NASH also can be associated with obesity, diabetes, and insulin resistance, all of which can contribute to an increased risk of HCC. Although our understanding of the pathogenesis of HCC in chronic viral infections such as hepatitis B or hepatitis C is improving, there is a complete lack of insight into the pathogenesis of NASH-associated HCC. Consequently, it is critical to delineate the molecular mechanisms involved in NASH-mediated hepatocarcinogenesis. An interesting and novel strategy is to determine changes in the expression profiles of specific microRNAs (miRs) and their target messenger RNAs (mRNAs) at different stages of liver tumorigenesis in an animal model. Such exploration is likely to provide important information regarding the miR signature and their target mRNAs at a very early stage of liver tumorigenesis and their relationship to the miRNA signature of primary human HCC that can be used in the diagnosis and prognosis of liver cancer.

MiRs are conserved small, noncoding RNAs identified in plants, animals, and viruses5 that, in general, negatively regulate gene expression by interacting with the 3′-untranslated region of protein coding genes.6 Primary miRs predominantly coded by RNA polymerase II are processed to precursor miRs (pre-miRs) by Drosha/DGCR8 in the nucleus.7 Pre-miRs are transported to the cytoplasm by exportin 5 to undergo further processing by DICER1 to mature miRs that are recruited by miR-induced gene silencing complex to exert their biological functions.

Recent studies have identified an important role for miR in several human cancers, including human HCC.8, 9 The expression profiling studies in human HCC have identified aberrant expression of several miRs.10 These signatures have the potential for use as markers of disease progression and prognosis, and they may serve as therapeutic targets. Elucidating the role of aberrant miR expression at early stages in hepatocarcinogenesis is likely to enhance understanding pathogenesis of the disease.

Although different animal models of hepatocarcinogenesis are reported, a dietary model of NASH associated with progressive disease resulting in HCC11, 12 is of particular interest. Rodents on this diet show well-defined pathological changes that are markedly similar to the progression of liver cancer in humans. We used a recently developed mouse model to identify temporal changes in miR expression with the goal of elucidating the role of specific miRs in the initiation and progression of hepatocarcinogenesis. Here, we show that some of the key oncomirs and their tumor suppressor targets are reciprocally regulated in a murine dietary NASH model that mimics altered miR expression profile in primary human HCCs.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Mice and Diet.

All animal experiments and diet are described in Supporting Methods.

Microarray Analysis.

Microarray analysis was performed as described.13 Briefly, 5 μg of total RNA was used for hybridization of miR microarray chips. The chips were hybridized in 6× SSPE (3.0 M Sodium Chloride 0.2 M Sodium Hydrogen Phosphate, 0.02 M EDTA pH 7.4) at 37°C. The miRs were labeled as biotin-containing transcripts and detected by streptavidin-Alexa647 conjugates. The processed slides were scanned using a microarray scanner. The miR nomenclature was according to miRBase (http://microrna.sanger.ac.uk).

The alteration in the level of miRs was considered statistically significant if their P-value was lower than 0.01. We also performed a global test to determine whether the expression profiles differed between the classes by permuting the labels of each array corresponding to each class. For each permutation, the P values were recomputed, and the number of genes significant at the 0.01 level was noted. The proportion of permutations that gave at least as many significant genes as with the actual data was the significance level of the global test: P = 0.003 for the randomized block design and P = 0.025 for the paired t test.

Real-Time Reverse-Transcription Polymerase Chain Reaction and In Situ Hybridization.

Real-time reverse-transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) are described in Supporting Methods.

Cell Lines and HCC Tumor Tissue.

HCC cell lines obtained from the American Type Culture Collection (Manassas, VA) were cultured as recommended by the supplier. Primary human hepatocellular tumor and adjacent normal tissue samples were obtained from the Cooperative Human Tissue Network at The Ohio State University James Cancer Hospital. Tissue specimens were procured in accordance with The Ohio State University Cancer Internal Review Board guidelines.

Preparation of the liver nuclear extract and electrophoretic mobility-shift assay (EMSA) were performed as described.14, 15 The detailed protocol and oligo sequences are provided in the Supporting Methods.

Imunohistochemical analysis were performed as described.16, 17

Transfections.

For miR precursor or anti-miR transfection, cells were plated in 60-mm dishes and transiently transfected with 50 nM pre–miR-155, negative control RNA, 60nM anti–miR-155 inhibitor, or control anti-sense RNA (Applied Biosystems, Foster City, CA).

Western Blot Analysis.

Whole cell or tissue extracts were prepared in sodium dodecyl sulfate lysis buffer followed by immunoblotting with specific antibodies (catalogue numbers are provided in the Supporting Material) as described.17

Cell Proliferation Assay.

Cell proliferation was assessed using a cell proliferation reagent kit I (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT]) (Roche Applied Science, Indianapolis, IN) as described.16

Statistical Analysis.

Statistical significance of differences between groups was analyzed by unpaired Student t test, and P ≤ 0.05 was considered to be statistically significant. Paired Student t test was used to analyze differences in expression of miR and mRNA levels among tumors and paired nontumor tissues in real-time RT-PCR analysis. Single and double asterisks indicate P ≤ 0.05 and P ≤ 0.01, respectively. The correlation between miR-155 and CCAAT/enhancer binding protein beta (C/EBPβ) mRNA levels was analyzed by two-tailed Pearson correlation test. All real-time RT-PCR (assayed in triplicate), western blotting, and transfection experiments were repeated twice, and reproducible results were obtained. Representative data are presented in each experiment.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Temporal Changes in Hepatic MicroRNA Expression Profile Occur at Early Stages of Hepatocarcinogenesis Induced by the Choline-Deficient, Low-Methionine, and Amino Acid–Defined Diet.

The choline-deficient, low- methionine, and amino acid–defined diet (CDAA diet) is known to induce liver tumors in C57BL/6 mice,11, 12 which are normally resistant to hepatocarcinogenesis. Mice on a CDAA diet develop NASH at early stages, leading to the formation of preneoplastic nodules after 65 weeks and hepatocellular adenomas and carcinomas after 84 weeks11, 12 (Supporting Fig. 1).

To identify miRs that may play a causal role in hepatocarcinogenesis, we performed microarray analysis of hepatic RNA in mice fed a CDAA diet for different periods. The result showed deregulation of 30 miRs (P ≤ 0.01) in mice fed a CDAA diet for 6, 18, 32, and 65 weeks compared with those fed a choline-sufficient and amino acid–defined (CSAA) (control) diet (Fig. 1). Among these miRNAs, 17 were up-regulated and 10 were down-regulated in at least one time point (Table 1). Three miRs, miR-17, miR-346, and miR-20b, were up-regulated at 32 weeks but decreased at 65 weeks. The up-regulated miRs in mice fed a CDAA diet can be broadly classified into four groups based on their expression: (1) microRNAs such as miR-155, miR-221, miR-222, miR-34a, miR-223, miR-342, and miR-16 consistently up-regulated and remained high from 18 to 65 weeks; (2) miR-181, miR-150, miR-99b, miR-214, miR-142, and miR-195 increased at 32 and 65 weeks; (3) miR-17, miR-346, and miR-20b up-regulated transiently at 32 weeks; and (4) miR-200 and miR-487a elevated only at 65 weeks. Based on this temporal pattern of expression of several miRs, it is conceivable that the targets of each miR are likely to be involved in precise control of diet-induced pathological changes in the liver. It is notable that some of these miRs, such as miR-221/222, miR-181b, miR-34a, miR-214, miR-16, and miR-99b, are also up-regulated in the livers of human NASH patients.18

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Figure 1. MicroRNA expression was dysregulated at early stages of hepatocarcinogenesis. (A) Clustering of the miRNA expression profiles at four time points (6, 18, 32, and 65 weeks) was performed by average linkage using correlation metrics. MicroRNAs were selected by class comparison using analysis of variance with randomized block design. The cluster tree with the fold change of 30 miRs (P ≤ 0.01) that varied with the time course was constructed.

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Table 1. Hepatic microRNAs Dysregulated in Mice Fed a CDAA Diet for 6, 18, 32, and 65 Weeks
Gene SymbolFold ChangeParametric P ValueFDR False Discovery Rate
6 Weeks18 Weeks32 Weeks65 Weeks
  1. Analysis of variance with randomized block design identified 30 hepatic miRNAs that are altered (P < 0.01) on feeding mice a CDAA diet compared with a CSAA diet. RNA from four mice on a CSAA diet and five mice on a CDAA diet at each time point was used for microarray analysis.

miR-200c1.000.841.002.670.0000.004
miR-200b1.001.001.002.530.0010.010
miR-181d0.770.454.212.400.0000.005
miR-1550.871.432.811.900.0000.000
miR-487a3.040.920.461.780.0060.040
miR-181b0.700.923.201.73<1e-07<1e-07
miR-2230.471.842.821.62<1e-07<1e-07
miR-342-3p0.511.541.731.400.0000.000
miR-1500.990.922.791.390.0000.005
miR-99b0.761.001.681.330.0000.001
miR-2140.800.981.731.320.0020.017
miR-2210.801.191.511.320.0090.057
miR-1950.901.021.671.290.0000.000
miR-142-5p0.541.032.191.280.0050.035
miR-2220.841.171.371.280.0020.017
miR-34a0.571.221.621.260.0040.032
miR-160.881.121.721.160.0000.000
miR-1071.010.690.670.920.0010.006
miR-30a1.020.920.810.880.0050.038
let-7a1.100.660.620.850.0050.038
miR-1031.000.711.080.810.0090.054
miR-30b0.990.730.960.740.0040.032
miR-30e0.960.830.700.700.0100.059
miR-323-5p1.340.750.520.600.0010.008
miR-27a1.061.010.970.400.0070.048
miR-8021.000.740.710.280.0000.000
miR-320.980.870.790.210.0000.001
miR-170.830.991.450.900.0000.003
miR-3461.501.191.340.410.0000.001
miR-20b0.770.921.200.760.0020.016

Real-Time RT-PCR Analysis Confirmed Up-regulation of Several miRNAs Including Oncogenic miR-155, miR-221, miR-222, miR-21, and Down-regulation of miR-122 in Mice Fed a CDAA Diet.

Next we confirmed dysregulation of a few critical miRs by real-time RT-PCR analysis of mature miRs. The result showed that hepatic miR-155 known to be induced by inflammatory mediators19 was up-regulated (approximately 2.3-fold) (P = 0.003) in animals fed a CDAA diet for 18 weeks and remained elevated after 32 weeks (P = 0.005) and 65 weeks (P = 0.005) compared with that in control mice (Fig. 2A). We also observed significant up-regulation of miR-221 (approximately 1.5-fold) (P = 0.0005) at an early stage (18 and 32 weeks) (Fig. 2A), suggesting that it may play a key role in the NASH. Interestingly, miR-222 processed from the same primary transcript of miR-221 was also elevated (approximately 1.5-fold) (P = 0.02) after feeding a CDAA diet for 18 and 32 weeks (Fig. 2A). Unlike miR-221, the miR-222 level remained elevated even after 65 weeks, which may be attributable to differential processing of these two miRs. Notably, maximal increase in most of these miRs' expression was observed after 32 weeks.

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Figure 2. (A) Validation of miRNA microarray data by real-time RT-PCR analysis of deoxyribonuclease I–treated total RNA. RNAs from five mice were used for RT-PCR, and each sample was analyzed in triplicate. Single and double asterisks denote P ≤ 0.05 and P ≤ 0.01, respectively. (B) Localization of miR-122 in livers by LNA-ISH. Tissue sections were hybridized to biotin-labeled oligo (anti–miR-122), which was captured with alkaline phosphatase–conjugated streptavidin, and the signal (blue color) was developed with nitro blue tetrazolium chloride/5-Bromo-4-chlor-3-indolyl phosphate (NBT/BCIP). The cell body was stained with nuclear fast red. Red arrows indicated mature miR-122. (C) Representative photographs of hematoxylin-eosin–stained liver sections from mice. (D) Localization of miR-155 in livers by LNA-ISH. Scrambled oligo probe was used as negative control. Green arrows indicate mature miR-155.

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Although microarray analysis did not reveal significant alteration in the expression of miR-21, we measured its level by real-time RT-PCR analysis because it is an oncomir frequently up-regulated in many solid tumors, including HCC.20 The results showed a small but significant increase in miR-21 after 18 weeks (P = 0.0006), which persisted after 32 (P = 0.006) and 65 weeks (P = 0.03) of feeding a CDAA diet (Fig. 2A). In contrast, the level of miR-122, the most abundant (70%) hepatic miR, which is frequently down-regulated in NASH18 and HCC,21 decreased by 40% (P = 0.006) at 65 weeks (Fig. 2A). Northern blot analysis confirmed the real-time RT-PCR data of these two miRs (Supporting Fig. 2A, B). In situ hybridization with locked nucleic acid (LNA)-modified anti–miR-122 probe also showed a decrease in miR-122–positive hepatocytes in mice fed a CDAA diet for 65 weeks (Fig. 2B). No signal was detected, with scrambled oligo demonstrating specificity of the probes (data not shown). The inability to detect alteration in miR-21 and miR-122 by microarray analysis is probably attributable to saturation of the signal of these abundant miRs and a higher threshold set for a specific analysis (such as P ≤ 0.01 in this microarray analysis). Taken together, these results demonstrate dysregulation of both oncogenic and tumor suppressor miRs at early stages of CDAA diet–induced hepatocarcinogenesis, which is consistent with their differential expressions in primary human HCC.20, 21

Histopathological Analysis Revealed NASH in Mice Fed a CDAA Diet That Correlated with a Higher miR-155 Level.

Mice fed a CDAA diet had a higher level of steatosis (90% as opposed to 30%-60% in the control livers) (Table 2). A representative histopathology profile is presented in Fig. 2C. Moderate inflammation occurred in the livers of mice fed a CDAA diet for 32 weeks. Mice fed a control diet had negligible NASH (score, 1-2 points), whereas mice fed a CDAA diet exhibited higher NASH (5 points) (Table 2). Although animals in both diet groups developed marked steatosis after 65 weeks (Fig. 2C), inflammation was more prominent in the CDAA diet group (Table 3). Dysplastic nodules were apparent in one liver (CDAA no. 2). Notably, three of four mice developed NASH after 65 weeks on a CDAA diet, whereas none from the CSAA-fed group exhibited NASH (Table 3).

Table 2. Liver Pathology and miR Expression in Mice Fed Diet for 32 Weeks
CaseHistologymiR-155-LNA-ISHmiR-155/18S rRNA (2−ΔCt × 104)
  1. Formalin-fixed, paraffin-embedded (FFP) liver sections were stained with hematoxylin-eosin. Steatosis, inflammation, and fibrosis were scored blindfolded by a liver pathologist. FFP tissue sections were also subjected to in situ hybridization with LNA-modified anti–miR-155 (LNA-ISH), and miR-155–positive cells were counted. The miR-155 was scored as follows: 1+ was between 1% and 20% positive, 2+ was 21%-50% positive, 3+ was >50% positive (the majority of cells). The numbers were derived from counting at least 250 hepatocytes. The miR-155 level was also determined in the liver by real-time RT-PCR.

CSAASteatosis (60%)  
 Inflammation (0)1+4.28
 Not NASH7% Hepatocytes + 
CSAASteatosis (50%)  
 Inflammation (0)2+6.2
 Not NASH22% Hepatocytes + 
CSAASteatosis (30%)  
 Inflammation (0)1+2.57
 Not NASH2% Hepatocytes + 
CSAASteatosis (30%)  
 Inflammation (0)1+2.68
 Not NASH5% Hepatocytes + 
CDAASteatosis (90%)  
 Inflammation (moderate)3+17.56
 NASH69% Hepatocytes + 
CDAASteatosis (90%)  
 Inflammation (moderate)3+16.3
 NASH57% Hepatocytes + 
CDAASteatosis (90%)Not determined11.38
 Inflammation (moderate)  
 NASH  
CDAASteatosis (90%)Not determined22.13
 Inflammation (moderate)  
 NASH  
Table 3. Liver Pathology and miR Expression in Mice Fed Diet for 65 Weeks
DietHistologymiR-155-LNA-ISHmiR-155/18S rRNA (2−ΔCt) × 104
CSAASteatosis (75%)  
 Inflammation (mild)2+4.14
 Not NASH21% Hepatocytes + 
CSAASteatosis (90%)  
 Inflammation (mild)2+5.25
 Not NASH24% Hepatocytes + 
CSAASteatosis (90%)  
 Inflammation (mild)Not determined1.46
 Not NASH  
CSAASteatosis (90%)  
 Inflammation (mild)1+3.37
 Not NASH6% Hepatocytes + 
CDAASteatosis (90%)  
 Inflammation (moderate to severe)3+7.25
 NASH52% Hepatocytes + 
CDAASteatosis (90%)  
 Portal tract inflammation (moderate)3+6.83
 NASH55% Hepatocytes + 
CDAASteatosis (75%)  
 Inflammation (moderate)1+3.33
 Not NASH19% Hepatocytes + 
CDAASteatosis (90%)  
 Inflammation (moderate)3+8.38
 NASH59% Hepatocytes + 

Next, we investigated whether diet-induced histopathological changes in the liver correlated with dysregulation of miR-155, known to be induced by inflammatory mediators,19 by LNA-ISH. The results showed high levels of miR-155 in the cytoplasm of hepatocytes and inflammatory cells in mice fed a CDAA diet for 32 weeks (Fig. 2D). Although miR-155 was detectable in only a few steatotic hepatocytes in the livers of age-matched mice fed the control diet, the number of miR-155–positive cells correlated with the extent of inflammation in each mouse (Tables 2, 3). No signal was detected with scrambled oligonucleotide, demonstrating specificity of the probes (Fig. 2D). Furthermore, real-time RT-PCR analysis of miR-155 expression level in individual mice correlated positively with the number of miR-155–positive cells as well as the extent of inflammation in these mice, suggesting that miR-155 could play a causal role in CDAA diet–induced pathogenesis.

CDAA Diet Induced Activation of Nuclear Factor Kappa B Up-regulated Hepatic miR-155.

Next, we sought to identify the transcription factor that plays a key role in up-regulation of hepatic miR-155 in mice fed a CDAA diet. Because miR-155 level correlated with diet-induced inflammation (Tables 2, 3), and nuclear factor kappa B (NF-κB) is known to activate miR-155 expression,22 we performed electrophoretic mobility-shift assay with liver nuclear extracts from mice on CSAA or CDAA diet for 32 weeks. A specific complex was detected with 32P-labeled NF-κB probe in the liver nuclear extracts from mice fed a CSAA diet that was twofold increased (P = 0.002) in the mice fed a CDAA diet (Fig. 3A, compare lanes 12-15 with 2-5; and Fig. 3B). The competition of the complex by an excess of unlabeled wild-type but not the mutant oligo (Fig. 3A, lanes 6, 7, and 16, 17), and supershift of this complex with antibodies specific for p65 and p50 subunits of NF-κB (Fig. 3A, lane 8, 9 and 18, 19) confirmed its identity with NF-κB. Furthermore, an excess of a duplex oligo encompassing the NF-κB cognate site in miR-155 promoter was also able to compete out the complex formation (Fig. 3A, lanes 10 and 20), indicating that NF-κB could bind to this site on the miR-155 promoter.

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Figure 3. (A) NF-κB was activated in the liver nuclear extract of mice fed a CDAA diet. An identical amount (3 μg) of the extract was incubated with 32P-labeled NF-κB oligo under optimal binding conditions. The protein DNA complex was resolved in a 5% polyacrylamide gel, dried, and subjected to phosphorimager analysis. For competition and supershift assays, the extracts were preincublated with 100-fold molar excess of unlabeled oligos and antibodies, respectively, for 30 minutes before adding labeled probe. (B) Quantitative analysis of the data in (A).

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The Tumor Suppressors C/EBPβ and Phosphatase and Tensin Homolog, Respective Targets of miR-155 and miR-21, Were Down-regulated in the Livers of Mice Fed a CDAA Diet.

Next, we identified the possible target of miR-155 that may potentially be involved in diet-induced hepatocarcinogenesis. C/EBPβ, a tumor suppressor frequently suppressed in HCC,23 is a validated target of miR-155.24 C/EBPβ harbors a conserved miR-155 site in its 3′ untranslated region (Fig. 4A). Western blot analysis showed reduced expression of C/EBPβ in Hep3B and HepG2 cells transfected with pre–miR-155 (Fig. 4B), indicating that miR-155 can target C/EBPβ in HCC cells. Next we checked C/EBPβ expression in the livers of mice fed a CDAA diet. Its mRNA level decreased by approximately 50% after 32 (P = 0.03) and 65 (P = 0.024) weeks in mice fed a CDAA diet compared with control mice (Fig. 4C). The C/EBPβ protein level decreased by approximately 40% (P = 0.02) after 32 weeks of feeding a CDAA diet (Fig. 4D) and was further reduced by approximately 80% (P = 0.003) after 65 weeks (Fig. 4D).

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Figure 4. Down-regulation of C/EBPβ and PTEN, respective targets of miR-155 and miR-21, in the livers of mice fed a CDAA diet. (A) Schematic representation of the conserved miR-155 site in C/EBPβ 3′ untranslated region. (B) Western blot analysis of C/EBPβ in HCC cells. Hep3B and HepG2 cells were transfected with pre–miR-155 (50 nM) followed by western blot analysis after 48 hours. (C) Real-time RT-PCR analysis of C/EBPβ in the liver of mice fed diet for 32 and 65 weeks. (D and E) Western blot analysis of C/EBPβ, PTEN, and glyceraldehyde 3-phosphate dehydrogenase in liver extracts. Equal amount of proteins were subjected to immunoblot analysis first with specific primary and secondary antibodies, and the signal was developed with enhanced chemiluminescence reagent. The signal was quantified using Kodak Imaging software, and the data were normalized to glyceraldehyde 3-phosphate dehydrogenase.

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Phosphatase and tensin homolog (PTEN), another tumor suppressor that is a target of the up-regulated miR-21 (Fig. 2A), was also decreased by approximately 50% in protein level after 32 weeks (P = 0.02) and 65 weeks (P = 0.03) in mice fed a CDAA diet (Fig. 4E). No significant change in PTEN mRNA level was observed (data not shown).

miR-155 Modulated Growth of HCC Cells.

MicroRNA-155, up-regulated in many primary cancers, demonstrated oncogenic properties when overexpressed in lymphocytes.25 We therefore investigated its growth regulatory property in HCC cells. Overexpression of miR-155 by precursor transfection accelerated growth in both Hep3B (P = 0.003) (Fig. 5A) and HepG2 cells (P = 0.006) (Fig. 5B). In contrast, depletion of endogenous miR-155 by transfecting anti–miR-155 resulted in reduced growth of SNU-182 cells (P = 0.0068 after 6 days) (Fig. 5C). These results demonstrate the growth stimulatory property of miR-155.

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Figure 5. (A,B) Ectopic expression of miR-155 promoted growth of Hep3B and HepG2 cells in culture. Cells were transfected with miR-155 precursor or control RNA (50 nM) followed by MTT assay. (C) Knockdown of endogenous miR-155 reduced SNU-182 cell growth. Cells were transfected with anti–miR-155 or control RNA (60 nM) followed by MTT assay. NC, negative control. The upper panels present real-time RT-PCR analysis of miR-155 in HCC cells at the last time point when cell growth was measured.

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miR-155 Was Up-regulated in Primary Human HCCs.

Next, we measured miR-155 levels in primary human HCCs and pair-matched normal liver tissues. Among the 20 HCC samples analyzed (listed in Supporting Table 5),the miR-155 level increased in 16 HCC samples (P = 0.0004) (Fig. 6A). Comparison of miR-155 and its target showed an inverse correlation between the two (n = 20, r = −0.51, P = 0.02) (Fig. 6B). Immunoblot analysis of the extracts from five HCC samples demonstrated a significant decrease in C/EBPβ protein levels in four HCCs compared with matching livers (Fig. 6C). Immunohistochemical analysis of three HCC samples showed that C/EBPβ expression was undetectable or very low (only in 5% cells), whereas in adjacent benign liver tissues 15% to 55% of the hepatocytes were positive for C/EBPβ (a representative photograph is shown in Supporting Fig. 3). These data showed reciprocal regulation of oncogenic miR-155 and its tumor suppressor target C/EBPβ in primary human HCCs, suggesting that miR-155 may play a causal role in transformation of hepatocytes.

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Figure 6. MicroRNA-155 and C/EBPβ levels were reciprocally regulated in primary HCCs. (A) Total RNA from 20 HCCs and pair-matched normal liver tissues was subjected to real-time RT-PCR analysis for miR-155 level. Expression of miR-155 in each sample presented in Supporting Table 5 is depicted as dot plots. Horizontal bars indicate median expression value. (B) Inverse correlation between C/EBPβ mRNA level and miR-155 expression in HCCs (r = −0.51, P = 0.02), determined by real-time RT-PCR analysis in 20 HCC samples. (C) Western blot analysis of C/EBPβ in whole tissue extracts (500 μg) from HCCs (T) and matching livers (N). Asterisks denote HCC samples in which C/EBPβ is reduced.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

It is now well established that the expression of miRs and their key targets is either elevated or reduced in almost all types of cancer. Although a few altered miRs exhibit oncogenic or tumor suppressor properties, the biological functions of most miRs remain to be elucidated. The current study is a comprehensive analysis of differential expression of miRs and their important targets during preneoplastic transformation of hepatocytes in a mouse model. A major advantage of this model is that the liver tumor is developed in the absence of potent chemicals or viruses. Although similar dietary deficiency also induces HCCs in rats, the development of the mouse model facilitates studies on the role of different genetic factors in the induction of hepatocarcinogenesis.

A significant observation of the current study is the dysregulation of specific miRs and their targets at early stages of hepatocarcinogenesis, long before preneoplastic transformation, implicating their role in the initiation of tumorigenesis. These altered miRs were almost identical to those observed in primary human HCCs. MicroRNA-21 and miR-221/222 have been reported to be up-regulated in various types of cancers, including HCC.20, 26 Notably, the expression of PTEN, a tumor suppressor target of miR-21, was significantly reduced in the livers of CDAA-fed mice at early stages of tumorigenesis.

MicroRNA-155, a common target of proinflammatory cytokines,19 is overexpressed in solid tumors and functions as an oncogene when overexpressed in B cells.25 The correlation of miR-155 level with CDAA diet–induced pathological changes in the liver suggests that miR-155 plays a causal role in this dietary model of hepatocarcinogenesis. Our study also revealed that diet-induced activation of NF-κB plays a key role in miR-155 expression. Suppression of miR-155 in Huh-7 cells by Bay 11, a potent inhibitor of NF-κB, confirmed the role of NF-κB in miR-155 expression in hepatocyte-derived cells (data not shown). It is conceivable that up-regulation of miR-155 with concomitant down-regulation of its tumor suppressor target C/EBPβ plays a causal role in diet-induced liver pathogenesis.

Although the current study focused on up-regulated miRNAs, a few miRNAs, including the most abundant liver miR-122, are suppressed in rodent and human primary HCCs.21 Interestingly, a recent study displayed a dramatic reduction of miR-122 level in human NASH patients.18 Microarray analysis has shown that down-regulation of miR-122 leads to re-expression of genes that are normally suppressed in the liver.27 Thus, the loss of miR-122 is likely to promote dedifferentiation of hepatocytes. It would be of considerable interest to generate miR-122 conditional knockout mice for exploring its role in hepatocarcinogenesis in vivo.

In conclusion, using a mouse model of NASH, we have shown that up-regulation of oncogenic miRs with concomitant suppression of their tumor suppressor targets is a very early molecular event that could play a causal role in hepatocarcinogenesis. Interestingly, very similar changes in miR profile in livers of NASH patients18 underscore the usefulness of the mouse model to test the therapeutic efficacy of microRNA mimetics (miR-122, let-7a) or anti–miR-155 in preclinical trials.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_23100_sm_SupDoc.doc187KSupplemental Materials and Methods
HEP_23100_sm_SupFig1.tif8760KFigure S1. Pathological changes associated with multistage hepatocarcinogenesis in mice fed CDAA diet. Arrows indicate liver tumors.
HEP_23100_sm_SupFig2.tif5678KFigure S2. A and B. Northern blot analysis of miR-21 and miR-122 in the liver. Total RNA (30 μg) was separated in 15% acrylamide gel denaturing (8M urea) gel, transferred to nylon membrane followed by hybridization to 32P-labeled anti-sense miR-21/122 probes. The signal was developed by autoradiography and quantified using Kodak Imaging software.
HEP_23100_sm_SupFig3.tif4395KFigure S3. Immunohistochemical analysis of C/EBPβ in a HCC and benign liver tissues. Formalin-fixed HCC sections were subjected to immunohistochemistry analysis with anti-C/EBPβ antibody using Ventana Ultraview Universal Red system. C/EBPβ signal is red (green arrow) and the cell body is light blue.

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