E XP E RI ME N TAL CE L L R ES E ARC H

328 (2014) 361 378

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

JMJD2A attenuation affects cell cycle and tumourigenic

inammatory gene regulation in lipopolysaccharide
stimulated neuroectodermal stem cells
Amitabh Dasa, Jin Choul Chaib, Kyoung Hwa Jungb, Nando Dulal Dasc,
Sung Chul Kangb, Young Seek Leeb, Hyemyung Seob, Young Gyu Chaia,b,n
a

Department of Bionanotechnology, Hanyang University, Seoul 133-791, Republic of Korea

Department of Molecular & Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do,
Republic of Korea
c
Clinical Research Centre, Inha University School of Medicine, Incheon 400-711, Republic of Korea
b

article information

abstract

Article Chronology:

JMJD2A is a lysine trimethyl-specic histone demethylase that is highly expressed in a variety of

Received 25 March 2014

tumours. The role of JMJD2A in tumour progression remains unclear. The objectives of this study

Received in revised form

were to identify JMJD2A-regulated genes and understand the function of JMJD2A in p53-null

21 July 2014

neuroectodermal stem cells (p53
/
NE-4Cs). We determined the effect of LPS as a model of

Accepted 19 August 2014

inammation in p53
/
NE-4Cs and investigated whether the epigenetic modier JMJD2A alter the

Available online 1 September 2014

expression of tumourigenic inammatory genes. Global gene expression was measured in JMJD2A

Keywords:

knockdown (kd) p53
/
NE-4Cs and in LPS-stimulated JMJD2A-kd p53
/
NE-4C cells. JMJD2A

Inammation

attenuation signicantly down-regulated genes were Cdca2, Ccnd2, Ccnd1, Crebbp, IL6r, and Stat3

JMJD2A

related with cell cycle, proliferation, and inammatory-disease responses. Importantly, some tumour-

Cell cycle

suppressor genes including Dapk3, Timp2 and TFPI were signicantly up-regulated but were not

Neuroectodermal stem cell

affected by silencing of the JMJD2B. Furthermore, we conrmed the attenuation of JMJD2A also

p53

down-regulated Cdca2, Ccnd2, Crebbp, and Rest in primary NSCs isolated from the forebrains of E15
embryos of C57/BL6J mice with effective p53 inhibitor pithrin- (PFT-). Transcription factor (TF)
motif analysis revealed known binding patterns for CDC5, MYC, and CREB, as well as three novel
motifs in JMJD2A-regulated genes. IPA established molecular networks. The molecular network
signatures and functional gene-expression proling data from this study warrants further investigation as an effective therapeutic target, and studies to elucidate the molecular mechanism of JMJD2Akd-dependent effects in neuroectodermal stem cells should be performed.
& 2014 Elsevier Inc. All rights reserved.

n
Corresponding author at: Department of Molecular & Life Science, Hanyang University, 1271 Sa 3-dong, Ansan 426-791, Gyeonggi-do, Republic
of Korea. Fax: 82 31 436 8173.
E-mail addresses:
amitabhdas.kn@gmail.com (A. Das), jincchai@gmail.com (J.C. Chai), khjung2@gmail.com (K.H. Jung), nando.hu@gmail.com (N.D. Das),
gujiju11@gmail.com (S.C. Kang), yslee@hanyang.ac.kr (Y.S. Lee), hseo@hanyang.ac.kr (H. Seo), ygchai@hanyang.ac.kr (Y.G. Chai).

http://dx.doi.org/10.1016/j.yexcr.2014.08.029
0014-4827/& 2014 Elsevier Inc. All rights reserved.

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Introduction
Post-translational modication of histone tails, such as phosphorylation, ubiquitination, acetylation, and methylation, are critical
for chromatin regulation, gene activity, and nuclear architecture.
Methylation of histone 3 lysine 4 (H3K4) and H3K36 is generally
linked to transcriptionally active genes, whereas methylation of
H3K9 and H3K27 is associated with repressed genes. Multiple
lysine methyl transferases are responsible for histone methylation
and exert control over gene expression [1]. The discovery of lysine
demethylases showed that lysine methylation is a reversible and
dynamic process. A large number of demethylases can demethylate specic histone lysine and arginine residues via amine
oxidation, hydroxylation, or deamination.
Jumonji Domain Containing 2A (JMJD2A), also known as JHDM3 or
KDM4A was identied and characterised in 2004 [2]. JMJD2A belongs
to the JmjC domain-containing family of JMJD2 proteins. These
enzymes are lysine trimethyl-specic histone demethylases that
catalyse the demethylation of trimethylated H3K9 (H3K9me3) and
H3K36 (H3K36me3) and are considered to be transcriptional repressors [2]. JMJD2A inhibit transcription by interacting with the tumour
suppressor retinoblastoma protein (Rb), histone deacetylases (HDACs),
and the co-repressor nuclear receptor corepressor (N-CoR) [3,4].
Because of Rb protein is essential in cell cycle; JMJD2A was reported
to play an important role in cell proliferation and oncogenesis.
Similar to other JmjC domain proteins, JMJD2A requires Fe (II) and
-ketoglutarate as cofactors for demethylation [5]. Structure of the
catalytic core region of JMJD2A mainly consists of a number of
individual domains (JmjN and JmjC domains) and structural motifs.
The JmjC domain has been shown to fold into eight -sheets, thus
forming an enzymatically active pocket that coordinates Fe(II) and
a-ketoglutarate. Three extremely conserved amino acid residues
within the JmjC domain connect to the Fe(II) cofactor and two water
molecules were replaced by two oxygen atoms from -ketoglutarate
[6]. JMJD2A family genes are also cancer-associated genes. JMJD2A is
widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA has been observed in several cell
types, including human T-cell lymphotropic virus 1-infected cell lines,
the HT1376 bladder carcinoma cell line, the U2OS osteosarcoma cell
line and a prostate cancer cell line [7,8]. This expression pattern
suggests that JMJD2A has tumour-promoting functions. However,
little is known about the expression and role of JMJD2A in neuronal
stem cells (NSCs).
Cell cycle control is essential to determining when a cell should
perform deoxyribonucleic acid (DNA) synthesis, proliferation, growth
arrest, DNA repair or apoptosis. Accordingly, cell cycle pathways are
regulated by both oncogenes and tumour suppressors and are
frequently deregulated in human cancers. Deregulation of cell cycle
regulatory genes may lead to independence from growth-regulating
signals, which are critical determinants of tumour progression.
Common causes in tumours include the aberrant expression of
positive regulators, such as cyclins, and the loss of function of negative
regulators, such as cyclin-dependent kinase inhibitors (CKIs) [9].
At present, many types of tumours occur due to aberrant expression
of cell cycle regulatory genes. Among them, medulloblastoma
(MB) and supratentorial neuroectodermal tumours are rare but
destructive forms that contribute signicantly to childhood cancerrelated morbidity and mortality and frequently occur because of
aberrant expression of cell cycle regulatory genes [1012]. Major

3 28 (2014) 3 61 37 8

tumour-suppressor pathways regulated by p53 are also involved in

MB and supratentorial neuroectodermal tumour development; both
tumours occur in families with constitutional p53 defects [13,14]. P53
was found to be expressed at elevated levels in NSCs compared to
other regions of the mouse brain [15,16]. Cells in the brain lateral
ventricle stem cell niche exhibit increased proliferation in p53knockout mice compared to wild type [15]. A p53 deciency in
neurospheres increases self-renewal capacity and cell proliferation
and reduces apoptosis [15,17]. The majority of tumour cells may
benet from having mutant p53 or no p53, but some tumour cells
might also thrive with wild-type p53. Given that the formation of
supratentorial neuroectodermal tumours might begin with mutant or
no p53, we chose to study NE-4Cs a p53-null neuroectodermal stem
cells. These cells were established from cerebral vesicles of 9-day-old
mouse embryos with decient p53 genes [18]. Extensive research on
the connection between inammation and cancer has shown the
context-dependent modulation of inammation-associated cancer
[19]. The mechanism by which an inammatory atmosphere inuences NSCs and thus alters NSCs self-renewal, survival, migration,
proliferation, and differentiation is unknown [20]. Several studies
focused on the effects of inammation on the regenerative capacity of
NSCs that undergo microglial activation after an acute injury or
lipopolysaccharide (LPS) exposure. Toll-like receptor 4 (TLR4) is
expressed by NSCs and directly modulates the self-renewal and
self-fate decisions of neural progenitor cells [21]. The connection
between brain inammation and NSC-mediated brain tumour formation and the function of the niche environment in the modulation
of neuronal differentiation under alternative conditions are under
intense investigation. Here, we investigated the effect of LPS as a
model of inammation on p53
/
NE-4Cs and primary NSCs
determined whether the epigenetic modier JMJD2A affected the
capacity of NE-4Cs as well as NSCs and altered the expression of cell
cycle mediated tumourigenic inammatory genes.
In the present study, a comparative gene expression signature
was determined for JMJD2A-attenuated p53
/
NE-4C cells to
identify molecular targets of JMJD2A-attenuated p53
/
NE-4Cs cells.
In particular, JMJD2A attenuation signicantly down-regulated key
cell-cycle mediated tumourigenic inammatory genes in NE-4C and
NSCs. Importantly, some tumour-suppressor genes were signicantly
up-regulated in JMJD2A-kd p53
/
NE-4Cs but were not affected by
silencing of the JMJD2A paralogue JMJD2B. Overall, the results
suggested that JMJD2A might be an effective therapeutic target with
possible research and clinical value. In addition, the signalling pathways and molecular networks induced by LPS-treated JMJD2Aattenuated p53
/
NE-4C cells were determined by Ingenuity pathways analysis (IPA). We also performed TF motif analysis of the
promoter region (
500oTTSo200) of genes that exhibited differential gene expression under our tested conditions and found binding
sequences for known TFs. Therefore, clarifying the function of JMJD2A
might help to identify novel therapeutic targets for tumourigenesis as
well as brain inammation.

Materials and methods

Cell culture and stimulation
The NE-4C cell line (CRL-2925) was obtained from the American
Type Culture Collection. Cultures were maintained subconuence
in a 95% air, 5% CO2 humidied atmosphere at 37 1C in Eagle's

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minimum essential medium supplemented with 10% foetal

bovine serum (catalogue # 26140), 100 IU/ml penicillin, and
10 mg/ml streptomycin (catalogue # 15140) from Invitrogen
(USA). NE-4C cells were incubated with LPS (1 mg/ml) for the
specied times under normal culture conditions. Medium containing the appropriate agents was replaced every other day.
Primary NSCs were isolated from the forebrains of E15 embryos
obtained from pregnant C57/BL6J mice for the neurosphere
cultures was performed as previously described [22,23]. Briey,
isolated cells were seeded in 100-mm dishes in Dulbecco's
modied Eagle's medium/nutrient mixture F-12 (DMEM/F12,
1:1), 2% B-27 Invitrogen (USA), penicillin 100 IU/ml penicillin,
and 10 mg/ml streptomycin, 20 ng/ml bFGF (Invitrogen) and 5 mg/
ml heparin, and they were maintained at 37 1C in a 5% CO2
atmosphere as the neurospheres formed. After formation of the
neurospheres, they were subcultured once per week using 0.25%
trypsin/EDTA. The neurosphere-forming NSCs were harvested,
seeded in polyl-lysine-coated culture dishes containing DMEM,
P/S, 2% B-27 and 1% FBS and were used for the following
experiments. The p53 inhibitor used was Pithrin- (PFT-)
(30 mM) from Santa Cruz Biotechnology (sc-45050) [24].

Cell viability assay

Cell proliferation was assayed using a tetrazolium salt colorimetric assay using PreMix WST-1 according to the manufacturer's
instructions (Takara Bio Inc., Shiga, Japan). Cells were seeded at
5  103 cells per well in 96-well plates in 100 ml and incubated for
the indicated time. PreMix WST-1 was added as indicated and
incubated for an additional 4 h, and absorbance was measured at
450720 nm.

Nitric oxide assay

Accumulation of nitric oxide (NO) in culture supernatants was
measured using the Griess reaction. NE-4C cells (5  103/well)
were plated into 96-well microtitre plates and treated with LPS
(1 mg/ml) for the indicated time. A 50 ml sample of the culture
supernatant was mixed with 50 ml Griess reagent (part I: 1%
sulphanilamide; part II: 0.1% naphthylethylene diamide dihydrochloride and 2% phosphoric acid) at room temperature. The
absorbance was determined at 540 nm after 15 min.

Gene silencing by small interfering RNA

Oligoduplexes specic for JMJD2A (ID # s106548) and JMJD2B
(ID # s101446) were from Ambion Applied Biosystems. JMJD2A
was attenuated with small interfering (si)RNA sense strand
50 -CCUUUAUCCUGAGGACAUAtt-30 and antisense strand 50 -UAUGUCCUCAGGAUAAAGGtt-30 , and JMJD2B was attenuated with
sense strand 50 -CCACUGCGCAGACAUUCUAtt-30 and antisense
strand 50 -UAGAAUGUCUGCGCAGUGGTG-30 siRNA. NE-4C and primary NSCs were transfected using siPORT NeoFX transfection
agent (Ambion; Applied Biosystems L/N: 1203023) with JMJD2A
or JMJD2B siRNA constructs following the Silencer Select siRNA
transfection protocol with non-targeting siRNAs (Ambion and
Applied Biosystems). JMJD2A siRNA was used at concentrations
of 5, 10, and 25 nM for 48 h using OptiMEM medium.

328 (2 014 ) 361 37 8

363

Cell-cycle analysis
The cell cycle was measured by FACS analysis. Cells (2.0  105
cells/well) were seeded in six-well plates with or without JMJD2A
attenuation (25 nM of siRNA), cultured for 48 h and were
collected after transfection and treatment with trypsin-EDTA by
trypsinization, washed with 1% cold phosphate-buffered saline
solution. The cell suspension was centrifuged at 3000 rpm for
4 min, and the supernatant was removed leaving approximately
50 ml of residual uid in the tube to avoid disturbing the pellet.
The cells were analysed using a commercially available kit
(CycleTEST PLUS DNA Reagent Kit; BD Biosciences, San Jose, CA,
USA), whereby propidium iodide (PI) stained nuclei were analysed by ow cytometry (FACscan, BD Biosciences, San Jose, CA,
USA). The cell-cycle phases were analysed using ModFit LT 3.0
software (Verity Software House, Topsham, ME, USA). The experiments were repeated thrice.

Total RNA extraction

Total RNA ( 8 mg) was extracted using TRIzol (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Two
hundred microliters of chloroform was added, and tubes with
lysing mix were inverted gently to mix for 5 min. The mix was
centrifuged at 12,000g for 15 min at 4 1C, and the clear top
solution was placed into a new tube to which 500 ml isopropanol
was added. Tubes were inverted to mix before incubation on ice
for 1 h. The lysis mix was centrifuged at 12,000g for 10 min at
4 1C, and the isopropanol was decanted. Ice-cold 70% ethanol was
added to the RNA pellet for gentle washing. After centrifuging as
above for 10 min, the ethanol was removed. RNA pellets were
dried at room temperature for 510 min before reconstitution in
20 ml RNase-free water. RNA was treated with RNase-free DNase
(Promega, Wisconsin, USA). The RNA quality was assessed using
an Agilent 2100 Bioanalyser with the RNA 6000 Nano Chip
(Agilent Technologies, Waldbronn, Germany), and the quantity
was determined using a spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Microarray data analysis

Genome-wide transcriptional analysis was performed on NC-siRNA
NE-4Cs (non-targeting), JMJD2A-kd NE-4Cs, 2 h LPS-stimulated
JMJD2A-kd NE-4Cs, and 10 h LPS-stimulated JMJD2A-kd NE-4Cs
via four individual arrays (Affymetrix Gene Chip Scanner 3000
7G). We performed three times of same siRNA mediated JMJD2A
knockdown analyses of same biological samples, and the JMJD2A
mRNA or proteins that showed highest knockdown efciency
compared with negative control (NC)-siRNA were selected for
genome-wide transcriptional analysis. Total RNA samples (10 mg)
were labelled with cyanine 3 (Cy3)- or cyanine 5 (Cy5)-conjugated dCTP (Amersham, Piscataway, NJ) by reverse transcription
with Super Script II (Invitrogen, Carlsbad, USA). The RNA quality
was assessed using an Agilent 2100 Bioanalyser with the RNA
6000 Nano Chip (Agilent Technologies, Waldbronn, Germany)
(Fig. 6B). Labelled cDNA mixtures were concentrated using an
ethanol precipitation method. Concentrated Cy3- and Cy5labelled cDNAs were resuspended in 10 ml hybridisation solution.
Microarray data were scanned using a laser confocal scanner
(Affymetrix). Global scaling normalisation was performed for

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signals. The relative signal intensities for each gene were generated using the robust multi-array average (RMA) algorithm. Data
were processed based on the RMA average normalisation method.
Normalised and log-transformed intensity values were analysed
(Affymetrix). Fold-change lters included the requirement that
genes were expressed at levels of at least 150% of the controls for
up-regulated genes and less than 66% of controls for downregulated genes. Hierarchical clustering data were clustered in
groups that behaved similarly across experiments using Gene
Spring GX 12.5 (Agilent technologies, Canada). The clustering
algorithm used was Euclidean distance, average linkage.

3 28 (2014) 3 61 37 8

Functional annotation
DAVID (Database for Annotation, Visualisation and Integrated
Discovery) version 6.7 software (http://david.abcc.ncifcrf.gov/
home.jsp) was used to determine the most functional annotation
of signicant genes in datasets as described previously [27].
DAVID calculates a modied Fisher's exact P value to demonstrate
gene ontology (GO) or molecular pathway enrichment. Values less
than 0.05 were considered to be strongly enriched in the annota
tion category.

Canonical pathway analysis of datasets

cDNA synthesis and quantitative real-time RT-PCR
Reverse transcription of the RNA samples was performed as
described by [25], using 2 mg of total RNA, 1 ml random hexamers
(per reaction) and Prime Script 1st-strand cDNA synthesis kits
(Takara, Japan). Random hexamers and RNA templates were
mixed and denatured at 65 1C for 5 min followed by cooling for
2 min on ice. Prime Script buffer (5  ), RTase and RNAse inhibitor
were added to the cooled template mix and incubated for 1 h at
50 1C before enzyme inactivation at 70 1C for 15 min. Quantitative
real-time RT-PCR (qRT-PCR) was performed using SYBR Green PCR
Master Mix (Takara Bio Inc., Shiga, Japan) and a 7500 fast realtime PCR system (Applied Biosystems, Foster City, USA).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used
as an internal control. Complementary DNA samples were diluted
1.5-fold, and qRT-PCT was run on a AB-7500 Real-time thermal
cycler (Applied Biosystems, Foster City, USA) using SYBR Premix
Ex-Taq II (Takara Bio, Shiga, Japan) according to the manufacturer's directions. The reactions were 20 ml with 0.4 mM of each
primer (Table 3). Each PCR run included a no-template control
with water instead of cDNA and a reverse transcriptase-negative
control for each gene. Triplicate measurements were performed
for all reactions. Different samples were run on 96-well plates for
gene expression experiments, while all samples were analysed on
a single plate for endogenous control determination. The results
were analysed using the critical threshold (CT) and the comparative critical threshold (CT) methods in AB-7500 software
with the Norm nder and the geNorm-plus algorithms. Primers
were designed by Primer Express (Applied Biosystems, Foster
City, USA).

IPA (Ingenuity W Systems, www.ingenuity.com, Mountain View,

CA, USA) was conducted to analyse canonical pathways that were
most signicant in datasets as described previously [26,28,29].
Genes from datasets that were associated with canonical path
ways in the Ingenuity Pathways Knowledge Base (IPAKB) were
considered for literary analysis. The signicance of associations
between datasets and canonical pathways was measured in the
following two ways: (1) the ratio of the number of genes from the
dataset that mapped to a canonical pathway divided by the total
number of genes that mapped to the same canonical pathway;
and (2) Fischer's exact test for a P value indicating the probability
that the association could be explained by chance. After uploading
the datasets, gene identiers were mapped to corresponding gene
objects, and genes were overlaid onto a global molecular network
in IPAKB. Gene networks were algorithmically generated based on
connectivity.

Graphical representation of networks and pathways

Graphical representation of molecular relationships between
genes and gene products was based on the following. Genes or
gene products were represented as nodes, and the biological
relationship between two nodes was represented as an edge
(line). All edges were supported by at least one reference from the
literature, textbook or canonical information in IPAKB. The node
colour intensity indicated the degree of up-regulation (red) or
down-regulation (green). Nodes were displayed by using shapes
to represent functional classes of gene products.

Transcription factor motif analysis

Western blot analysis
Western blots were performed according to standard procedures
[26]. Whole cell lysates (20 mg) were resolved by electrophoresis
on 12% polyacrylamide gels. Proteins were transferred to polyvinylidene diuoride membranes (Schleicher & Schuell
Bioscience, Inc., Keene, NH) by electro-blotting using a semi-dry
method. Membranes were blocked with 5% skim milk in 1% TBSTween for 1 h and incubated overnight with primary antibodies at
4 1C. After washing, membranes were incubated for 1 h with the
appropriate secondary antibody. Blots were visualised with
enhanced chemiluminescence (ECLTM plus Western Blotting
Detection Kit; GE Healthcare, Piscataway, NJ). The antibodies used
were JMJD2A (NB110-40585) from Novus Biologicals and -actin
(sc-8432) from Santa Cruz Biotechnology.

TF motifs were analysed using enriched groups of 700 base pair (bp)
sequences around promoter regions (
500otranscription start site
(TSS)o200) and Amadeus PBM v1.0 software. Sequences from the
Ensembl project included 3 kb upstream of the TSS and the rst two
exons and introns. Sequence databases and analysis programs were
from Ron Shamir's computational genomics group at Tel Aviv
University (http://acgt.cs.tau.ac.il/). Known TF motif data from the
TRANScriptionFACtor (TRANSFAC) database were used. Data were
analysed as described previously [30].

Statistical analysis
Statistical analysis used SPSS 17.0 (SPSS Inc., IL, and USA). Data
were tested by one-way ANOVA followed by Tukey's HSD post hoc
test. nPo0.05 and nnPo0.001 were considered signicant.

E XP ER I ME NTAL C E LL RE S E ARCH

Results
Effect of LPS on cell viability and NO induction
The effect of LPS (1 mg/ml) on NE-4Cs was investigated after incubation from 2 h to 48 h. LPS treatment resulted in a signicant reduction
in MTT absorbance and NE-4Cs viability after 24 h, 36 h, and 48 h.
However, LPS treatment of NE-4Cs for 24 h or less did not result in
severe cell death (Fig. 1A). LPS treatment resulted in a multiple-fold,
time-dependent increase in NO production compared with controls
(Fig. 1B).

LPS-induced specic gene expression (histone

demethylase, cell cycle, transcription factor, and
inammatory response-related genes)
To identify genes controlled by histone demethylases, cell cycle, or
inammation in NE-4Cs, we performed expression analysis of NE-4Cs
treated with LPS for 224 h and compared the results with the
expression in untreated cells under normal culture conditions. We
found signicant, time-dependent up-regulation of JMJD2A mRNA
with up to 10 h of LPS treatment. However, other jumonji domaincontaining genes were not signicantly induced, except for a slight
increase in JMJD2B mRNA after LPS treatment (Fig. 2A). We also
examined the expression of cell cycle-response and inammatory
response-related genes in NE-4Cs after short-term LPS exposure and
observed that mRNA levels were markedly and time-dependently
increased except for the 12 and 24 h time points (data not shown).
Genes related to inammatory response that were up-regulated by
LPS treatment of NE-4Cs included interleukin 6 (Il6), and Il6r. Cell
cycle genes up-regulated by LPS in NE-4Cs included Ccnd2, Ccnd1,
and Cdk6, and TF genes were p65, cREL (dominant NF-B gene), and
Stat3 (Fig. 2B) as assessed by qRT-PCR.

Silencing JMJD2A and its effect on other key genes

NE-4C cells and primary NSCs were treated with JMJD2A-siRNA at
10 and 25 nM with or without LPS for 48 h. Treatment with 25 nM

328 (2 014 ) 361 37 8

365

had the highest silencing activity. Expression after silencing was

measured by real-time RT-PCR and Western blot (Fig. 3AC).
JMJD2A mRNA expression was signicantly reduced in cells treated
with siRNA (25 nM), which had expression levels of less than 12% of
JMJD2A levels in NE-4C cells. Since mRNAs are less stable than
proteins [31], we predict that treatment of LPS 10 h the JMJD2A
protein is stable when we knock-down JMJD2A. Nevertheless, in the
presence of chronic LPS exposures we need further study to actually
see a change in the JMJD2A protein levels with JMJD2A siRNA and
their target proteins as well. In NSCs treatment with 25 nM
JMJD2A-siRNA had the signicant silencing activity less than 20%
of JMJD2A mRNA levels (data not shown). There were no observed
differences in cell viability after JMJD2A (Fig. 3D) or JMJD2B
silencing by siRNA (data not shown). Importantly, we also found
that JMJD2A attenuation inhibited the gene expression of Ccnd1,
Stat3 and p65 (Fig. 4A). Interestingly, some of the tumoursuppressor genes, such as Gadd45gip1 and Dapk3, were signicantly up-regulated in JMJD2A-kd NE-4C cells (Fig. 4B). These genes
are involved in cellular processes such as apoptosis, cell cycle,
proliferation, migration, transcription, and inammatory disease
progression. However, these genes were not affected when the
JMJD2A paralogue JMJD2B was silenced (Supplementary Figs. S1
and S2).

Silencing of JMJD2A induces in G2/M and S phase arrest

We next investigated the effects of JMJD2A depletion on the cell
cycle distribution as analysed by ow cytometry. Histograms of
ow cytometric data are shown in (Fig. 5). The results show that
in comparison with the control group, JMJD2A depleted NE-4C
cells signicantly (nPo0.01) increased cell population at the G2
and S phase accompanied with the decreased G0/G1 phase cell
populations, which suggested that the cell cycles were arrested at
G2 and S phase induced by JMJD2A. The amount of cells in the S
phase increased from 45% in untreated cells to 54%, 50% and 48%
in JMJD2A depleted NE-4C, LPS 2 h treated JMJD2A depleted
NE-4C and LPS 10 h treated JMJD2A depleted NE-4C cells respectively and G2 phase increased from 13% in untreated cells to 22%
in JMJD2A depleted NE-4C cells. However it should be noted that

Fig. 1 Effects of LPS on cell viability and NO production in NE-4C cells. (A) NE-4C cells were treated with LPS (1 lg/ml) for 248 h.
Cell viability was determined using a WST assay. Before 24 h, LPS had a negligible effect on cell viability; after 24 h, LPS resulted in
cell death. (B) NO production determined using a Griess reagent assay kit on cells treated with LPS (1 lg/ml) for the indicated
times. Data represent three independent experiments, *Po0.05, compared with untreated and LPS-treated cells. Values are
mean7SD of triplicate wells.

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3 28 (2014) 3 61 37 8

genes were up-regulated. Fold-change lters included the

requirement that genes were expressed at levels at least 150%
of controls for up regulated genes and less than 66% of controls
for down regulated genes. We generated a more stringent lists of
genes altered as a result of JMJD2A silencing (Tables 1 and 2).
Down-regulated genes included genes encoding regulators of the
cell cycle, proliferation, differentiation, adhesion, migration, the
immune response, and transcription, including structural maintenance of chromosomes 2 (Smc2), N (alpha)-acetyltransferase 15,
NatA auxiliary subunit (Naa15), Ncor1, pleckstrin homology
domain-interacting protein (Phip), chromo domain helicase DNA
binding protein 4 (Chd4), Crebbp, Ccnd2, Ccnd1, Il20, Stat3, and a
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue (Kras).
Up-regulated transcripts in JMJD2A-kd NE-4Cs included tumoursuppressor genes such as Dapk3, Gadd45gip1, Timp2, Aen and
Vhl. Analysis of differentially expressed genes after 2 h and 10 h of
LPS of JMJD2A-kd NE-4Cs found that in the presence of LPS, genes
were signicantly expressed compared with untreated JMJD2A-kd
NE-4C cells (Tables 1 and 2).

Correlation and cluster analysis of differentially expressed

genes

Fig. 2 Induction of histone demethylase (JMJD2A), cell cycle,

transcription factor, and inammatory response-related genes
in LPS-stimulated NE-4C cells. (A) Quantitative real-time
reverse transcriptase-PCR analysis of expression of JmjC genes
in NE-4C cells stimulated with LPS (1 lg/ml). JMJD2A was
signicantly up-regulated (417-fold) in cells treated with LPS
compared to untreated cells (*Po0.05 and **Po0.001). (B) Cell
cycle (Ccnd1, Ccnd2, Cdk6), transcription factor (p65, cREL,
Stat3), and inammatory response-related (Il6, Il6r) genes
were signicantly up-regulated in LPS-stimulated NE-4C cells
at the indicated times. Gene expression was normalised to
GAPDH transcript levels. Data represent three independent
experiments. Values are mean7SD of triplicate wells.

cells were not arrested at G2 phase in LPS 2 h and LPS 10 h treated

JMJD2A depleted NE-4C cells from the control.

Global analysis of expression differences in JMJD2Asilenced NE-4Cs

Heat map correlation and hierarchical clustering analysis were

performed with GPlot Software for the following three different
comparisons: untreated control samples vs. JMJD2A-kd NE-4Cs,
untreated control samples vs. 2 h LPS-treated JMJD2A-kd NE-4Cs,
and untreated control samples vs. 10 h LPS-treated JMJD2A-kd
NE-4Cs. The hierarchical clustering analysis showed that the
genes expressed in JMJD2A-kd NE-4Cs were signicantly different
from the genes expressed in control NE-4Cs. Interestingly, in the
presence of LPS, JMJD2A-kd NE-4Cs demonstrated a signicant
change in the gene expression pattern when compared with
untreated JMJD2A-kd NE-4Cs (Fig. 6A). Differentially expressed
genes (fold-change cutoff of 1.5) were functionally analysed using
DAVID Informatics Resources by classication into GO categories
(FDR 0.05) by the biological process (BP) and molecular functions
(MF) categories and into KEGG (Kyoto Encyclopaedia of Genes and
Genomes) pathways in JMJD2A-kd NE-4Cs. Genes that were
differentially expressed in JMJD2A-kd NE-4Cs were involved in
several BPs and MFs. We observed that the largest groups of 86
genes were involved in the regulation of gene expression and
cellular biosynthetic processes. Other pathways, such as the
regulation of transcription (76 genes), regulation of programmed
cell death (34 genes), regulation of apoptosis (34 genes), chromatin modication (27 genes) and M phase of mitotic cell cycle
(15 genes) were also identied in the analysis of differentially
expressed genes in JMJD2A-kd NE-4Cs (Fig. 6C).

Pathways affected by JMJD2A silencing

JMJD2A is widely expressed in human tissues and cell lines, and
high endogenous expression of JMJD2A mRNA is found in
numerous cell types, including human T-cell lymphotropic virus
1-infected cell lines, the HT1376 bladder carcinoma cell line, the
U2OS osteosarcoma cell line and a prostate cancer cell line [7,8].
We used silencing to modify mRNA patterns in cells to identify
novel JMJD2A therapeutic targets. Microarray analysis revealed
differentially expressed genes in JMJD2A-kd NE-4Cs and LPStreated JMJD2A-kd NE-4Cs. In total, 221 annotated genes were
differentially expressed; 192 genes were down-regulated, and 29

To gain further insights into the function of JMJD2A, we performed IPA to identify gene networks that were highly enriched
by genes that are positively regulated by JMJD2A. These pathways
have the potential to dene molecular targets associated with
JMJD2A-attenuated p53
/
NE-4Cs. Molecular networks characterised by IPA analysis showed that JMJD2A-attenuated p53
/

NE-4Cs down-regulated G2e3, Ccnd2, Cdk6, Crebbp, nuclear receptor
co-repressor 1 (Ncor1), TGF-beta-activated kinase 1 (Tab2), and ralA
binding protein 1 (Ralbp1) and up-regulated Dapk3, Gadd45gip1,

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328 (2 014 ) 361 37 8

367

Fig. 3 Silencing of JMJD2A in LPS-stimulated NE-4C cells. NE-4C cells were transiently transfected with JMJD2A siRNA or nontargeting siRNA (NC-siRNA). (A, C) Real-time RT-PCR was performed when JMJD2A expression was knocked down by approximately
90% with 25 nm JMJD2A-siRNA. **Po0.001 compared with NC-siRNA-treated cells. (B) Western blot for JMJD2A after treatment
with 25 nm JMJD2A-kd siRNA. (D) NE-4C cells were treated with different concentrations of JMJD2A-siRNA. Cell viability was
determined using a WST assay. Concentrations of 10 nM or 25 nM JMJD2A-siRNA either presence or absence of LPS had a negligible
effect on cell viability.

large tumour suppressor 2 (Lats2), SIVA1, apoptosis-inducing factor

(SIVA1). Genes with altered expression patterns were involved in the
cell cycle, proliferation, adhesion, migration, transcription, and apoptosis (Fig. 7A and B; Supplementary Figs. S3 and S4). IPA analysis
showed that JMJD2A kd mainly affected genes responsible for the cell
cycle; cellular assembly and organisation; DNA replication, recombination, and repair; cellular movement; cell-to-cell signalling and
interaction; cell death and survival; post-translational modication;
and cancer (Fig. 7C). Interestingly, genes enriched in JMJD2A-kd NE4Cs were in networks; NF-kB, Smarca4, Ccnd2, Crebbp, and Stat3
formed the central node of an interconnected regulatory system
(Fig. 7A and B; Supplementary Figs. S3 and S4). Among these genes
were NF-kB target genes such as Timp2, Tab2, and Smarca4 and
Ccnd2 target genes such as Phip, Rho-associated coiled-coil containing protein kinase 1 (Rock1), Cdk6, and S-phase kinase-associated
protein 2 (skp2), which are implicated in tumour progression. The
IPA-identied biological networks and pathways that were most

involved in JMJD2A-kd NE-4Cs complemented the ndings from

Pathway Express. The most signicant canonical pathways were
involved in the molecular mechanism of cancer (Fig. 7D). Other
notable pathways include the cell cycle, G1/S checkpoint regulation, acute phase response signalling, and cyclins and cell cycle
regulation in JMJD2A-kd NE-4Cs cells. The top ve molecular and
cellular functions and functional annotations identied for JMJD2Akd NE-4Cs are shown in Tables S1 and S2.

Expression and regulation of transcription factor motifs in

JMJD2A-kd NE4Cs cells
We carried out TF motif analysis of promoter regions (
500o
TTSo200) for differentially expressed genes in JMJD2A-kd
NE-4Cs. Many TFs bind close to the TSS of target genes as opposed
to distant promoter regions [32]. Some elements that directly

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3 28 (2014) 3 61 37 8

Fig. 4 Silencing effects of JMJD2A on key genes in LPS-stimulated NE-4C cells. (A) Effect of JMJD2A attenuation on cell cycle,
transcription factor, and inammatory response-related genes after 2 h and 10 h of exposure of JMJD2A-kd NE-4C cells to LPS. (B)
Attenuation of JMJD2A signicantly increased tumour-suppressor gene expression. Gene expression was normalised to GAPDH
transcript levels.*Po0.05 and **Po0.001 compared with NC-siRNA-treated cells. Data represent three independent experiments.

Fig. 5 JMJD2A induces G2/M and S phase arrest in NE-4C cells. (A) NC-siRNA, or (B)) LPS treated NC-siRNA, (C) JMJD2A si-RNA, (D)
LPS 2 h treated JMJD2A si-RNA and (E) LPS 10 h treated JMJD2A si-RNA for 48 h and were collected by centrifugation and stained by
PI, (F) cell cycle distribution in percentages of the different groups. The DNA contents of the cells were determined with the Aria
FACS ow cytometry system and cell cycle distribution was analysed with ModFit LT 3.0 software (Verity Software House, Topsham,
ME, USA). Data were obtained by ow cytometry and presented as mean (n 3).

Table 1 Most down-regulated genes in JMJD2A-kd NE-4C cells.

LPS-2 h JMJD2A-kd/NC
intensity

LPS-10 h JMJD2A-kd/NC
intensity

Function (biological process)

NM_001185020
NM_008017
NM_010123
NM_053089
NM_011308
NM_001081216
NM_145979
NM_175238
NM_133780
NM_001080773
NM_001025432
NM_009211
NM_009706
NM_001110162
NM_053124
NM_001025566
NM_001174078
NM_001015099
NM_009847
NM_008702
NM_177224
NM_008448
NM_011263
NM_009071
NM_001037726

Wnk1
Smc2
Eif3a
Naa15
Ncor1
Phip
Chd4
Rif1
Tpr
Pdpk1
Crebbp
Smarcc1
Arhgap5
Cdca2
Smarca5
Chka
Smarca4
G2e3
Cd2ap
Nlk
Chd9
Kif5b
Rest
Rock1
Creb1

0.14615

0.16559

0.19847

0.21468

0.26440

0.26655

0.27007

0.27273

0.28695

0.28958

0.31094

0.32080

0.33714

0.33836

0.35346

0.361

0.3632

0.36335

0.36419

0.39913

0.40579

0.41105

0.41206

0.41676

0.43192

0.41352

0.58641

0.69007

0.80181

0.84392

0.96564

0.67370

0.72441

0.59911

0.88062

0.68157

0.50635

0.74139

0.91612

0.78965

0.57172

0.810

0.93733

0.74249

0.90576

0.74573

0.87528

0.70831

0.83462

0.88344

0.56977

0.41431

0.30259

0.42154

0.57386

0.74168

0.65747

0.39551

0.66386

0.48711

0.82894

0.59179

0.42814

0.61014

0.61613

0.44828

0.771

0.65323

0.50297

0.78929

0.53672

0.62314

0.62821

0.61213

0.68645

NM_009829
NM_010638
NM_007658
NM_026201
NM_010615
NM_001164057
NM_009067
NM_019827

Ccnd2
Klf9
Cdc25a
Ccar1
Kif11
Pola2
Ralbp1
Gsk3b

0.44694

0.45957

0.46172

0.46660

0.47872

0.47977

0.48117

0.50673

0.96142

0.81515

0.84971

0.88302

0.86071

0.76404

0.61629

0.97062

0.80812

0.63641

0.84723

0.69018

0.55668

0.65608

0.64589

0.90810

NM_010233
NM_145436
NM_013787
NM_021380
NM_007631
NM_010559
NM_026484
NM_008443
NM_053123
NM_138667

Fn1
Cdc27
Skp2
Il20
Ccnd1
Il6r
Ccny
Kif3a
Smarca1
Tab2

0.50878

0.53608

0.53935

0.54365

0.54959

0.57457

0.57843

0.57981

0.58516

0.59166

0.6482

0.95624

0.94733

0.78503

0.79056

0.68252

0.84954

0.76305

0.95350

0.94889

0.94010

0.68225

0.54968

0.66772

0.58342

0.79929

0.63854

0.56109

0.53537

0.96383

Signal transduction, transport

Cell cycle
Translation
Cell differentiation, transcription
Cell growth, homoeostasis, transcription
Apoptosis, cell cycle, cell proliferation
Transcription
Cell cycle, response to stress
Cell cycle, immune response, translation
Cell adhesion, signal transduction
Cell migration, transcription
Signal transduction, transcription
Cell migration, signal transduction
Cell cycle
Response to stress, transcription
Lipid metabolism
Cell cycle, growth
Apoptosis
Cell adhesion, cell cycle cell, migration
Signal transduction, transcription
Transcription
Transport
Cell differentiation, transcription
Apoptosis, cell adhesion, cell migration inammatory response
Apoptosis, cell differentiation, growth signal transduction,
transcription
Cell cycle, cell proliferation, cell differentiation,
Signal transduction, transcription
Cell cycle
Cell cycle, transcription
Cell differentiation, transport
Transport
Cell cycle, cell proliferation, signal transduction, transport
Apoptosis, cell cycle, cell differentiation, cell migration, cell
proliferation
Immune response, response to stress, apoptosis, cell adhesion
Cell cycle
Cell cycle, apoptosis, cell proliferation
Immune response, signal transduction
Cell cycle, cell proliferation, cell differentiation, signal transduction
Immune response, signal transduction, apoptosis, cell proliferation
Cell cycle, signal transduction
Signal transduction
Transcription, cell differentiation
Signal transduction

369

JMJD2A- kd/NC
intensity

328 (2 014 ) 361 37 8

Gene
symbol

E XP ER I ME NTAL C E LL RE S E ARCH

Gene
accession_ID

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Table 1 (continued )
Gene
symbol

JMJD2A- kd/NC
intensity

LPS-2 h JMJD2A-kd/NC
intensity

LPS-10 h JMJD2A-kd/NC
intensity

Function (biological process)

NM_001039079
NM_033563
NM_009873
NM_013672
NM_152810
NM_009238

Prkcz
Klf7
Cdk6
Sp1
Cdc5l
Sox4

0.59395

0.59868

0.60179

0.60569

0.60832

0.62976

0.75215

0.77259

0.65138

0.97869

0.79906

0.80025

0.66053

0.67713

0.81848

0.63303

0.63651

0.70563

NM_021284
NM_011486
NM_009424
NM_023243

Kras
Stat3
Traf6
Ccnh

0.64052

0.64158

0.68114

0.68962

0.74731

0.69883

0.66200

0.82832

0.70304

0.69299

0.91441

0.56400

Apoptosis, cell proliferation, cell adhesion

Cell differentiation, cell migration
Cell adhesion, cell cycle, cell proliferation, cell differentiation
Transcription, cell differentiation
Cell cycle, transcription
Cell differentiation, cell proliferation, signal transduction,
transcription
Apoptosis, cell adhesion, cell cycle, transcription
Immune response, homoeostasis, growth
Apoptosis, cell differentiation, cell proliferation
Cell cycle

Gene
accession_ID

Gene
symbol

JMJD2A- kd/NC
intensity

LPS-2 h JMJD2A-kd/NC
intensity

LPS-10 h JMJD2A-kd/NC
intensity

Function (biological process)

NM_023524
NM_007523
NM_001190473
NM_183358
NM_011594
NM_007544
NM_011349
NM_001161737
NM_001162939
NM_001177319
NM_019676
NM_009507
NM_007909
NM_011348
NM_013657

Tfpt
Bak1
Dapk3
Gadd45gip1
Timp2
Bid
Sema3f
Siva1
Aen
Tfpi
Plcd1
Vhl
Efna2
Sema3e
Sema3c

3.172515
2.333912
2.068146
1.856187
1.739395
1.683675
1.668045
1.63806
1.584418
1.57019
1.566998
1.535595
1.521655
1.531078
1.525556

1.58484
1.097543
1.317619
1.062044
1.509739
1.251268
1.436322
1.062275
1.223225
1.071396
1.116439
1.119968
1.264369
1.501091
1.508277

1.145183
1.676165
1.359414
1.149264
1.086614
1.498087
1.515213
1.302107
1.68561
2.302508
1.487532
1.437071
1.626428
2.744747
2.252428

Apoptosis
Apoptosis, cell proliferation, homoeostasis, response to stress
Apoptosis, cell differentiation, signal transduction
Cell cycle
Cell cycle, cell differentiation, cell proliferation
Apoptosis, transport
Cell migration, signal transduction
Apoptosis, homoeostasis
Apoptosis, response to stress
Response to stress
Cell proliferation, homoeostasis, signal transduction
Cell differentiation, transcription
Cell differentiation, cell migration
Cell differentiation, signal transduction
Cell differentiation, cell migration

3 28 (2014) 3 61 37 8

Table 2 Most up-regulated genes in JMJD2A-kd NE-4C cells.

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Gene
accession_ID

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371

Table 3 List of primers used in q-RT-PCR studies.

Gene designation
JMJD1A
JMJD1B
JMJD1C
JMJD2C
JMJD3
JMJD4
JMJD5
JMJD2D
JMJD2B
JMJD2A
Il6
Il6ra
Ccnd1
Ccnd2
Cdk6
cREL
Stat3
p65
Gadd45g
Dapk3
Cdca2
Crebbp
Rest
Tipm2
Tfpi
Gapdh

Forward sequence (5'-3')

Reverse sequence (5'-3')

CACATTTAGGTTCCCAGTCACA
TTCTGCTGGAAGGCTCACTT
AGAAGAGAAAGGCGAGGTC
CACGGAGGACATGGATCTCT
CCCCCATTTCAGCTGACTAA
CTCAAGGACTGGCATCTGTG
TGTCATGTTAGAGCGGATGG
TGGGAAGACTCAACAGTGGAA
GGCTTTAACTGCGCTGAGTC
GACCACACTCTGCCCACAC
TAGTCCTTCCTACCCCAATTTCC
GCCACCGTTACCCTGATTTG
CCATGACTCCCCACGATTTC
GCAGAAGGACATCCAACCGT
TCTCACAGAGTAGTGCATCGT
AGAGGGGAATGCGGTTTAGAT
CACCTTGGATTGAGAGTCAAGAC
AGGCTTCTGGGCCTTATGTG
TTTCACGTTGATTCAGGCGTT
ACATTCAGGCAAGAGGATGTTG
GTTCACACGACAAGCCTCTCT
TTCTCCGCGAATGACAACACA
GGCAGATGGCCGAATTGATG
GCAACCCCATCAAGAGGATTC
GGGCCACTGTGTGTCTGTT
TGCGACTTCAACAGCAACTC

GCCACGATGTTAACACAGGA
GATGCATCCCATTAGCATCC
TTGGGACCTATCTCACAGCA
CGAAGGGAATGCCATACTTC
CTGGACCAAGGGGTGTGTT
CTGAGGAGCGGAAGATGTC
TGTACCTTGAGCCCACTTCC
CCCTCAATCACTATGCCACATT
GTGTGGTCCAGCACTGTGAG
TCCTGGGGTATTTCCAGACA
TTGGTCCTTAGCCACTCCTTC
TCCTGTGGTAGTCCATTCTCTG
GCTACCATGGAGGGTGGGTT
ATGGGTCTTAGGAGTCGGGAC
CGAGGTAAGGGCCATCTGAAAA
CTACCTGCTGATCGCCCTTC
AGGAATCGGCTATATTGCTGGT
TGCTTCTCTCGCCAGGAATAC
AAATGAGGATGCAATGCAGGT
CTCACCTCGCGTTCGATCT
GGAGTCACAAACGGTTCAGTT
CCTGGGTTGATGCTAGAGCC
CTTTGAGGTCAGCCGACTCT
GGGGCCGTGTAGATAAACTCG
GCACAAAATGTATGTAGCGGTTT
CTTGCTCAGTGTCCTTGCTG

interact with or are part of the basal transcriptional machinery,

such as the TATA box and initiator sequences, are found mainly in
core promoters, spanning several dozen bases around the TSS
[33]. We used a localisation score that measures the tendency of a
motif to occur at specic locations along promoters (
500oTTS
o200). Applying this score to the promoters of differentially
expressed genes revealed binding sites of known TFs including
CDC5, MZF1, CP2, and TRAB1 and prominent TFs (e.g., MYC, CREB),
as well as three novel motifs (Fig. 8).

Conrmation of differentially expressed genes by q-RT-PCR

To conrm the microarray and pathway analysis results, we
measured differentially expressed genes by real-time qRT-PCR
for the same sample used for microarray studies. To measure gene
expression, mRNA was reverse transcribed into cDNA using Prime
Script TM Reverse Transcriptase (Takara Bio Inc., Shiga, Japan).
In total, 68 genes were selected from the list of differentially
expressed genes and key pathways affected by JMJD2A-kd NE4Cs. Among the analysed genes, we conrmed the downregulation of 6 genes and the up-regulation of 2. We focused on
IPA-identied genes because of the known or presumed functions
of their pathways in tumour development (Fig. 9A and B). To
conrm the JMJD2A effect on gene regulation in primary NSCs, we
incubated NSCs under inammatory conditions (LPS 1 mg/ml),
which induced inammatory genes with effective p53 inhibitor
PFT- (data not shown). More importantly, JMJD2A attenuation
signicantly down-regulated Cdca2, Ccnd2, Crebbp, and Rest in
NSCs with the presence of PFT- and LPS (Fig. 9C and D). However
it should be noted that il6ra gene was not affected by silencing of
the JMJD2A.

Discussion
A previous study reported that tumour suppressor pathways
regulated by p53 are involved in MB and supratentorial neuroectodermal tumour development; both tumours occur in families
with constitutional p53 defects [13]. Brain tumour development
in mice can begin with a p53 knockout or mutation of the p53
gene, which increases NSC proliferation and self-renewal capacity
and reduces apoptosis [15,16]. Here, we used p53
/
NE-4Cs to
study the involvement of JMJD2A in regulating the expression of
tumourigenic inammatory genes. We determined whether
changes in gene expression affect the cell cycle, proliferation,
adhesion, migration, transcription, and apoptosis as well as
signalling pathways that involve NF-B, cell cycle regulators,
and other transcription factors.
We found that JMJD2A attenuation signicantly downregulated Cdca2, Ccnd2, Cdk6, Ccnd1, Ccny, G2e3, cell division
cycle and apoptosis regulator 1 (Ccar1), S-phase kinase-associated
protein 2 (Skp2), and cyclin H (Ccnh) in p53
/
NE-4Cs in the
presence or absence of LPS (Table 1). These results were consistent with the observations of Krasnoselsky et al. [34] on Cdca2, a
well-known cell cycle-related protein that is over-expressed in
aggressive neuroblastoma tumours and melanoma cell lines.
Recently, Uchida et al. [35] reported that attenuation of CDCA2
signicantly inhibits cellular proliferation and promotes apoptosis
by arresting the cell cycle at the G1 phase in oral squamous cell
carcinoma. Ccnd1 and Ccnd2 are important in proliferation in vivo
and in vitro. Recently, Koyama-Nasu et al. [36] determined a
specic role for cyclin D2 in cell cycle progression and the
tumourigenicity of glial stem cells. In addition, Li et al. [11]

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3 28 (2014) 3 61 37 8

Fig. 6 Hierarchical cluster analysis and functional annotation of differentially expressed genes. (A) Hierarchical cluster analysis of
differentially expressed genes in groups. (B) Electropherogram of RNA samples on Agilent 2100 Bioanalyser. The determination of RNA
quality is based on the separation of RNA samples by gel electrophoresis. Each sample is separated into two distinct bands (18S and 28S
subunits), and the data are detected through a laser-uorescent beam and translated into a gel-like image (bands). (C) Functional
annotation of JMJD2A-associated genes. Analysis of GO term enrichment for the biological process category of JMJD2A-associated genes.
The top GO terms are ranked by the number of counts. The X-axis shows the number of genes in each functional category.

Fig. 7 Effect of JMJD2A-kd on NF-kB signalling and CREBBP-signalling networks. (A, B) Ingenuitys Bioinformatics pathway analysis of
gene network with connections to NF-kB, CREBBP and differentially expressed genes in JMJD2A-kd NE-4C cells. Symbols for genes in
specic categories and interactive relationships are depicted in the legend. Node colour intensity indicates the degree of up-regulation
(red) or down-regulation (green). (C) IPA of the top gene networks enriched in differentially expressed genes after JMJD2A depletion in
NE-4C cells. (D) The most highly represented canonical pathways of genes differentially expressed in JMJD2A-kd NE-4C cells. Line, ratio of
the number of genes represented in each pathway to total genes in the pathway; a, b, c, d, e, f, g, h, I, and j indicate molecular mechanism
of cancer, prolactin signalling, prostate cancer signalling, role of macrophages, broblasts, and endothelial cells in rheumatoid arthritis,
cell cycle:G1/S checkpoint regulation, glucocorticoid receptor signalling, mouse embryonic stem cell pluripotency, acute phase response
signalling, NGF signalling, and cyclins and cell cycle regulation, respectively.

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Fig. 8 Transcription factor-motif analysis. Amadeus was used in

a de novo search of promoters of differentially expressed genes
in JMJD2A-kd NE-4C cells for motifs that were overrepresented at
a particular distance from the TSS. Promoters were considered to
be the 700 bp sequences around promoter regions (
500oTSS
o200). Both known and novel motifs were found. Sequences
from the Ensembl project include 3 kb upstream of the TSS and
the rst two exons and introns.

demonstrated that attenuation of CDK6 or CCND1 decreased pRB

phosphorylation and impaired cell cycle progression in both
medulloblastoma and supratentorial neuroectodermal tumour
cell lines. It has been reported that attenuation of Ccny inhibits
cell proliferation, colony formation, and cell cycle progression in
glioma cells [12]. Skp2 belongs to the F-box family of proteins and
is required for G1S transition. Down-regulation of Skp2 using
siRNA induces p27 and causes growth arrest and apoptosis in
T98G glioblastoma cells [37]. In this study, we also performed cell
cycle distribution as analysed by ow cytometry. Our data
demonstrated that cell cycles were arrested at G2 and S phases
in JMJD2A depleted NE-4C cells compared to the control (Fig. 5).
Based on these results and those from our study, we propose that
down-regulation of JMJD2A inhibited cell proliferation and
tumourigenicity as well as promoted apoptosis. Therefore, JMJD2A
could be a target for tumour suppression in p53
/
NE-4Cs.
However, the mechanism by which JMJD2A attenuation suppresses key cell cycle regulatory genes requires further study.
Microarray analysis revealed that Il6r and Stat3 were signicantly down-regulated in JMJD2A-attenuated p53
/
NE-4Cs. IL-6
is a pleiotropic cytokine primarily implicated in regulating the
immune and inammatory responses, and it is extensively
expressed by a variety of malignant tumours, including prostate,
breast, lung cancer and glioblastoma [38]. A number of IL-6related signalling pathways are connected with increased proliferation, migration and invasion of tumour cells. IL-6 binds to an
IL6r and triggers dimerisation of a signal-transducing receptor,
leading to the activation of signal transduction pathways such as
the STAT, Ras-MAPK and PI-3 kinase pathways [39]. The STAT3
signalling pathway is one of the largest studied cytokine

3 28 (2014) 3 61 37 8

signalling systems. Stat3, a key cytoplasmic transcription factor

involved in inammation, is activated in response to a variety of
cytokines, chemokines and growth factors. Stat3 is constitutively
active in human malignancies, including breast and pancreatic
cancer and glioblastoma [40]. Activation of Stat3 is linked to
clinically more aggressive glioblastoma phenotypes [41]. Constitutive Stat3 signalling contributes to cancer development by
regulating target genes involved in the regulation of critical
cellular processes that affect the genesis of glioma such as
promoting cell cycle progression, angiogenesis, cell migration/
invasion and immune evasion, and the prevention of apoptosis
[40]. Moreover, Ccnd1 is transcriptionally regulated by Stat3 and
is usually deregulated in various cancers [42]. Therefore, Stat3 can
be considered an oncogenic transcription factor because it promotes malignancy. In this study, we showed that JMJD2A attenuation signicantly down-regulated Il6r and Stat3 and their target
gene Ccnd1. Thus, down-regulation of Il6r, Stat3 and Ccnd1
through JMJD2A attenuation could inhibit tumourigenesis as well
as brain inammation in p53
/
NE-4Cs.
We found that siRNA-mediated reduction of JMJD2A downregulated CREB, a SWI/SNF-related, matrix-associated, actindependent regulator of chromatin, as well as subfamily a member
4 (SMARCA4) and the REST, TRAF6, and NF-kB genes (p65 and
cREL) in p53
/
NE4Cs. CREB is a nuclear transcription factor that
is over-expressed in several neoplastic cancers. CREB1 is an
oncogene involved in the proliferation, survival, and metastasis
of tumour cells [43]. Recently, Peng et al. [44] reported that
ectopic expression of CREB1 attenuated growth suppression by
glioma cells. SMARCA4/BRG1 is observed in several human
cancers including brain tumours [45]. Further investigation
revealed that attenuation of SMARCA4 in glioma cell lines inhibits
cell growth due to G1-phase arrest by down-regulation of Ccnd1
[46]. REST is a transcriptional repressor that regulates the
expression of approximately 2000 neuronal genes in neural and
non-neural tissues, including embryonic and NSCs [47]. In neoplasia, heightened REST function in medulloblastoma tumour
cells contributes to their tumourigenicity by preventing their
differentiation in mouse models of the disease [48]. In addition,
Conti et al. [49] reported that REST is highly expressed in selfrenewing tumourigenic-competent glioblastoma multiform cells,
and attenuation strongly reduces self-renewal in vitro and
tumour-initiating capacity in vivo. TRAF6 possesses a unique
receptor-binding specicity as the signalling mediator for the
TNF receptor super family and TLR super family induced by NF-B
activation. The NF-B TF is important in inammation, immune
response, cellular proliferation, apoptosis, tumourigenesis, and
invasion [50]. Previous studies demonstrated that constitutive
activation of NF-B is an important regulator of genes involved in
tumourigenesis, invasion, and migration. Inhibition of NF-B
activation restrains invasion and migration [51]. Tao et al. [51]
reported that TRAF6 might be important in tumourigenesis,
metastasis, and invasion by suppressing NF-B activation. Furthermore, Peng et al. [52] reported that kd of TRAF6 promoted glioma
cell apoptosis via the NF-B pathway. Therefore, the coordinated
reduction of these genes after JMJD2A silencing suggested that
JMJD2A regulated-molecular circuitries could be targets for
therapeutic intervention in tumourigenic development. Finally,
real-time RT-PCR analysis of Ccnd2, Ccnd1, Il6r, Stat3, Cdk6,
Cdca2, and REST (Figs. 4A and 9A) illustrated a decrease in the
above- mentioned mRNA in JMJD2A-kd NE-4C cells compared to

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328 (2 014 ) 361 37 8

375

Fig. 9 Conrmation of differentially expressed genes by quantitative reverse transcription-polymerase chain reaction. (A) Crebbp,
Cdca2, Rest, Ccnd2, IL6R and Cdk6 genes were signicantly down-regulated in JMJD2A-kd NE-4C cells. (B) Timp2 and Tfpi were
up-regulated in JMJD2A-kd NE-4C cells. (C, D) Crebbp, Cdca2, Rest, Ccnd2, and Cdk6 genes were signicantly down-regulated and
Timp2 and Tfpi were up-regulated in JMJD2A-kd NSC cells with the presence of PFT- (30 lM) and LPS. Gene expression was
normalised to GAPDH transcript levels. *Po0.05 and **Po0.001 compared with NC-siRNA-treated cells. Data represent three
independent experiments.

NC-siRNA NE-4Cs. Furthermore JMJD2A down-regulates above mentioned genes in primary NSCs, under inammatory conditions (LPS
1 mg/ml) with effective p53 inhibitor PFT- (Fig. 9C and D).
Dapk3, which is an important regulator of cellular processes
including apoptosis, adherence, and cell cycle progression and
proliferation [53], was induced in JMJD2A-kd NE-4Cs (Table 2).
DAPK3 is suggested to be a tumour suppressor and is a proapoptotic protein [53]. In addition, Brognard et al. [54] reported
that mutation of DAPK3 promotes increased cell survival, proliferation, aggregation and resistance to chemotherapy. Thus, upregulation of Dapk3 in JMJD2A-kd NE-4Cs might affect tumour
suppression in JMJD2A-kd NE-4Cs. Another signicantly up-

regulated gene in JMJD2A-kd NE-4C cells was Timp2, an endogenous and bi-functional inhibitor of angiogenesis that mediates
transcription, pro-enzyme activation, and Timp2 inhibition, inhibits matrix metalloproteinase activity and modulates proliferation
and apoptosis [55]. In addition, Hiraoka et al. [56] showed that
Timp2 inhibits tumour growth and invasion in murine models
and inhibits neoangiogenesis in collagen and brin matrices.
Therefore, up-regulated Timp2 might inhibit tumour growth in
JMJD2A-kd NE-4Cs. We also observed that Tfpi dramatically
increased in JMJD2A-kd NE-4Cs. Tfpi is an endogenous inhibitor
that antagonises the activity of proteases such as plasmin, trypsin,
chymotrypsin and cathepsin G. Tfpi expression is inversely related

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to the degree of malignancy in several tumours. Methylation of

Tfpi promoter CpG islands is proposed to result in enhanced
invasiveness and tumour growth [57]. Furthermore, Tfpi might
exert a tumour suppressor function by inhibiting matrix metalloproteinases in glioma cell lines [58]. Thus, up-regulation of Tfpi in
JMJD2A-kd NE-4Cs could be involved in tumour suppression. The
up-regulation of DAPK3, Timp2, and Tfpi was validated in JMJD2Akd NE-4Cs by real-time RT-PCR (Figs. 4B and 9B).
To gain further insights into the function of JMJD2A, we performed IPA analysis and identied several gene networks, canonical
pathways, functional annotation and molecular and cellular functions in JMJD2A-kd NE-4Cs (Fig. 7AD; Supplementary Figs. S3 and
S4). IPA analysis showed that in JMJD2A-kd NE-4Cs, the affected
genes were mostly involved in the cell cycle, cellular assembly and
organisation, and DNA replication, recombination, and repair
(Fig. 7C). Genes enriched in cell cycle, movement, death and cancer
responses were in networks with an interconnected regulatory
system with NF-kB, Ccnd2, Crebbp, and Stat3 as a central node
(Fig. 7A and B; Supplementary Figs. S3 and S4). Down-regulation of
Ccnd2, Stat3 and NF-kB after JMJD2A-kd was further illustrated by
gene expression analysis (Figs. 4A and 9A). JMJD2A-kd NE-4Cs
down-regulated Smarca4, ralA binding protein 1 (Ralbp1), Ccnd2,
Cdk6, Ncor1, Crebbp, Il6r and Stat3 and up-regulated Timp2, Dapk3,
Gadd45gip1, Tfpi and other genes, which was signicant for the
establishment of JMJD2A-kd NE-4Cs. Among the up-regulated genes
were the NF-kB target gene SMARCA4, the Crebbp target gene
CREB1 and the Stat3 target gene Il6r, which have been implicated
in brain cancer oncogenesis [38,44,46]. Functional analysis of
differentially expressed genes in JMJD2A-kd NE-4Cs using a foldchange cutoff of 1.5 showed that the largest group of 86 genes were
involved in the regulation of gene expression and cellular biosynthetic processes. Analysis by DAVID informatics resources into KEGG
pathways showed that other pathways such as regulation of
transcription (76 genes), regulation of programmed cell death (34
genes), regulation of apoptosis (34 genes), chromatin modication
(27 genes) and the M phase of the mitotic cell cycle (15 genes)
pathways were also affected in JMJD2A-kd NE-4Cs (Fig. 6C). We also
performed TF motif analysis on JMJD2A-regulated genes. The
promoters of differentially expressed genes revealed binding sites
of known TFs including CDC5, MZF1, CP2, and TRAB1 and other TFs
such as MYC and CREB as well as three novel motifs in JMJD2A-kd
NE-4Cs (Fig. 8).
Overall, Gene Chips Mouse Genome 430_2.0 arrays, IPA, TF-motif
analysis and DAVID informatics resources analysis identied key
genes and signalling networks that may be responsible for suppressing genes involved in the cell cycle, proliferation, migration, and
inammatory disease response when JMJD2A was attenuated in
NE-4Cs. Although we found evidence for gene up- and downregulation through gene chip analysis, extensive in vivo experiments
using JMJD2A mutant or knockout mice would validate our
hypotheses.

Conclusion
In summary, we demonstrated that a wide spectrum of cellular
responses is induced in LPS-stimulated JMJD2A-kd NE-4Cs. Global
connections between JMJD2A with signalling pathways were established using IPA analysis, and novel JMJD2A targets were predicted.
The JMJD2A-kd NE-4Cs used in this study down-regulated genes

3 28 (2014) 3 61 37 8

such as Cdca2, Ccnd2, Ccnd1, Ccny, SMARCA4, Crebbp, IL6r, and

Stat3 in the presence or absence of LPS. This down-regulation might
inhibit the cell cycle, proliferation, migration, tumour formation and
inammatory cellular processes, and up-regulation of the tumour
suppressors Dapk3, Timp2, and Tfpi after JMJD2A attenuation is
important in tumourigenic development processes. Moreover,
TF-motif analysis identied binding patterns of known TFs including
CDC5, MYC, and CREB, as well as three novel motifs in JMJD2Aregulated genes in NE-4Cs. We conclude that the genes identied by
expression proling could be important regulators of tumourigenesis in JMJD2A-attenuated p53 null NE-4Cs.

Conict of interest
The authors declare that they have no conict of interest.

Author contributions
Conception and design: Amitabh Das, Nando Dulal Das.
Performed the experiments: Amitabh Das, Kyoung Hwa Jung,
Jin Choul Chai, Sung Chul Kang.
Wrote the paper: Amitabh Das.
Analysed the data: Amitabh Das, Young Seek Lee, Nando Dulal
Das, Hyemyung Seo.
Study supervision: Young Gyu Chai.

Acknowledgments
We would like to thank Hyung Tae Lee, Dalmuri Han, and Se Kye
Kim for their technical assistance. This work was supported by the
National Research Foundation of Korea (NRF) Grant funded by the
Korea government (MSIP) 2011-0030049.

Appendix A.

Supporting information

Supplementary data associated with this article can be found in

the online version at http://dx.doi.org/10.1016/j.yexcr.2014.08.029.

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