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abstract
Article Chronology:
tumours. The role of JMJD2A in tumour progression remains unclear. The objectives of this study
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
inammation in p53 / NE-4Cs and investigated whether the epigenetic modier JMJD2A alter the
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
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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E X PE R IM ENTA L C ELL R E S EA RC H
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
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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.
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E X PE R IM ENTA L C ELL R E S EA RC H
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.
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).
365
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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E X PE R IM ENTA L C ELL R E S EA RC H
3 28 (2014) 3 61 37 8
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,
E XP ER I ME NTAL C E LL RE S E ARCH
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.
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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).
LPS-10 h JMJD2A-kd/NC
intensity
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
369
JMJD2A- kd/NC
intensity
Gene
symbol
E XP ER I ME NTAL C E LL RE S E ARCH
Gene
accession_ID
370
Table 1 (continued )
Gene
symbol
JMJD2A- kd/NC
intensity
LPS-2 h JMJD2A-kd/NC
intensity
LPS-10 h JMJD2A-kd/NC
intensity
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
Gene
accession_ID
Gene
symbol
JMJD2A- kd/NC
intensity
LPS-2 h JMJD2A-kd/NC
intensity
LPS-10 h JMJD2A-kd/NC
intensity
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
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Gene
accession_ID
E XP ER I ME NTAL C E LL RE S E ARCH
371
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
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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3 28 (2014) 3 61 37 8
E XP ER I ME NTAL C E LL RE S E ARCH
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
376
E X PE R IM ENTA L C ELL R E S EA RC H
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
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
references
[1] T. Kouzarides, Chromatin modications and their function, Cell
128 (2007) 693705.
[2] R.J. Klose, K. Yamane, Y. Bae, D. Zhang, H. Erdjument-Bromage,
P. Tempst, J. Wong, Y. Zhang, The transcriptional repressor
JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine
36, Nature 442 (2006) 312326.
[3] H.G. Yoon, D.W. Chan, Z.Q. Huang, J. Li, J.D. Fondell, J. Qin,
J. Wong, Purication and functional characterization of the
human N-CoR complex: the roles of HDAC3, TBL1 and TBLR1,
EMBO J. 22 (2003) 13361346.
[4] D. Zhang, H.G. Yoon, J. Wong, JMJD2A is a novel N-CoRinteracting protein and is involved in repression of the human
transcription factor achaete scute-like homologue 2 (ASCL2/
Hash2), Mol. Cell. Biol. 25 (2005) 64046414.
[5] J.R. Whetstine, A. Nottke, F. Lan, M. Huarte, S. Smolikov, Z. Chen,
E. Spooner, E. Li, G. Zhang, M. Colaiacovo, Y. Shi, Reversal of
E XP ER I ME NTAL C E LL RE S E ARCH
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
377
378
E X PE R IM ENTA L C ELL R E S EA RC H
3 28 (2014) 3 61 37 8
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
A.H. Rahman, B.J. Conti, T.K. Eitas, B.H. Koller, J.P. Ting, The innate
immune sensor NLRC3 attenuates Toll-like receptor signaling via
modication of the signaling adaptor TRAF6 and transcription
factor NF-kappaB, Nat. Immunol. 13 (2012) 823831.
T. Tao, C. Cheng, Y. Ji, G. Xu, J. Zhang, L. Zhang, A. Shen, Numbl
inhibits glioma cell migration and invasion by suppressing
TRAF5-mediated NF-kappaB activation, Mol. Biol. Cell 23 (2012)
26352644.
Z. Peng, Y. Shuangzhu, J. Yongjie, Z. Xinjun, L. Ying, TNF
receptor-associated factor 6 regulates proliferation, apoptosis,
and invasion of glioma cells, Mol. Cell. Biochem. 377 (2013)
8796.
D. Gozuacik, A. Kimchi, DAPk protein family and cancer, Autophagy 2 (2006) 7479.
J. Brognard, Y.W. Zhang, L.A. Puto, T. Hunter, Cancer-associated
loss-of-function mutations implicate DAPK3 as a tumorsuppressing kinase, Cancer Res. 71 (2011) 31523161.
D.E. Gomez, D.F. Alonso, H. Yoshiji, U.P. Thorgeirsson, Tissue
inhibitors of metalloproteinases: structure, regulation and biological functions, Eur. J. Cell Biol. 74 (1997) 111122.
N. Hiraoka, E. Allen, I.J. Apel, M.R. Gyetko, S.J. Weiss, Matrix
metalloproteinases regulate neovascularization by acting as
pericellular brinolysins, Cell 95 (1998) 365377.
S.D. Konduri, K.S. Srivenugopal, N. Yanamandra, D.H. Dinh,
W.C. Olivero, M. Gujrati, D.C. Foster, W. Kisiel, F. Ali-Osman,
S. Kondraganti, S.S. Lakka, J.S. Rao, Promoter methylation and
silencing of the tissue factor pathway inhibitor-2 (TFPI-2), a gene
encoding an inhibitor of matrix metalloproteinases in human
glioma cells, Oncogene 22 (2003) 45094516.
F. Gessler, V. Voss, V. Seifert, R. Gerlach, D. Kogel, Knockdown
of TFPI-2 promotes migration and invasion of glioma cells,
Neurosci. Lett. 497 (2011) 4954.