proteomic analysis of liver tissue from hbx-transgenic mice at early stages of hepatocarcinogenesis

RESEARCH ARTICLE

Proteomic analysis of liver tissue from HBx-transgenic

mice at early stages of hepatocarcinogenesis

Sun-Young Kim1,2�, Phil Young Lee1�, Hye-Jun Shin3, Do Hyung Kim1, Sunghyun Kang1,Hyung-Bae Moon4, Sang Won Kang2, Jin-Man Kim5, Sung Goo Park1, Byoung Chul Park1,Dae-Yeul Yu3��, Kwang-Hee Bae1�� and Sang Chul Lee1

1 Medical Proteomics Research Center, KRIBB, Daejeon, South Korea2 Department of Life Sciences and Center for Cell Signaling and Drug Discovery, Ewha Womans University, Seoul,

South Korea3 Disease Model Research Center, KRIBB, Daejeon, South Korea4 Department of Pathology and Institute of Medical Science, Wonkwang University, School of Medicine, Iksan,

Chonbuk, South Korea5 Department of Pathology, College of Medicine, Chungnam National University, Daejeon, South Korea

Received: October 2, 2008

Revised: August 17, 2009

Accepted: August 18, 2009

The hepatitis B virus X-protein (HBx), a multifunctional viral regulator, participates in the

viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously

reported a high incidence of HCC in transgenic mice expressing HBx. In this study,

proteomic analysis was performed to identify proteins that may be involved in hepatocarci-

nogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from

the liver tissue of HBx-transgenic mice at early stages of carcinogenesis (dysplasia and

hepatocellular adenoma) were separated by 2-DE, and quantitative changes were analyzed. A

total of 22 spots displaying significant quantitative changes were identified using LC-MS/MS.

In particular, several proteins involved in glucose and fatty acid metabolism, such as mito-

chondrial 3-ketoacyl-CoA thiolase, intestinal fatty acid-binding protein 2 and cytoplasmic

malate dehydrogenase, were differentially expressed, implying that significant metabolic

alterations occurred during the early stages of hepatocarcinogenesis. The results of this

proteomic analysis provide insights into the mechanism of HBx-mediated hepatocarcino-

genesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and

novel drug development for treatment of the disease.

Keywords:

Animal proteomics / Dysplasia / Hepatitis B virus X-protein / Hepatocellular adenoma /

Hepatocellular carcinoma

1 Introduction

Hepatocellular carcinoma (HCC) is a common malignancy

responsible for a quarter of a million deaths annually. Chronic

hepatitis B virus (HBV) infection is one of the major causes of

HCC in humans. Chronic carriers of HBV display a 200- to

300-fold greater risk of HCC than the general population

[1, 2]. However, the precise mechanisms underlying HBV-

mediated carcinogenesis are poorly understood at present.

Among the proteins encoded by HBV, HBV X-protein (HBx)

is a multifunctional protein that inhibits p53, transactivates

several transcription factors (including AP-1, CREB and NF-

kB) and is essential for HBV replication. Additionally, HBx

expression correlates with the activation of various signal

transduction pathways, such as RAS/RAF/MAPK, JAK/STAT,

Abbreviations: FABP2, fatty acid binding protein; HBV, hepatitis

B virus; HBx, HBV X-protein; HCA, hepatocellular adenoma; HCC,

hepatocellular carcinoma; MDH, malate dehydrogenase; RKIP,

raf kinase inhibitory protein

� These authors contributed equally to this work.��Additional corresponding authors: Dr. Kwang-Hee Bae

E-mail: [email protected]

Dr. Dae-Yeul Yu; E-mail: [email protected]

Correspondence: Dr. Sang Chul Lee, Medical Proteomics

Research Center, KRIBB, 52 Eoeun-Dong, Yusung-Gu, Daejeon

305-806, South Korea

E-mail: [email protected]

Fax:182-42-860-4593

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

5056 Proteomics 2009, 9, 5056–5066DOI 10.1002/pmic.200800779

MEKK1/JNK and PI3K/AKT [3]. Malignant transformation

has been reported in specific cell lines transfected with the

HBx gene and in the corresponding transgenic mouse model

[4–6]. Therefore, this protein appears to play a key role in the

neoplastic transformation of hepatocytes in HBV-infected

liver. However, the details of the mechanisms whereby HBx

induces HCC require elucidation.

Proteomics is the large-scale characterization of proteins

expressed from the genome. This technique provides a global,

systematic and comprehensive approach for elucidating

biochemical processes, pathways and networks in both normal

and disease states at the protein level. The procedure facilitates

the identification of a range of protein markers potentially

indicative of specific diseases [7–10]. However, proteomic

analysis of HCC remains problematic due to insufficient

understanding of the molecular pathogenesis, use of samples

from patients with heterogeneous genetic backgrounds and

life styles and difficulty in detecting the disease at the early

stages [11–19]. Therefore, suitable animal models of HCC that

allow genetic background and environmental conditions to be

controlled and that permit longitudinal studies from preneo-

plastic stages are extraordinarily useful in the search for

biomarkers and proteins involved in hepatocarcinogenesis by

proteomics. We previously generated HBx-transgenic mice

showing a high incidence of HCC [6]. We also recently

reported that increased HBx expression causes lipid accumu-

lation in hepatic cells mediated by sterol regulatory element-

binding protein 1 and peroxisome proliferator-activated

receptor g, which may be a mechanism mediating the patho-

physiology of hepatocarcinogenesis during chronic HBV

infection [20]. Thus, the HBx-transgenic mouse is an optimal

model for the mining of biomarkers and proteins involved in

hepatocarcinogenesis. HCC is a multifactorial and progressive

disease that requires early detection for effective treatment [2].

Therefore, it is critical to identify biomarkers for early diag-

nosis and to mine novel therapeutic targets. Here, proteins of

liver tissue from HBx-transgenic mice at early stages of

hepatocarcinogenesis (dysplasia and hepatocellular adenoma

(HCA)) were separated by 2-DE, and their quantitative altera-

tions were extensively analyzed. A number of proteins showing

differential expression patterns were identified. Several

proteins within this group related to glucose and fatty acid

metabolism were differentially expressed in early stages of

hepatocarcinogenesis, which is consistent with our previous

report [20]. Our proteome data provide a useful list of proteins

that may be employed as biomarkers for diagnosis and/or

constitute targets for the development of novel drugs.

2 Materials and methods

2.1 Transgenic mice

The production of HBx-transgenic mice was described in

earlier reports [6, 20]. Briefly, we generated HBx homo-

zygous (1/1) transgenic mice by mating HBx heterozygous

transgenic mice with each other. To generate HBx homo-

zygous transgenic mice in a mixed background of C57BL/6

and CBA strains, HBx homozygous C57BL/6 mice were

crossed with CBA wild-type mice. The heterozygous trans-

genic offspring with a mixed background of C57BL/6 and

CBA strains were mated to each other. Among the offspring,

HBx homozygous transgenic mice were selected by geno-

typing. Selected mice were intercrossed up to F12 for study

as an incipient inbred strain with a mixed genetic back-

ground of two strains (C57BL/6 and CBA). HBx (1/1)

transgenic mice were verified by PCR using specific primer

sets. Wild-type mice were derived from littermates between

HBx heterozygous transgenic male mice and female mice

with a mixed genetic background of the two strains (C57BL/

6 and CBA). Mice were housed in a pathogen-free envir-

onment and maintained in accordance with the guidelines

of the Institutional Animal Care and Use Committee, Korea

Research Institute of Bioscience and Biotechnology (KRIBB,

Daejeon, South Korea).

2.2 Histology and sample preparation

Liver tissue samples were fixed in 10% neutral buffered

formalin, embedded in paraffin, sectioned and stained with

H&E according to standard methods. Histopathologic diag-

noses were based on criteria described by Frith and Ward

[21]. The livers of wild-type and transgenic mice with

dysplasia, HCA or HCC were collected. Fresh liver samples

were homogenized in buffer A (50 mM Tris-HCl, pH 7.1,

100 mM KCl, 20% glycerol and protease inhibitors) and

sonicated for 1 min. Next, homogenates were centrifuged

twice at 50 000 rpm (226 000� g) for 1 h at 41C. The protein

concentration was measured in supernatant fractions using

the Bradford assay, with BSA as the standard.

2.3 IEF and electrophoresis

The liver homogenate (150mg of protein) was mixed with

rehydration buffer (9 M urea, 4% CHAPS, 2 M thiourea,

40 mM DTT and 2% IPG buffer). Protein samples were

directly applied to IPG strips (pH 4–7, 13 cm) and rehydrated

for 14 h at room temperature. Next, IEF was performed using

the Multiphor II (GE Healthcare, Uppsala, Sweden) apparatus

[22–24]. The initial voltage was maintained at 300 V for 1 min

and linearly increased from 300 to 3500 V within 1.5 h. The

voltage was then maintained at 3500 V for 8 h. The plate

temperature was kept constant at 251C during IEF. Focused

IPG strips were briefly equilibrated for 15 min with equili-

bration solution (50 mM Tris-HCl (pH 8.8), 6 M urea, 2% SDS

and 30% glycerol) containing 1% DTT, and it was equilibrated

again with the same solution containing 5% iodoacetamide

instead of DTT for 15 min. Equilibrated strips were directly

loaded onto 13% polyacrylamide gels (150� 150� 1.5 mm3) or

stored at �801C until use. Polyacrylamide gels loaded with

Proteomics 2009, 9, 5056–5066 5057


IPG strips were run constantly at 30 mA per gel with the

PROTEAN II Xi/XL system (Bio-Rad).

2.4 Staining and image analysis

After electrophoresis, gels were fixed and protein spots were

visualized by silver staining (PlusOne Silver Staining Kit,

GE Healthcare). The 2-DE images were scanned and

processed with Progenesis SameSpots v3.0 software

(Nonlinear Dynamics). To confirm the variations, at least

three gels were prepared for every case. Spot volumes were

normalized based on the total spot volume of each gel.

Protein spot intensity was defined as the normalized spot

volume, i.e. the ratio of the single spot volume to the total of

the spot volumes on the 2-DE gel (total spot normalization).

Computer analysis facilitated the automatic detection and

quantification of protein spots, as well as matches between

gels of controls and dysplasia or HCA samples. Spots

displaying reliable and significant differences (7over

twofold, po0.05) were selected for MS analysis.

2.5 In-gel digestion and identification by LC-MS/MS

Spots of interest were manually excised from 2-DE gels and

destained with chemical reducers to remove silver [7].

Briefly, 50–100 mL of the freshly prepared reducing solution

(1:1 mixture of 30 mM potassium ferricyanide and 100 mM

sodium thiosulfate) was added to the gel plugs and mixed.

After the brown color disappeared, gel plugs were rinsed

with water, and 200 mM ammonium bicarbonate was

added for 20 min. Subsequently, gel plugs were cut into

small pieces, washed with water and dehydrated repeatedly

with ACN until the pieces turned opaque white. Next,

gel pieces were dried in a vacuum centrifuge for 30 min, and

the proteins were digested with 20 ng/mL of sequencing

grade-modified trypsin (Promega) for 16–24 h at 371C.

Digested peptides were extracted with extraction solution

(50% ACN and 5% TFA), and the extracted peptides were

dried using a vacuum drier. Samples were subjected to MS

analysis.

Peptides were analyzed using a Synapt High Definition

Mass Spectrometer (Waters, Manchester, UK) equipped with a

nanoACQUITY Ultra Performance LC system (Waters,

Milford, MA, USA). In brief, 2mL of peptide solution was

injected onto a 75 m� 100 mm Atlantis dC18 column (Waters,

USA). Solvent A consisted of 0.1% formic acid in water, and

Solvent B was composed of 0.1% formic acid in ACN. Peptides

were initially separated using 100 min gradients and electro-

sprayed into the mass spectrometer (fitted with a nanoLock-

Spray source) at a flow rate of 300 nL/min. Mass spectra were

acquired from m/z 300–1600 for 1 s, followed by four data-

dependent MS/MS scans from m/z 50–1900 for 1 s each. The

collision energy used to perform MS/MS was varied according

to the mass and charge state of the eluting peptide. (Glu1)-

Fibrinopeptide B was infused at a rate of 350 nL/min, and an

MS scan was acquired for 1 s every 30 s throughout the run. A

database search was performed with MASCOT (Matrix Science,

London, UK) using the following parameters: NCBInr.08.03.26

database, Mus musculus species and maximum number of

missed cleavage by trypsin at 1. Mass tolerance ranged from

750 to 7100 ppm. The peptide modification allowed was

carbamidomethylation in the fixed modification mode.

2.6 Target validation using real-time PCR and

Western blot analysis

Total RNA samples were isolated from the liver tissues of wild-

type and transgenic mice using TRIzol (Invitrogen), according

to the manufacturer’s instructions. Total cellular RNA (2mg)

was denatured at 651C for 5 min, and first-strand cDNA was

synthesized by incubating with M-MLV reverse transcriptase at

371C for 60 min in the presence of 0.5mg oligo(dT), 10 mM

dNTP and 0.1 M DTT (Bioneer, South Korea) in a total volume

of 40mL. The RT reaction was terminated by heating at 751C

for 15 min, and double-stranded cDNA fragments of the target

candidate gene were obtained. SYBR Premix Ex Tag (TaKaRa)

was employed for real-time PCR. The primers used to amplify

the genes were as follows: alanyl-tRNA synthase, 50- AGGAC-

CATGTGCAATACTTGGTG-30 and 50- GGAGTCTGG-

GAGTCTATTCGGTGA-30; ubiquitin-activating enzyme E1,

50- TAGTTCAAGGGCACCAACAGCTC-30 and 50- AAAGC-

GATCCCACAATGTCCA-30; thyroid hormone-responsive

protein, 50-GTGACGCGGAAATACCAGGAA-30 and 50-CC-

AAGTCCACAGATGCACTCAGA-30; thiopurine methyl-

transferase, 50-CACATCTCATTCCATCAGGAGCA-30 and

50-CGCAGTCCACTCTGGCCTTTA-30; and b-actin, 50-AGGC-

CCAGAGCAAGAGAGG-30 and 50-TACATGGCTGGGGTG

TTGAA-30. Statistical analysis was performed using an inde-

pendent Student’s t-test, and p-values of o0.05 were consid-

ered statistically significant.

Protein samples (20 mg) were separated on a 13% SDS-

PAGE gel and transferred to an NC membrane using

standard procedures. The membrane was blocked with

5% v/v skim milk in TBS-T buffer (20 mM Tris-HCl, pH 7.6,

0.1369 M NaCl and 0.1% Triton X-100) and then incubated

with the primary antibody for 12 h on a rocking platform at

41C. The membrane was then washed three times with TBS-

T buffer for 15 min and incubated with 5% skim milk in

TBS-T buffer containing HRP-conjugated secondary anti-

body (diluted to 1:3000) for 1 h. The hybridized membrane

was washed in TBS-T buffer and visualized using a chemi-

luminescent ECL detection kit (GE Healthcare).

2.7 Statistical analysis

Experimental differences were tested for statistical signifi-

cance using ANOVA and Student’s t-test. P-values o0.05

were regarded as statistically significant.

5058 S.-Y. Kim et al. Proteomics 2009, 9, 5056–5066


3 Results and discussion

3.1 Pathophysiological properties of HBx-transgenic

mice

As summarized in Table 1, HBx-transgenic mice exhibited

progressive morphological changes that commenced with

the development of dysplasia. This was followed by the

appearance of benign adenomas and eventually, HCC

(Fig. 1). Furthermore, transgenic mice also showed signifi-

cant changes in the levels of both micro-fatty acids and

macro-fatty acids (Table 1) [20]. Accordingly, our HBx-

transgenic mice constitute a valuable animal HCC model

suitable for analyzing its molecular mechanisms and

mining for biomarkers of hepatocarcinogenesis. In this

study, proteome alterations during the early stages of

hepatocarcinogenesis were monitored by quantitative 2-DE

analysis of HBx-transgenic mice.

3.2 Comparative 2-DE analysis between control and

transgenic mice at early stages of

carcinogenesis

To establish proteomic changes during the early stages of

hepatocarcinogenesis, liver proteins from mice showing

dysplasia or HCA were separated by 2-DE. Experiments

were performed using samples from at least three individual

mice. More than 1500 protein spots were detected on gels

after silver staining, automatic spot detection, background

subtraction and volume normalization. In three sets of

experiments, protein spots displaying significant changes

(greater than twofold in magnitude compared with control

mice) were scored and identified. In total, 22 spots exhibited

changes during dysplasia (ten spots) or HCA (12 spots)

(Fig. 2). Among these, five proteins were up-regulated (two

and three spots in dysplasia and HCA, respectively) and 17

were down-regulated (eight and nine spots in dysplasia and

HCA, respectively). Protein spot numbers in Table 2 corre-

spond to those in Fig. 2. Additionally, most differentially

expressed protein spots showed consistent changes among

individual mice (Fig. 3 and Supporting Information Fig. 1).

3.3 Identification and classification of differentially

expressed proteins by LC-MS/MS

The proteins identified were classified into the following

functional groups: metabolism, apoptosis, molecular

chaperones, signal transduction, cytoskeletal, catalytic,

RNA-related, proteolysis and redox regulation. Among

these, a number of proteins related to metabolism were

differentially expressed during the early stages of hepato-

carcinogenesis. Cancer cells display high rates of aerobic

glycolysis for energy generation, known as the Warburg

effect [25]. In addition, cancers show high levels of lipo-Tab

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Proteomics 2009, 9, 5056–5066 5059


genesis, and hepatic lipid accumulation is correlated with

hepatic fibrosis, apoptosis and cancer [11, 26, 27]. Recently,

we reported that elevated HBx expression causes lipid

accumulation in hepatic cells that is mediated by sterol

regulatory element-binding protein 1 and peroxisome

proliferator-activated receptor g [20]. Our proteomic results

revealed that several proteins involved in glycolysis/gluco-

neogenesis and lipolysis/lipogenesis, including fatty acid-

binding protein 2 (FABP2), cytoplasmic malate dehy-

drogenase (MDH) and thyroid hormone-responsive protein,

were differentially expressed (greater than twofold in

magnitude compared with control mice). Additionally,

several other proteins involved directly or indirectly in

critical metabolic processes, including ketohexokinase (1.80-

fold up-regulation in dysplasia), aldolase B (1.50-fold down-

regulation in dysplasia), hydroxypyruvate isomerase homo-

log (1.70-fold down-regulation in dysplasia) and fructose

bisphosphatase 1 (1.50-fold down-regulation in HCA), were

reliably up- or down-regulated during early stages of hepa-

tocarcinogenesis (data not shown). These results strongly

suggest that significant metabolic dysregulation occurs at

the early stages of hepatocarcinogenesis.

3.4 Validation of proteins by real-time PCR and

Western blot

To validate the 2-DE results and assess the expression

changes of several proteins showing differential patterns at

early stages of hepatocarcinogenesis, Western blotting and

real-time PCR analysis were performed (Figs. 4A and B).

With the exception of serum albumin precursor, HSP70,

HSP90, major urinary protein 2 precursor and intermediate

filament protein, we tested all of the identified proteins by

Western blot analysis using available commercial anti-

bodies. The proteins that failed validation by Western blot

analysis (possibly due to low antibody specificity and/or

sensitivity) were assayed again for mRNA expression levels

by real-time PCR. For the most part, the Western blot and

real-time PCR results correlated well with the 2-DE data

(Figs. 4A and B).

Among the identified proteins, aldehyde dehydrogenase

1 family member L1 (ALDHL1) was down-regulated at both

the dysplasia and the HCA stages. ALDHL1 is a tumor

suppressor protein that is selectively cytotoxic to cancer cells.

Furthermore, it is significantly and ubiquitously down-

regulated in tumors [28]. A recent report shows that ectopic

expression of ALDHL1 in A549 lung cancer cells induces

p53-dependent G1 arrest and apoptosis [29]. As expected, the

reduced expression of ALDHL1 observed in the 2-DE

analysis was confirmed by Western blot analysis (Table 2

and Fig. 4A). Furthermore, down-regulation of ALDHL1

was detected in clinical samples of human HCC patients by

immunostaining with anti-ALDHL1 antibody (Fig. 4C). This

finding confirms that our proteomic analysis of liver

samples from HBx mice has relevance for human hepato-

carcinogenesis.

According to the 2-DE findings, cytoplasmic MDH1

levels were lower at the HCA stage. Western blot analysis

consistently revealed significant down-regulation of MDH1

at both the HCA and the HCC stages (Fig. 4A). As

mentioned above, considerable metabolic changes may

occur during early hepatocarcinogenesis. MDH1 catalyzes

the conversion of oxaloacetate and malate utilizing the

NAD1/NADH coenzyme system, and it participates

in the malate/aspartate shuttle [30]. This shuttle

exchanges reducing equivalents across mitochondrial

membranes in the form of malate/oxaloacetate rather

than NAD1/NADH. HBx induces oxidative stress in liver

cells, leading to significant changes in the redox balance [31,

32]. To date, no involvement of MDH1 in hepatocarcino-

genesis has been documented. Our data showing MDH1

down-regulation at the HCA and HCC stages (Fig. 4A)

support the possibility of a connection between hepato-

carcinogenesis and the malate/aspartate shuttle via differ-

ential expression of MDH1. Recently, it was reported that

MDH1 regulates p53 transcriptional activity in response to

metabolic stress [33]. Specifically, MDH1 binds and stabi-

lizes p53 and concomitantly transactivates p53 targets by

binding to p53-responsive promoter elements upon glucose

deprivation [33]. Our results suggest that MDH1 down-

regulation in HBx-transgenic mice may cause p53 instability

and thereby decrease its apoptotic and tumor suppressive

activities.

Significantly increased expression of intestinal FABP2

was also detected during dysplasia (Fig. 4A), which is

consistent with the 2-DE result. FABPs, known as intracel-

lular lipid chaperones, are a group of proteins that coordi-

nate lipid responses and are linked to metabolic and

inflammatory pathways [34]. FABPs bind fatty acids and

other small hydrophobic molecules and consequently

participate in the uptake, intracellular metabolism and/or

transport of long-chain fatty acids [34–36]. It is notable that

A B C

D E F

Figure 1. Histopathological analysis by H&E staining of HBx-

transgenic mice. (A), (B) and (C) are wild-type control mice at the

ages of 25, 27 and 36 wk. (D), (E) and (F) are HBx-transgenic mice

at the ages of 25, 27 and 36 wk. Dysplasia was found in (D), HCA

in (E) and HCC in (F). Magnification: 400� .



exacerbated lipogenesis appears early in carcinogenesis

[20, 37]. In mammalians, there are several distinct FABP

genes, but they differ with respect to tissue distribution and

ligand-binding specificity [34]. FABP2 is expressed in the

small intestine and liver. Knockdown of FABP2 resulted in

an enlarged liver, significantly higher triglyceride levels and

weight gain in male mice [34, 38]. Although it is not known

in detail how these proteins impact hepatocarcinogenesis,

the differential expression patterns of these proteins at early

stages of HCC may reflect their role in altering the meta-

bolic balance.

Mitochondrial 3-ketoacyl-CoA thiolase (HADHA) was

down-regulated in the dysplasia stage (Fig. 3A and Table 2).

Western blot validation showed significantly reduced expres-

sion in both the dysplasia and the HCA stages, although there

was no dramatic change in protein level at the HCC stage

(Fig. 4A). HADHA is the a-subunit of the mitochondrial

trifunctional protein, which catalyzes the mitochondrial

b-oxidation steps of long chain fatty acids. This protein has

3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase

activities. Defects in HADHA cause trifunctional protein

deficiency, long-chain 3-hydroxyl-CoA dehydrogenase defi-

ciency and acute maternal fatty liver during pregnancy [39].

Based on these reports, we speculate that the reduced expres-

sion of HADHA may be related to a metabolic disorder during

early stages of hepatocarcinogenesis.

Several proteins not involved in metabolic pathways

were also detected and validated. Among them, raf kinase

pI 4 pI 7 pI 4 pI 7

pI 4 pI 7pI 4 pI 7

MW (kDa)DysplasiaWildA

B

170130100

72

55914

1619

1619

914

40

33

24

190

337

98209 190

337

98 209

19

11117

11117

11

MW (kDa)HCAWild

170130100

72

55

795152

72 133

795152

72133

40

33

24

60 60

19

4

87 528

119 9196

87 528

119 9196

114 4

Figure 2. Representative 2-DE gel images of liver proteins from wild-type and dysplasic mice. (A) Total protein lysates from wild-type

control (left panel) and dysplasic (right panel) mice were separated on pH 4–7 non-linear IPG strips in the first dimension, followed by 13%

SDS-PAGE in the second dimension and subsequent visualization by silver staining. Differentially expressed protein spots are indicated

with circles (down-regulated) or rectangles (up-regulated). Spots were identified using LC-MS/MS as outlined in Table 2. (B) Total protein

lysates from wild-type control mice (left panel) and mice showing the HCA phenotype (right panel) were separated on pH 4–7 non-linear

IPG strips in the first dimension, followed by 13% SDS-PAGE in the second dimension and subsequent visualization by silver staining.

Differentially expressed protein spots are indicated with circles (down-regulated) or rectangles (up-regulated). Spots were identified using

LC-MS/MS, as outlined in Table 2.

Proteomics 2009, 9, 5056–5066 5061


inhibitory protein (RKIP) was significantly down-regulated

at all stages of hepatocarcinogenesis (Fig. 4A). RKIP is a

cellular inhibitor of the MAP kinase cascade that binds

and blocks the phosphorylation of regulatory sites on

Raf-1, thereby inhibiting Raf-1 activation and downstream

signal transduction. This protein is a member of the

ubiquitously expressed and evolutionarily conserved

phosphatidylethanolamine-binding protein family and

functions as a suppressor of cancer metastasis [40]. After

stimulation, RKIP is phosphorylated at S153 by protein

kinase C, inducing its dissociation from Raf-1. The HBx

protein triggers activation of the Raf-1/MEK kinase cascade

[41, 42], which is essential for HBV gene expression. Recent

reports show that RKIP protein and mRNA are down-

regulated in HCC [43, 44]. In our experiments, RKIP

expression was considerably lower at all stages of HCC,

implying that its down-regulation is involved in HBx-

mediated hepatocellular carcinogenesis.

3.5 Protein validation in human clinical samples

Next, to determine whether the identified proteins can be

used as diagnostic markers and/or drug targets in human

patients, we performed immunohistochemical analysis of

liver tissue samples from patients with HCC. Although the

number of patients was small due to the difficulty of

detecting early-stage HCC, we confirmed the down-regula-

tion of MDH1, up-regulation of FABP2 and the reduced

level of HADHA in HCC regions compared with non-

neoplastic controls in HBV-associated HCC patients

(Fig. 4C). A dramatically lower level of HADHA was seen in

the HCC samples, whereas MDH1 was moderately lower

and FABP2 was moderately higher. To validate whether

these proteins can be effectively utilized as biomarkers for

early diagnosis and/or recurrence of HCC in humans, more

extensive studies utilizing a larger number of patients will

be required.

Table 2. List of identified increased or decreased proteins during dysplasia or HCA

Spotno.

Accessionno.

Protein name Function No. ofMatchedpeptides

MOSCOTscore

Coverage(%)

Alteration

D-9 gi|163310765 Serum albumin precursor Signal transduction 28 794 26.2 3.70#D-11 gi|6679737 Fatty acid-binding

protein 2, intestinalMetabolism 8 221 10.9 2.93"

D-14 gi|14917005 Heat shock 70 kDa protein 9 Molecular chaperone 13 587 10.2 2.40#D-16 gi|124486712 Ribosome-binding

protein 1 isoform aDNA-related protein 8 66 3.7 2.20#

D-19 gi|27532959 Aldehyde dehydrogenase1 family, member L1

Metabolism 22 804 21.0 2.13#

D-98 gi|6756060 Annexin A5 Apoptosis 13 567 20.7 2.83#D-117 gi|6755911 Thioredoxin 1 Redox regulation 8 228 38.1 2.42"D 190 gi|121956694 3 ketoacyl-CoA thiolase,

mitochondrialMetabolism 3 158 10.2 2.40#

D-209 gi|12859782 Intermediate filament protein Cytoskeleton 18 296 17.9 2.08#D-337 gi|6753060 Annexin A5 Apoptosis 16 304 17.8 2.50#A-4 gi|6678345 Thyroid hormone-

responsive proteinMetabolism 16 207 25.3 5.70#

A-51 gi|26336489 Alanyl-tRNA synthase RNA-related protein 8 117 2.6 3.00#A-52 gi|6678483 Ubiguitin-activating

enzyme E1 (UBA1)Proteolysis 3 144 4.9 3.00#

A-60 gi|12229867 Acyl CoA thioesterase 2,mitochondrial precursor

Metabolism 21 406 28.9 2.85"

A-72 gi|194027 Heat-shock protein 90 Molecular chaperone 5 184 3.5 2.60#A-79 gi|23271467 Aldehyde dehydrogenase

1 family, member L1Metabolism 4 166 4.1 2.50#

A 87 gi|127527 Major urinary protein 2precursor (MUP2)

Signal transduction 12 235 30.5 2.40#

A-91 gi|4104621 Thiopurine methyltransferase Catalytic protein 6 71 12.5 2.35#A-96 gi|129729 Protein disulfide isomerase

b polypeptideCatalytic protein 7 279 18.1 2.30"

A-119 gi|387129 Cytoplasmic MDH1 Metabolism 15 310 27.5 2.12#A-133 gi|26340966 Serum albumin precursor

(Albumin 1)Signal transduction 85 1101 35.0 2.00"

A-528 gi|74222953 RKIP Signal transduction 19 305 28.3 2.00#



Dysplasia (spot No. 11) Dysplasia (spot No. 117)

Fat

ty a

cid

b

ind

ing

pro

tein

2

No

rmal

izat

ion

Vo

lum

e

No

rmal

izat

ion

Vo

lum

eN

orm

aliz

atio

n V

olu

me

No

rmal

izat

ion

Vo

lum

eN

orm

aliz

atio

n V

olu

me

No

rmal

izat

ion

Vo

lum

eN

orm

aliz

atio

n V

olu

me

No

rmal

izat

ion

Vo

lum

eN

orm

aliz

atio

n V

olu

me

No

rmal

izat

ion

Vo

lum

eN

orm

aliz

atio

n V

olu

me

No

rmal

izat

ion

Vo

lum

eWild Transgenic Wild Transgenic

Wild Transgenic

Wild Transgenic

TransgenicWild

TransgenicWild

TransgenicWild

Th

iore

do

xin

1

Dysplasia (spot No. 19)Dysplasia (spot No. 190)

Ald

ehyd

e d

ehyd

rog

enea

se 1

3-ke

toac

yl-C

oA

th

iola

se

Dysplasia (spot No. 98) Dysplasia (spot No. 337)

An

nex

in A

5

An

nex

in A

5

HCA (spot No. 4) HCA (spot No. 91)

Th

yro

id h

orm

on

e-r

esp

on

sive

pro

tein

HCA (spot No. 52)

Th

iop

uri

ne

met

hyl

tran

sfer

ase

HCA (spot No. 119)

Ub

iqu

itin

-act

ivat

ing

en

z-1

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

A

B

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Tra

nsg

enic

Wild

Wild Transgenic

Wild Transgenic

Wild Transgenic

Wild Transgenic

HCA (spot No. 79)

Cyt

op

lasm

ic m

alat

e d

ehyd

rog

enas

e

Wild Transgenic

HCA (spot No. 528)

Ald

ehyd

e d

ehyd

rog

enas

e 1

Wild

Tra

nsg

enic

Raf

kin

ase

inh

ibit

or

pro

tein

Figure 3. Zoom-in images of selected spots showing consistent expression variations in three replicates. (A) Spots ]11, ]19, ]98, ]117,

]190 and ]337 (corresponding to FABP2, aldehyde dehydrogenase 1 family L1, annexin A5, thioredoxin 1, 3-ketoacyl-CoA thiolase and

annexin A5, respectively) were significantly up- or down-regulated in liver samples showing the dysplasia phenotype relative to wild type.

The remaining selected spots are shown in Supporting Information Fig. 1A. (B) Spots ]4, ]52, ]79, ]91, ]119 and ]528 (corresponding to

thyroid hormone-responsive protein, uniquitin-activating enzyme E1, aldehyde dehydrogenase 1 family L1, thiopurine methyltransferase,

cytoplasmic MDH and RKIP, respectively) were significantly up- or down-regulated in liver samples showing the HCA phenotype relative

to wild type. The remaining selected spots are shown in Supporting Information Fig. 1B.

Proteomics 2009, 9, 5056–5066 5063


4 Concluding remarks

We performed expression profiling of liver tissue from HBx-

transgenic mice with early-stage HCC. Through this

proteomic approach, we identified 22 proteins that may be

involved in HBx-mediated HCC, particularly at the early

stages. The majority of the identified proteins, including

MDH1, FABP2 and HADHA, are involved directly or

indirectly in critical metabolic processes such as glycolysis

and lipogenesis, indicating that significant metabolic chan-

ges occur at the early stages of hepatocarcinogenesis. In

future studies, we aim to investigate in detail the

roles of these proteins during early hepatocarcinogenesis

and to determine whether they can be effectively utilized as

biomarkers for early diagnosis and/or recurrence of

HCC in human patients. Our proteomic findings,

together with further characterization of proteins

involved in HBx-mediated hepatocarcinogenesis, should

provide valuable new information about the development of

HCC.

The authors thank Dr. Seung-Wook Chi, Dr. Sang J. Chungand Dr. Do Hee Lee for careful reading of our manuscript. Thiswork was supported by KRIBB and the 21st Century FrontierProgram in the Functional Human Genome Project of Korea.

The authors have declared no conflict of interest.

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proteomic analysis of liver tissue from hbx-transgenic mice at early stages of hepatocarcinogenesis

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