Expression profiling reveals dysregulation of cellular cytoskeletal genes in HBx-induced hepatocarcinogenesis.
ABSTRACT The molecular mechanisms underlying hepatitis B virus encoded HBx protein-mediated tumorigenesis are not fully understood. In order to gain a better view of the effects of HBx on transcriptional regulation and hepatocarcinogenesis, the expression profiles of liver and tumor tissues from 6- and 18-month-old p21-HBx transgenic and control mice were monitored using oligo microarrays. Data analysis demonstrated that 42 genes were deregulated in both 6- and 18-month-old HBx transgenic mouse tissues. Gene ontology assisted analysis classified these genes into functionally related clusters that encode proteins related to metabolism, signal transduction, transcription regulation and stress responses. Among them, cytoskeletal genes, including microtubule genes tubulinbeta2 (Tubb2), tubulinbeta3 (Tubb3) and tubulinbeta6 (Tubb6), intermediate filament genes periplakin, keratin 8 (K8) and keratin 18 (K18) and actingamma1 (Actg1), were closely clustered and upregulated in liver tissues. These results were validated by semi-quantitative RT-PCR in both mouse and human HCC tissues. The upregulation of K8 and K18 was only detected in p21-HBx but not p21-HBsAg liver tissues, suggesting that the global change in the expression of cellular cytoskeletal genes was correlated with the expression of HBx transgene. These findings propose for the first time that systemic dysregulation of cellular cytoskeletal genes is involved in HBx-induced hepatocarcinogenesis.
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ABSTRACT: Hepatocellular carcinoma (HCC) is the fifth most common cancer, but the third leading cause of cancer death, in the world, with more than 500,000 fatalities annually. The major etiology of HCC/liver cancer in people is hepatitis B virus (HBV), followed by hepatitis C virus infection (HCV), although nonviral causes also play a role in a minority of cases. Recent molecular studies confirm what was suspected: that HCC tissue from different individuals have many phenotypic differences. However, there are clearly features that unify HCC occurring in a background of viral hepatitis B and C. HCC due to HBV and HCV may be an indirect result of enhanced hepatocyte turnover that occurs in an effort to replace infected cells that have been immunologically attacked. Viral functions may also play a more direct role in mediating oncogenesis. This review considers the molecular and cellular mechanisms involved in primary hepatocellular carcinoma, using a viral perspective.Oncogene 09/2003; 22(33):5093-107. · 7.36 Impact Factor
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ABSTRACT: Cell growth, an increase in mass and size, is a highly regulated cellular event. The Akt/mTOR (mammalian target of rapamycin) signalling pathway has a central role in the control of protein synthesis and thus the growth of cells, tissues and organisms. A striking example of a physiological context requiring rapid cell growth is tissue repair in response to injury. Here we show that keratin 17, an intermediate filament protein rapidly induced in wounded stratified epithelia, regulates cell growth through binding to the adaptor protein 14-3-3sigma. Mouse skin keratinocytes lacking keratin 17 (ref. 4) show depressed protein translation and are of smaller size, correlating with decreased Akt/mTOR signalling activity. Other signalling kinases have normal activity, pointing to the specificity of this defect. Two amino acid residues located in the amino-terminal head domain of keratin 17 are required for the serum-dependent relocalization of 14-3-3sigma from the nucleus to the cytoplasm, and for the concomitant stimulation of mTOR activity and cell growth. These findings reveal a new and unexpected role for the intermediate filament cytoskeleton in influencing cell growth and size by regulating protein synthesis.Nature 06/2006; 441(7091):362-5. · 38.60 Impact Factor
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Despite? the? great? progress? achieved? in? studying? HBx? oncogenic? mechanisms,? the?
1Genetic Laboratory of Development and Diseases; Institute of Biotechnology;
Beijing, P.R. China
2Cancer Institute and Hospital; Chinese Academy of Medical Science and Peking
Union Medical College; Beijing, P.R. China
*Correspondence to: Xiao Yang; Institute of Biotechnology; 20 Dongdajie;
Fengtai; Beijing 100071 P.R. China; Tel./Fax: +86.10.63895937(O); Email:
Original manuscript submitted: 11/07/06
Manuscript accepted: 02/03/07
Previously published online as a Cancer Biology & Therapy E-publication:
transgenic? mouse,? hepatocelluar? carcinoma,?
on? writing.? This? work? was? supported? by?
Chinese? National? Key? Program? on? Basic?
Science? Foundation? of? China? (30430350;?
30671078),? National? High-Tech? Research?
Expression Profiling Reveals Dysregulation of Cellular Cytoskeletal
Genes in HBx-Induced Hepatocarcinogenesis
The molecular mechanisms underlying hepatitis B virus encoded HBx protein‑
mediated tumorigenesis are not fully understood. In order to gain a better view of the
effects of HBx on transcriptional regulation and hepatocarcinogenesis, the expression
profiles of liver and tumor tissues from 6‑ and 18‑month‑old p21‑HBx transgenic and
control mice were monitored using oligo microarrays. Data analysis demonstrated
that 42 genes were deregulated in both 6‑ and 18‑month‑old HBx transgenic mouse
tissues. Gene ontology assisted analysis classified these genes into functionally related
clusters that encode proteins related to metabolism, signal transduction, transcription
regulation and stress responses. Among them, cytoskeletal genes, including microtu‑
bule genes tubulinb2 (Tubb2), tubulinb3 (Tubb3) and tubulinb6 (Tubb6), intermediate
filament genes periplakin, keratin 8 (K8) and keratin 18 (K18) and acting1 (Actg1),
were closely clustered and upregulated in liver tissues. These results were validated
by semi‑quantitative RT‑PCR in both mouse and human HCC tissues. The upregulation
of K8 and K18 was only detected in p21‑HBx but not p21‑HBsAg liver tissues,
suggesting that the global change in the expression of cellular cytoskeletal genes was
correlated with the expression of HBx transgene. These findings propose for the first
time that systemic dysregulation of cellular cytoskeletal genes is involved in HBx‑induced
HBV, hepatitis virus B; HCC, hepatocellular carcinoma; Hprt, hypoxanthine phospho‑
ribosyltransferase; RT‑PCR, reverse transcription polymerase chain reaction; APOBEC‑1,
apo‑B mRNA‑editing enzyme catalytic polypeptide 1; ALAS2, aminolevulinate synthase
2; HSP70, heat shock protein 70; Nfyb, nuclear transcription factor‑Y beta; Crem, cAMP
responsive element modulator; Copeb, core promoter element‑binding protein; HKII,
type II hexokinase; ROS, reactive oxygen spieces; k8, cytokeratin8; k18, cytokeratin18;
ppl, periplakin; AN, accession number; TM, melting temperature; bp, base pair
mammalian? hepadnaviruses.? Increasing? evidence? suggests? that? HBx? is? an? oncogenic?
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Western blot.? Western? blots? were? carried? out? on? liver/HCC?
extracts? prepared? as? described.4? Fifty? milligrams? of? proteins? were?
electrophoresed? on? 12%? SDS-PAGE? and? transferred? onto? PVDF?
study? of? liver? tissues? from? the? p21‑HBx?
transgenic? mice? aged? six? months? (for? ?
investigating? early? phase? gene? changes)? ?
investigating? pre-cancerous? phase? gene?
cytoskeletal? genes? is? involved? in? the?
mAteriAL ANd metHodS
Mouse tissue preparation and RNA
isolation.? The? liver? tissues? of? wild? type? ?
(WT),? p21HBx/‑ (X/-),? p21HBx/HBx? (X/
X),? p21HBsAg/‑ (S/-),? p21HBsAg/HBsAg? (S/S)? ?
transgenic? mice? were? collected? at? the? age?
tissues? using?TRIzol? reagent? (Life? technologies,? California,? USA)?
agarose? gel? electrophoresis? in? the? presence? of? ethidium? bromide.?
with? same? genotype? were? pooled? as? one? sample.? All? animals?
Human sample collection.?Four?pairs?of?HCC?specmens?and?the? ?
Gene expression profiling and data analysis.?This? study? used?
commercially? available? high-density? oligonucleotide? microarray?
of? cRNA,? hybridization,? scanning,? and? image? analysis? of? the?
Microarray? Suite? 5.0? software? (Affymetrix,? Santa? Clara,? CA)? was?
with? more? than? two? fold? change? (X/X:? WT)? were? regarded? as?
edu/clustering)? were? used? for? hierarchical? clustering? analysis? and?
RT‑PCR.? Reverse? transcription? PCR? was? performed? by? using?
Immunofluorescence staining.? The? liver? tissues? were? fixed? in?
4˚C? overnight? and? embedded? for? frozen? sections.? Sections? ?
4',6'-diamidino-2-phenylindole? (DAPI)? to? visualize? the? nucleus.?
Photo? capture? was? performed? using? a? Nikon? laser? microscope?
reSuLtS ANd diSCuSSioN
Identification of HCC‑related genes deregulated in HBx
transgenic livers.? We? performed? ? microarray? analysis? on? 6-? and? ?
24-month-old? HCC? tissues? to? represent? the? early,? pre-cancerous? ?
and? cancerous? phases? of? gene? alterations,? respectively.? Two? gene? ?
signal? screening? criteria? were? employed? to? identify? differentially? ?
expressed? genes:? (1)?The? fold? changes? of? both? homozygous? HBx
transgenic? liver? tissues? were? ≥2;? and? (2)? Scaling? signals? of? both? ?
homozygous? HBx? transgenic? liver? tissues? were? ≥100? in? order? to?
analysis? demonstrated? that? 150? transcripts? in? the? 6-month-old?
were? deregulated.? We? hypothesized? that? the? genes? that? were? ?
deregulated? in? the? 6-month-old? liver? tissues? might? reflect? the? ?
direct? effects? of? HBx? expression,? while? those? deregulated? in?
the? 18-month-old? liver? tissues? might? represent? the? pre-cancerous?
gene? alterations? during? hepatocarcinogenesis.? The? dramatically?
increased? deregulated? genes? in? the? 18-month-old? liver? tissues? ?
over? that? of? the? 6-month-old? livers? may? reflect? accumulated? ?
biological? and? pathological? changes? with? aging? and/or? secondary? ?
by? HBx? and? contributing? to? hepatocarcinogenesis,? we? selected? ?
them,? nine? genes? were? downregulated? and? 33? were? upregulated.?
255 bp NM_013556
60˚C 217 bp
55˚C 238 bp
60˚C 285 bp
55˚C 295 bp
60˚C 236 bp
60˚C 243 bp
58˚C 297 bp
AN, accession number; AT, annealing temperature; bp, base pair.
©2007 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
groups? demonstrate? that? overexpression? of? Apobec‑1? in? the? liver?
could? induce? HCC? and? dysplasia? in? transgenic? mice? and? rabbits?
transgenic? livers? and? HCC? tissues.? Mutations? in? Alas2? are? often?
Gene? ontology? assisted? analysis? classified? these? genes? into? eight?
signal? transduction,? transcription? regulation? and? stress? responses?
mainly? in? various? pathological? injuries? to? the? liver,? or? the? ?
Among? these? identified? genes,? several? have? been? reported? as?
important? hepatocarcinogenic? factors.? Apolipoprotein? B? (apo-B)?
Global analysis of gene expression in HBx transgenic livers.
as? a? cytoprotective? molecule? that? enhances? tumor? growth? and? ?
Although? not? directly? reported? in? HCC? pathogenesis,? ?
deregulated? expression? of? transcription? factor? nuclear? factor-Y? b
(Nfyb),? cAMP? responsive? element? modulator? (Crem)? and? core?
of? tumors.20-24? A? recent? study? suggests? that? Crem? and? Nfy? are? ?
responsible? for? the? overexpression? of? the? proto-oncogenic? protein?
type?II?hexokinase?(HKII )?in?many?cancers.25?However,?the?exact? ?
functions? of? these? genes? during? hepatocarcinogenesis? need? to? be? ?
A? number? of? studies? have? implicated? oxidative? stress? in? the?
development? of? hepatitis-induced? HCC.26,27? In? this? work,? two?
genes? encoding? the? antioxidant? proteins? GTP? binding? protein? 4? ?
(Gtpbp4)? and? sulfiredoxin? were? identified? to? be? upregulated? in? ?
Figure 1. Identification of genes deregulated in HBx transgenic liver and HCC tissues. (A) The number of genes changed significantly in 6‑month‑old
(150 genes) and 18‑month‑old (292 genes) HBx transgenic liver tissues. Forty five genes representing for 42 unique mRNAs were deregulated at both time
points. (B) Hierarchical clustering of the 42 significantly changed genes. Genes were divided into two subgroups as up or downregulation. Totally nine genes
were downregulated and 33 genes were upregulated. (C) Heat map representation of cytoskeletal genes. As indicated by the arrows, seven genes involved
in cytoskeleton organization and biogenesis were closely clustered together. Each row represents a single gene, and each column represents one sample.
Green color, downregulation; Red color, upregulation; X/‑(6) and X/X(6), 6‑month‑old HBx heterozygous and homozygous mice; X/‑(18) and X/X(18),
18‑month‑old HBx heterozygous and homozygous mice; X(T), HCC form HBx transgenic mice.
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oxidative? damage? induced? by? HBx,? which? has? been? documented?
infected? with? HBV.26,28? Although? both? of? these? genes? were? not?
reported? to? be? involved? directly? in? tumorigenesis,? it? is? believed?
that? their? upregulation? might? aid? the? damaged? hepatocytes? to? ?
escape? from? apoptosis,? possibly? induced? by? increased? reactive?
oxygen? species? (ROS),? and? to? favor?
Previous? works? demonstrate? that?
a? relatively? small? number? of? genes? ?
Upregulation of cytoskeletal genes
in HBx transgenic livers and HCC
are? potentially? coregulated? and? may?
act? synergistically? to? promote? HCC?
we? performed? a? hierarchical? cluster?
according? to? their? expression? simil-?
of? these? genes? was? validated? by?
(Fig.? 2B)? and? immuno-histochemical?
staining? of? transgenic? liver? tissues?
In? order? to? exclude? the? effects? of?
p21? knockout? on? the? expression? of?
cytoskeletal? genes,? we? examined? the?
expression? of? K8? and? K18? in? liver?
tissues? of? p21‑HBsAg? transgenic?
mouse.8?K8 and? K18 expressed? at? a?
comparable? level? in? both? p21‑HBsAg
that? p21 deficiency? and? the? overex-
pression? of? HBsAg? didn’t? influence?
the? expression? of? these? two? genes?
(Fig.? 2C).? These? results? support? a?
Notably,? these? cytoskeletal? genes?
limited? gene? lists,? as? compared? to?
previous? reports.29,30? Specifically,?
they? represent? 1/6? of? the? identified?
genes,? suggesting? that? deregulated?
among? the? major? molecular? changes?
implicated? in? the? process? of? hepato-?
carcinogenesis,? we? next? investigated? the? expression? of? these?
genes? in? the? HCC? tissues? obtained? from? four? HBx transgenic?
samples,? and? found? that? these? two? genes? were? also? upregulated?
regulation of transcription
Cell Cycle/ growth
Cytoskeleton organization and biogenesis
gene title X/X(6) X/X(18)
S100 calcium binding protein A10 (calpactin)
phospholipid scramblase 1
apolipoprotein B editing complex 1
solute carrier family 10, member 2
aminolevulinic acid synthase 2, erythroid
deleted in polyposis 1
phospholipase A2, group XIIA
nuclear transcription factor‑Y b
cAMP responsive element modulator
Kruppel‑like factor 6 (Klf6), mRNA
polymerase (RNA) II (DNA directed) polypeptide K
homeobox only domain
SRY‑box containing gene 18
caspase 8 associated protein 2 0.39 4.95
growth arrest and DNA‑damage‑inducible 45 g
insulin‑like growth factor binding protein 1
GTP binding protein 4
WD repeat and SOCS box‑containing 1
gap junction membrane channel protein a 1
dual specificity phosphatase 6
Down syndrome critical region homolog 1 (human)
heat shock protein 1B
zinc finger protein 36, C3H type‑like 1
chemokine (C‑X‑C motif) ligand 1
sulfiredoxin 1 homolog (S. cerevisiae)
immunoglobulin kappa chain, constant region
actin, g, cytoplasmic 1
tubulin, b 2
tubulin, b 3
tubulin, b 6
RIKEN cDNA 3110001A13 gene
RIKEN cDNA 1300007C21 gene
RIKEN cDNA 1810015C04 gene
RIKEN cDNA 6230421P05 gene
expressed sequence AA536743
X/X(6): fold changes of 6-month-old HBx homozygous mice as compared with 6 month-old wild type mice. X/X(18): fold changes of 18-month-old HBx
homozygous mice as compared with 18 month-old wild type mice.
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in? human? HCC? tissues? (Fig.? 4C).?These?
role? in? the? development? of? HCC.? This?
opinion? is? supported? by? previous? studies?
on? cytoskeletal? proteins.? Altered? actin?
for? cellular? malignant? transformation.31?
to? be? crucial? for? the? proliferation? and?
transition? of? tumor? cells? due? to? their?
prerequisite? role? in? cell? division,? cell?
signalling? and? locomotion.32? Therefore,?
a? growing? number? of? anti-cancer? drugs?
Altered? expression? of? the? hepatocytic?
K8? and? K8? hyperphosphorylation? has? ?
been? demonstrated? a? progression? marker?
for? human? liver? diseases.35? Supportively,?
heterozygous? mutations? in K8? and? K18?
alcohol-induced? cirrhosis,? primary? biliary?
In? addition,? proteomic? analysis? has?
and? K8? induced? autoantibodies? among?
In? this? study,? we? provided? the? first?
evidence? to? show? that? the? upregulation?
Figure 2. Expression validation of cytoskeletal genes by RT‑PCR (A) or Western blot (B). Cytoskeletal
genes were upregulated in both 6 and 18 month old HBx transgenic mouse but not HBsAg transgenic
mouse livers. Hprt and beta‑actin were selected as internal controls. GAPDH was also selected as internal
control to demonstrated that beta‑actin was a suitable internal control to reflected the changes of other
cytoskeletal genes’. Wt, wild type mice; X/‑, HBx heterozygous mice; X/X, HBx homozygous mice;
S/‑, HBsAg heterozygous mice; S/S, HBsAg homozygous mice.
Figure 3. Upregulated expres‑
sion of K8 and K18 in HBx
transgenic liver and tumor
tissues. Frozen sections of
18 month old livers (A) and
tumor tissues (B) were used for
The K8 and K18 were stained
red with corresponding anti‑
bodies, and the nuclei were
stained blue with DAPI. The
magnification is 400 x (A)
or 100 x (B). Wt, wild type
mice; X/‑, HBx heterozygous
mice; X/X, HBx homozygous
mice; H&E, hematoxylin and
eosin staining; N, non can‑
cerous tissues; C, cancerous