Original paper

Aim of the study: To analyse the ex-

pression profile of hepatocellular car-
cinoma compared with normal liver by
using bioinformatics methods.
Bioinformatics analysis of the gene
Material and methods: In this study,
we analysed the microarray expression expression profile of hepatocellular
carcinoma: preliminary results
data of HCC and adjacent normal liv-
er samples from the Gene Expression
Omnibus (GEO) database to screen for
differentially expressed genes. Then,
functional analyses were performed
using GenCLiP analysis, Gene Ontolo-
gy categories, and aberrant pathway
identification. In addition, we used the Jia Li1,2, Zhongxi Huang3*, Lixin Wei1*
CMap database to identify small mole-
cules that can induce HCC. 1
Department of Pathology, Chinese PLA General Hospital, Beijing, China
Results: Overall, 2721 differentially 2
Medical College, Nankai University, Tianjing, China
expressed genes (DEGs) were identi- 3
Institute of Oncology, Nanfang Medical University, Guangzhou, Guangdong, China
fied. We found 180 metastasis-relat-
ed genes and constructed co-occur- *These authors contributed equally to this work.
rence networks. Several significant
pathways, including the transforming
growth factor β (TGF-β) signalling
pathway, were identified as closely re- Introduction
lated to these DEGs. Some candidate Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide
small molecules (such as betahistine)
and is also the third leading cause of cancer-related mortality [1, 2]. The World
were identified that might provide
a basis for developing HCC treatments Health Organisation (WHO) has estimated that there are nearly 56,400 new
in the future. cases of HCC around the world per year [3], and the incidence is much higher
Conclusions: Although we functionally in men than in women. The highest liver cancer rates are found in developing
analysed the differences in the gene countries, especially in East Asia and Malaysia, South Africa, and Sub-Saharan
expression profiles of HCC and normal Africa, whereas rates are lower in Europe, North and South America, Austra-
liver tissues, our study is essentially
preliminary, and it may be premature
lia, and New Zealand [4]. HCC can be induced by several risk factors, such as
to apply our results to clinical trials. chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), hepatic
Further research and experimental cirrhosis, alcoholic liver disease, and exposure to aflatoxins [5–7].
testing are required in future studies. Hepatocellular carcinoma always results from accumulative, long-term
interactions between environmental and genetic factors. The multifactorial
Key words: hepatocellular carcinoma, progression of HCC involves the activation of oncogenes, the inactivation
bioinformatics analysis, differentially
expressed genes, functional analysis.
of tumour suppressor genes, gene mutations, and irreversible cell damage.
Many studies have focused on the genetic mutations and the overexpres-
sion of abnormal genes that promote malignant progression, such as Cyclin
Contemp Oncol (Pozn) 2016; 20 (1): 20–27 D1 (CCND1), v-raf murine sarcoma viral oncogene homolog B (BRAF), epider-
DOI: 10.5114/wo.2016.58497
mal growth factor receptor (EGFR), c-myc, Ras, AKT, Yap, and baculoviral IAP
repeat containing 2 (BIRC2) [8, 9], as well as on the deletion or loss of het-
erozygosity (LOH) in the chromosomal regions of tumour suppressor genes,
such as CDKN2A, RB1, TP53, and PTEN [9, 10].
Although many genes that can promote or suppress HCC have been iden-
tified, the molecular mechanisms underlying HCC initiation, progression,
metastasis, or targeted therapy remain unclear. High-throughput microarray
technology, which enables investigators to obtain massive expression data
sets, has been demonstrated to be a useful approach for identifying new
tumour marker genes for tumour diagnosis or targeted treatment [11–13].
The aim of this study was to analyse the expression profile of hepatocellu-
lar carcinoma compared with normal liver by using bioinformatics methods.

Material and methods

Affymetrix microarray data from hepatocellular carcinoma
and adjacent normal liver tissues
To investigate the change in expression profile between hepatocellular
carcinoma tissues and adjacent normal liver tissues and to explore the
mechanisms that may be involved in hepatocarcinogenesis, we download-
ed and analysed the gene expression profile of GSE33006 from the Gene
Expression Omnibus – a public functional genomics data repository (http://
www.ncbi.nlm.nih.gov/geo/). The dataset, which was submitted by Huang
Bioinformatics analysis of the gene expression profile of hepatocellular carcinoma: preliminary results 21

et al. (2011), contains three HCC tissue chips and three CMap database, to identify the small molecules associat-
adjacent normal liver tissue chips from patients who ed with these DEGs. First, we divided the DEGs into two
underwent surgery, and it is based on the Affymetrix groups with an upper limit of 500 probe sets per group:
GPL570 Platform (Affymetrix GeneChip Human Genome the up-regulated group and the down-regulated group.
U133 Plus 2.0 Array). Total RNA was extracted from biop- Then, the probe sets from the two groups were preload-
sied samples using TRIzol reagent for further individual ed into their sandbox with the GRP format for gene set
on-chip analysis. enrichment analysis. Finally, the enrichment scores, which
ranged from +1 to –1, were calculated.
Screening of differentially expressed genes The enrichment value represented the association be-
The original CEL files were downloaded and analysed tween the preloaded query signature and the gene profile
using the R package (3.0.2) (http://www.r-project.org/). for a small molecule treatment. A high positive up score
The robust multi-array average (RMA) method and Affy- (close to +1) indicates that the corresponding small mole-
metrix Microarray Suite version 5 (MAS5) were used for cule induces the expression of the probe sets in the up tag
data normalisation and background correcting. We used list (hepatocellular carcinoma), whereas a high negative
a classical t-test to identify differentially expressed genes up score (close to –1) indicates greater similarity between
with a variation > 2-fold, and we defined p < 0.05 to be the genes induced by the small molecule and the probe
statistically significant. The probe set ID list of selected sets in the down tag list (adjacent normal liver).
differentially expressed genes was then uploaded to the
Results
NetAffx™ Analysis Centre (http://Affymetrix.com/analy-
sis/index/affx) to obtain the corresponding gene symbols Identification of differentially expressed genes
and gene titles. The expression data were deleted if there Using bioinformatics analysis we found 4233 probe set
was no corresponding gene symbol for the probe set or if IDs that differed between the HCC and adjacent normal
more than one gene symbol corresponded to a probe set. liver tissues. After the gene symbols from Affymetrix data-
Only probe sets that mapped to a unique gene were suit- base were matched and the substandard expression data
able for further analysis. If there were multiple probe sets were removed, altered expression was identified for 2721
corresponding to the same gene, the expression values of probe set IDs (corresponding to 2721 genes); these genes
these probe sets were then averaged. were marked for further analysis.

Functional analysis of differentially expressed GenCLiP analysis of the differentially expressed

genes genes
GenCLiP (a software program for clustering gene lists Of the 2721 analysed genes, 2701 had related literature,
by literature profiling, and for constructing gene co-oc- with an average of 661 literature matches per gene; these
currence networks related to keywords of interest; http:// genes were subsequently used for cluster analysis (Fig. 1).
ci.smu.edu.cn/GenCLip/) [14, 15] was used to analyse the We used the “literature mining gene networks” func-
differentially expressed genes, explore the pathogenesis, tion of GenCLiP to search for related genes and to con-
and construct gene networks related to important biologi- struct co-occurrence gene networks among the DEGs
cal processes in tumours, such as metastasis, cell growth, using the keyword “metastasis”. A total of 180 known
and cell cycle progression. metastasis-related genes were identified, and 180 genes
Gene Ontology Analysis (GO) is a common useful ap- formed 268 related gene pairs (Fig. 2A). To determine
proach for annotating genes and gene products and for whether the 180 known metastasis-related genes were
predicting gene function for high-throughput genome or identified randomly, PubMed was used to search for the
transcriptome data [16, 17]. To better investigate the func- occurrence of each gene set of the microarray with the
tion of these DEGs, a functional annotation tool, DAVID keyword “metastasis”, and then 300 random simulations
(Database for Annotation, Visualisation, and Integrated were performed. The resulting distribution of the number
Discovery) v6.7 (http://david.abcc.ncifcrf.gov/), was used of metastasis-related genes and gene pairs derived from
to cluster enriched function-related gene groups according random genes was similar to the normal distribution, and
to gene ontology (GO) terms, including molecular function, the probability that a set of 2701 randomly selected genes
biological process, and cellular component [18]. We also contained more than 180 metastasis-related genes or 268
used DAVID to visualise genes on KEGG (Kyoto Encyclopae- gene pairs was p = 0.00000 for both distributions (Fig. 2B).
dia of Genes and Genomes) pathway maps to investigate Furthermore, gene networks related to the keywords “cell
the dysregulated biological pathways in which the DEGs growth” and “cell cycle” were also constructed (Supple-
may participate. The cut-off criteria were that a pathway ment Fig. S1).
must contain at least two differentially expressed genes
and have a p-value < 0.05. Gene Ontology analysis of differentially expressed
genes
Small molecule identification These 2721 differentially expressed genes were func-
The Connectivity Map (CMap, build version build O2, tionally classified into three Gene Ontology (GO) cate-
http://www.broadinstitute.org/cmap/) was used to com- gories using the online analysis tool DAVID. For the bio-
pare the differentially expressed genes with those in the logical process category, a cut-off level of p < 0.001 was
22 contemporary oncology

Fig. 1. Literature profiling of differentially expressed genes. Clustergram generated from analysing the keyword occurrence of the analysed
genes. Green represents the corresponding gene-term associations that were reported to be positive

used to identify significant enrichment of genes with the some enzymes changed, including the activities of ox-
corresponding GO terms. Analysis revealed changes in the idoreductase, endopeptidase inhibitor, transmembrane
biological processes of the immune system, such as the receptor protein tyrosine kinase, etc. The binding abilities
positive regulation of immune system processes, leuko- of some materials (polysaccharides, glycosaminoglycans,
cyte activation, innate immune response, T cell activation, etc.) were also changed.
B cell-mediated immunity, and immunoglobulin-mediat-
ed immune response. The DEGs also showed significant KEGG pathway enrichment analysis
enrichment in processes related to the regulation of cell The changed gene expression profile of hepatocellu-
growth, such as the regulation of cell proliferation, the cell lar carcinoma may result in many dysregulated signal
cycle, cell differentiation, nuclear division, and M phase pathways. We used DAVID to cluster the DEGs for KEGG
of the mitotic cell cycle. Changes in the expression profile pathway enrichment analysis, and p < 0.05 was set as the
also affected the biological processes of angiogenesis and cut-off criteria for statistical significance. As shown in Ta-
signal transduction (Table 1). ble 4, 20 dysregulated pathways were identified from the
In the category of cellular component, the most en- changes in HCC; of these pathways, the complement and
riched GO term was cytoplasm (1089 genes). In addition, coagulation cascades and cell adhesion molecules (CAMs)
the DEGs were also enriched in cellular components relat- showed the most significant enrichment (P = 2.79E-18 and
ed to the plasma membrane, extracellular region, chro- P = 3.17E-04, respectively). As previously reported, some
mosome, secretory granules, and the cytoplasmic mem- altered pathways were highly related to the initiation or
brane-bound vesicle lumen (p < 0.01) (Table 2). Table 3 progression of malignant tumours; these pathways includ-
shows the clustered GO terms in the molecular function ed the TGF-β signalling pathway, T cell/B cell receptor sig-
category for the differentially expressed genes (p < 0.01). nalling pathway, and pathways related to DNA replication
The HCC expression profile indicated that the activity of and cytokine-cytokine receptor interactions.
Bioinformatics analysis of the gene expression profile of hepatocellular carcinoma: preliminary results 23

Fig. 2. Constructing a gene co-occurrence network related to the keyword “metastasis” from the differentially expressed genes. A) The net-
work of analysed genes. The orange node represents known hepatocellular carcinoma-related genes. B) The distribution of the number of
metastasis-related genes and gene pairs derived from the random genes was similar to the normal distribution

Identification of candidate small molecules with a highly significant positive score and could partial-
To identify candidate small molecules that could re- ly imitate the status of hepatocellular carcinoma. These
verse the gene expression changes of hepatocellular car- small molecules may be strong induction factors for HCC.
cinoma, the DEGs were divided into two groups: up-reg- In contrast, cromoglicic acid (enrichment score = –0.927)
ulated and down-regulated, which were uploaded to the and ranitidine (enrichment score = –0.837) were associ-
CMap database for Gene Set Enrichment Analysis and then ated with highly significant negative scores and may imi-
matched to treatments with small molecules. The 20 most tate the normal liver status. These small molecules could
significant small molecules are listed in Table 5 with their reverse the tumoral status of HCC and therefore provide
enrichment scores and p-values. As shown in Table 5, the novel ideas and molecular mechanisms for developing new
small molecules vorinostat (enrichment score = 0.973) and drugs for treating HCC in the future. However, these candi-
trichostatin A (enrichment score = 0.895) were associated date small molecules still require further detailed research.
24 contemporary oncology

Table 1. Gene Ontology enrichment of differentially expressed genes in biological process

Term Count p-value

GO:0006955~immune response 182 9.01E-18
GO:0002376~immune system process 239 1.60E-17
GO:0002526~acute inflammatory response 49 2.01E-16
GO:0006952~defense response 157 7.08E-14
GO:0048518~positive regulation of biological process 402 1.34E-13
GO:0002460~adaptive immune response based on somatic recombination of immune receptors built 39 2.32E-13
from immunoglobulin
GO:0002682~regulation of immune system process 110 3.04E-13
GO:0002541~activation of plasma proteins involved in acute inflammatory response 27 3.09E-12
GO:0045087~innate immune response 53 6.50E-12
GO:0002449~lymphocyte-mediated immunity 35 7.52E-12
GO:0019724~B cell-mediated immunity 30 3.50E-11
GO:0016064~immunoglobulin-mediated immune response 29 7.34E-11
GO:0002455~humoral immune response-mediated by circulating immunoglobulin 20 1.96E-09
GO:0022610~biological adhesion 152 5.46E-08
GO:0007155~cell adhesion 151 8.58E-08
GO:0042110~T-cell activation 41 4.11E-07
GO:0008283~cell proliferation 98 3.28E-06
GO:0045595~regulation of cell differentiation 107 5.46E-06
GO:0001944~vasculature development 62 1.37E-05
GO:0050848~regulation of calcium-mediated signalling 12 2.32E-05
GO:0001568~blood vessel development 59 4.95E-05
GO:0007049~cell cycle 150 5.66E-05
GO:0007059~chromosome segregation 25 2.69E-04
GO:0009966~regulation of signal transduction 162 3.13E-04
GO:0007169~transmembrane receptor protein tyrosine kinase signalling pathway 52 3.76E-04
GO:0000087~M phase of mitotic cell cycle 52 3.76E-04
GO:0040011~locomotion 88 3.93E-04
GO:0007067~mitosis 51 4.46E-04
GO:0007167~enzyme linked receptor protein signalling pathway 72 5.78E-04
GO:0006928~cell motion 94 7.27E-04
GO:0001525~angiogenesis 37 7.33E-04
GO:0040007~growth 43 9.98E-04

Discussion action of CD44 and its association molecules, CD44 can

regulate cancer cell proliferation, adhesiveness, migra-
By analysing the differentially expressed genes us-
tion, and metastasis [22]. CD44 was also demonstrated
ing GenCLiP software, 180 metastasis related genes
to be closely associated with the extrahepatic metasta-
were identified and used to construct co-occurrence
sis of HCC [23]. Co-occurrence networks involving large
gene networks. Of the metastasis-related genes, TGFB1 numbers of related genes enable researchers to identify
and EGFR had the largest number of co-occurring genes critical genes and their possible interactional networks,
(34 and 33, respectively) and were located in the centre of which may provide a new direction for the diagnosis and
the network. As recognised dysregulated growth factors, targeted therapy of HCC.
TGFB1/EGFR and their downstream signalling pathway Gene Ontology (GO) analysis revealed that these DEGs
components contribute to the proliferation and invasive were closely related to multiple biological processes in-
behaviour of liver cancer cells [19–21]. As a transmem- volved in the mechanism of most malignant tumours,
brane glycoprotein, CD44 was shown to interact with such as the regulation of the immune system, cell growth,
28 genes. Reports have revealed that through the inter- the cell cycle, angiogenesis, and signal transduction. Sev-
Bioinformatics analysis of the gene expression profile of hepatocellular carcinoma: preliminary results 25

Table 2. Gene Ontology enrichment of differentially expressed genes in cellular component

Term Count p-value

GO:0044421~extracellular region part 212 2.83E-13
GO:0005615~extracellular space 160 3.50E-12
GO:0031226~intrinsic to plasma membrane 248 1.60E-11
GO:0005887~integral to plasma membrane 242 3.67-11
GO:0005576~extracellular region 366 8.21E-11
GO:0009897~external side of plasma membrane 52 2.24E-08
GO:0009986~cell surface 86 3.78E-08
GO:0031093~platelet alpha granule lumen 21 7.13E-08
GO:0060205~cytoplasmic membrane-bounded vesicle lumen 21 3.09E-07
GO:0031983~vesicle lumen 21 7.49E-07
GO:0000793~condensed chromosome 37 1.54E-05
GO:0005737~cytoplasm 1089 2.58E-05
GO:0005886~plasma membrane 593 2.81E-05
GO:0005579~membrane attack complex 7 4.00E-05
GO:0043235~receptor complex 32 1.41E-04
GO:0005694~chromosome 91 2.32E-04
GO:0030141~secretory granule 43 2.92E-04
GO:0044433~cytoplasmic vesicle part 41 0.0024
GO:0005604~basement membrane 21 0.0034
GO:0000777~condensed chromosome kinetochore 17 0.0039
GO:0031012~extracellular matrix 66 0.0041
GO:0000775~chromosome, centromeric region 29 0.0046
GO:0031410~cytoplasmic vesicle 112 0.0048
GO:0034358~plasma lipoprotein particle 12 0.0054
GO:0000779~condensed chromosome, centromeric region 18 0.0064
GO:0030055~cell-substrate junction 26 0.0082

Table 3. Gene Ontology enrichment of differentially expressed genes in molecular function

Term Count p-value

GO:0005515~protein binding 1282 5.58E-10
GO:0030247~polysaccharide binding 49 4.16E-08
GO:0005539~glycosaminoglycan binding 45 1.15E-07
GO:0030246~carbohydrate binding 87 1.71E-07
GO:0003823~antigen binding 20 1.46E-04
GO:0008201~heparin binding 30 1.49E-04
GO:0019955~cytokine binding 31 1.80E-04
GO:0019838~growth factor binding 30 2.14E-04
GO:0046983~protein dimerization activity 105 5.49E-04
GO:0030234~enzyme regulator activity 151 0.001
GO:0004896~cytokine receptor activity 18 0.0011
GO:0019825~oxygen binding 15 0.0017
GO:0004866~endopeptidase inhibitor activity 35 0.0017
GO:0030414~peptidase inhibitor activity 36 0.0023
GO:0004497~monooxygenase activity 25 0.0037
GO:0003824~catalytic activity 787 0.0051
GO:0019899~enzyme binding 96 0.0052
GO:0019842~vitamin binding 30 0.0074
GO:0019901~protein kinase binding 33 0.0074
GO:0008172~S-methyltransferase activity 5 0.0097
GO:0016491~oxidoreductase activity 120 0.0099
26 contemporary oncology

Table 4. The altered KEGG pathways

Term Description Count p-value

hsa04610 complement and coagulation cascades 44 2.79E-18
hsa04514 cell adhesion molecules (CAMs) 38 3.17E-04
hsa00071 fatty acid metabolism 15 0.0027
hsa04640 hematopoietic cell lineage 25 0.0037
hsa00380 tryptophan metabolism 14 0.0077
hsa04672 intestinal immune network for IgA production 16 0.0081
hsa04060 cytokine-cytokine receptor interaction 58 0.0084
hsa04660 T cell receptor signalling pathway 28 0.011
hsa04110 cell cycle 31 0.013
hsa00330 arginine and proline metabolism 16 0.017
hsa05214 glioma 18 0.019
hsa03030 DNA replication 12 0.022
hsa04512 ECM-receptor interaction 22 0.023
hsa00590 arachidonic acid metabolism 16 0.028
hsa04350 TGF-β signalling pathway 22 0.033
hsa04810 regulation of actin cytoskeleton 46 0.034
hsa05322 systemic lupus erythematosus 24 0.040
hsa00280 valine, leucine, and isoleucine degradation 13 0.041
hsa04662 B cell receptor signalling pathway 19 0.048
hsa04650 natural killer cell mediated cytotoxicity 30 0.049

Table 5. List of top 20 identified small molecules esis [20]. Interactions between the extracellular matrix
(ECM) receptor and cells play a vital role in cell adhesion
Cmap name Enrichment score p-value
and form a crucial step in tumour cell migration and inva-
vorinostat 0.973 0 sion into the extracellular matrix [24].
trichostatin A 0.895 0 Analysis using the CMap database identified a set of
geldanamycin 0.705 0 small molecules that may imitate the status of hepato-
fluphenazine 0.629 0 cellular carcinoma or a normal liver. The candidate small
molecules that were associated with highly significant
trifluoperazine 0.625 0
negative enrichment scores may reverse the abnormal
thioridazine 0.599 0 gene expression profile of HCC; this information will be
sirolimus 0.491 0 beneficial to investigators who may develop new tar-
valproic acid 0.359 0 get therapeutic drugs against HCC. Histamine has been
pioglitazone –0.646 0.00004 demonstrated to be involved in cell proliferation and
tumour growth by the activation of histamine receptors
6-bromoindirubin-3’-oxime –0.769 0.00008
[25]. As an agonist of histamine that interacts with the
withaferin A 0.896 0.0001 H1 and H3 receptors [26], betahistine may play a role in
wortmannin 0.501 0.0001 tumour biology through the regulation of histamine re-
ivermectin 0.858 0.00012 ceptors.
prochlorperazine 0.524 0.00014 In conclusion, we identified 2271 differentially ex-
pressed genes in hepatocellular carcinoma, and the co-oc-
suloctidil 0.888 0.00016
currence networks related to “metastasis”, “cell growth”,
cephaeline 0.848 0.00018 and “cell cycle” were constructed. Furthermore, we iden-
PNU-0293363 –0.954 0.00022 tified significant biological processes and abnormally al-
ranitidine –0.837 0.0003 tered pathways that were related to the development of
meptazinol –0.825 0.00177 HCC. We also screened a set of candidate small molecules,
some of which may induce the initiation of HCC, and some
betahistine –0.822 0.00189
reversed the expression profile of HCC. These small mol-
ecules may be candidates for therapeutic drugs that are
eral significant altered pathways were identified by KEGG capable of targeting hepatocellular carcinoma. However,
pathway analysis. The TGF-β signal pathway has been re- the number of samples involved in this study was limited,
ported to be functionally impaired in hepatocarcinogen- and the analysed results contained a massive amount of
Bioinformatics analysis of the gene expression profile of hepatocellular carcinoma: preliminary results 27

information, which requires thorough research and must 22. Marhaba R, Zoller M. CD44 in cancer progression: adhesion, mi-
be experimentally validated in future studies. gration and growth regulation. J Mol Histol 2004; 35: 211-31.
23. Hirohashi K, Yamamoto T, Uenishi T, et al. CD44 and VEGF expres-
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ma. Hepatogastroenterology 2004; 51: 1121-23.
The authors declare no conflict of interest. 24. Lara-Pezzi E, Majano PL, Yáñez-Mó M, Gómez-Gonzalo M, Carrete-
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