Downregulation and DNA methylation of ECRG4 in gastric cancer
OncoTargets and Therapy
Downregulation and Dna methylation of ecrg4 in gastric cancer
Peng Deng 1
Xiao-Jing chang 1
Zi-Ming gao 0
Xiao-Yang Xu 1
an-Qi sun 0
Kai li 0
Dong-Qiu Dai 1
0 Department of s urgical Oncology and g eneral s urgery, The First a ffiliated h ospital of c hina Medical University , shenyang, china
1 Department of g astrointestinal s urgery and c ancer c enter, The Fourth a ffiliated h ospital of c hina Medical University , shenyang, china
8 1 0 2 - l u J - 3 1 n o 7 1 1 . 2 2 . 8 3 . 4 5 y b / m o c . s s re . .vdoep lsyeon PowerdbyTCPDF(ww.tcpdf.org) Background: Esophageal cancer-related gene 4 (ECRG4) is a novel candidate tumor suppressor gene. Our study investigated the expression and function of ECRG4 in gastric cancer and highlighted the role of DNA hypermethylation at the promoter in silencing the ECRG4 expression. Methods: The GSE63089 data set was obtained from the Gene Expression Omnibus and analyzed for differentially expressed genes. Carcinoma and para-carcinoma tissues of 102 patients with gastric cancer were collected from January 2010 to July 2011. Immunohistochemistry, real-time polymerase chain reaction (PCR), and western blot analyses were performed to evaluate the expression of ECRG4. After measuring the change in the level of ECRG4 expression, CCK-8, Transwell, and flow cytometric cell cycle assays were performed. In addition, methylation-specific PCR was performed to detect the methylation state of ECRG4, and 5-aza-2′-deoxycytidine was used for demethylation of ECRG4. All statistical analyses were performed using the SPSS 17.0 software. Results: We found that ECRG4 expression was downregulated in gastric cancer, and this was closely related to lymph node metastasis. After ECRG4 was silenced using a specific small interfering RNA, the BGC-823 cell line became highly aggressive and proliferative. In addition, we verified whether downregulation of ECRG4 was highly correlated with DNA methylation of the ECRG4 promoter and found that the demethylating agent 5-aza-2′-deoxycytidine could effectively enhance ECRG4 expression. Conclusion: The aberrant expression of ECRG4 is associated with hypermethylation in the promoter region and plays an important role in the malignancy of gastric cancer. Therefore, ECRG4 may be a potential biomarker for molecular diagnosis of gastric cancer, and the use of 5-Aza-dC to reverse the hypermethylation of ECRG4 may be a new approach to the treatment of gastric cancer.
tumor stage, and even survival time.12–14 However, the role
of ECRG4 in GC is debatable.
In this study, we have highlighted the role of ECRG4
expression and its DNA methylation level in GC
tumorigenesis and have provided evidence that ECRG4 might be a new
biomarker and treatment target for GC in the future.
Materials and methods
Public data sets of gene expression profiles were obtained
from the Gene Expression Omnibus (GEO; https://www.ncbi.
nlm.nih.gov/geo/). The GSE63089 data set was identified,
which includes the expression data for 45 paired GC tissues
and gastric normal tissues.15 Differential analysis was
performed by using GEO2R, and partial results were presented
as a heatmap by using the Morpheus tool (https://software.
broadinstitute.org/morpheus). We screened the data for
Tissue specimens and clinicopathological characteristics
GC carcinoma and paracarcinoma tissues were collected from
102 patients after surgical resection at the First Affiliated
Hospital of China Medical University from January 2010 to
July 2011. This research was approved by the Ethics Committee
of China Medical University, and written informed consent
for this study was obtained from each patient. No neoadjuvant
radiotherapy, chemotherapy, or targeted therapy was applied.
The clinical characteristics included age, gender, tumor
size, differentiation state, depth of invasion, tumor location,
Borrmann type, and lymph node metastasis.
A real-time PCR reverse transcriptase kit was obtained from
Fermentas (Vilnius, Lithuania). ECRG4 and glyceraldehyde
3-phosphate dehydrogenase (GAPDH) real-time PCR primers
and ECRG4 methylation and demethylation primers were
synthesized by BGI Company (Shenzhen, China). A rabbit
antihuman ECRG4 monoclonal antibody (RabMAb) and ECRG4
isotope antibody were acquired from Santa Cruz Biotechnology
Inc. (Dallas, TX, USA). A ready-to-use immunohistochemistry
(IHC) hypersensitive Ultra-Sensitive™ SP kit (KIT-9701)
and 3,3′-diaminobenzidine kit were purchased from MXB
biotechnologles (MXB, Fuzhou, China). A bicinchoninic acid
(BCA) protein assay kit was provided by Beyotime (Shanghai,
China), and Cell Counting Kit-8 (CCK-8) was from Fanbo
Biochemicals Co. Ltd. (Beijing, China). A Wizard DNA
cleanup system kit was purchased from Promega Corporation
(Fitchburg, WI, USA). Ribonuclease was purchased from
Hoffman-La Roche Ltd. (Basel, Switzerland) and nylon mesh
from BD Biosciences (San Jose, CA, USA).
IHC staining of ECRG4 in carcinoma and paracarcinoma
gastric tissues was performed by using ECRG4 RabMAb.
Global ECRG4 was scored using a semiquantitative
integration method as follows: 1) the percentage of positive
cells: 0 (#5%), 1 (6%–25%), 2 (26%–50%), 3 (51%–75%),
and 4 (.75%); 2) the intensity of staining: 0 (no staining),
1 (faint yellow), 2 (yellow), and 3 (brown). After multiplying
the scores for percentage of positive cells and intensity of
staining, the combined staining score was determined as
follows: 0 (negative, −), 1–4 (weakly positive, 1+), 5–8
(positive, 2+), and 9–12 (strongly positive, 3+). The data
were used for statistical analysis.
Western blotting (WB)
Cells were lysed in a protein lysis buffer, and total protein
concentrations were determined by using the BCA assay.
Protein lysates were separated by 10% sodium dodecyl
sulfate polyacrylamide gel electrophoresis, and proteins
were electrotransferred onto membranes. The membranes
were probed with the RabMAb (1:1,000) as a primary
antibody, and the mouse anti-human GAPDH primary antibody
(1:1,000) was used as a loading control. After washing three
times with Tris-buffered saline containing Tween 20, a
corresponding secondary antibody was used. Each experiment
was performed in triplicate.
Quantitative real-time polymerase chain reaction (qr T-Pcr)
For qRT-PCR, total RNA was isolated from cells and tissues
by using the TRIzol reagent. cDNA was synthesized from
the RNA templates by using the M-MuLV reverse
transcriptase and oligo (dT)18 primers. The sense and
antisense primers for ECRG4 were 5′-GGTACCAGCAGTTT
CTCTACATG-3′ and 5′-CAGCGTGTGGCAAGTCATGGT
TAGT-3′, respectively. GAPDH was used as an internal
control, and its sense and antisense primers were 5′-AGTCCT
TCCACGATACCAAAGT-3′ and 5′-AGTCCTTCCACGA
TACCAAAGT-3′, respectively. The PCR mixture (25 μL)
was pre-denatured at 95°C for 10 min, followed by 40 cycles
of denaturation at 95°C for 15 s, annealing at 56°C for 30
s, and extension at 72°C for 30 s. Each experiment was
repeated three times. Analysis was performed by using the
2−ΔΔCt relative quantitation method.
An ECRG4-specific siRNA and pcDNA3.1-ECRG4
vector were transfected into BGC-823 cells to silence and
enhance the expression of ECRG4, respectively. Groups
transfected with an empty vector and without a vector were
used as controls. Transfection was performed by using the
Lipofectamine 2000 transfection reagent (Thermo Fisher
Scientific, Waltham, MA, USA). The expression level of
ECRG4 was confirmed by WB.
cell proliferation analysis
Cell proliferation was determined by using the CCK-8 assay.
A cell suspension (5×105) was seeded into 96-well plates,
and the plates were incubated at 37°C, 5% CO2 overnight.
The CCK-8 reagent (10 μL) was added to each well, and
the plates were incubated for another 3 h. Cell viability was
determined by measuring absorbance values at 450 nm.
A total of 1×104 cells were added to 200 μL of RPMI-1640
medium with 1% newborn calf serum (NBCS), and the
suspension was seeded in the upper chamber of a Transwell
device. To the lower chamber, 500 μL of RPMI-1640 medium
with 20% NBCS was added as a chemoattractant. After
incubation for 24 h, the cells on the top surface of the insert were
removed by using a cotton swab. Then, the insert was placed
in a stationary liquid for 30 min, stained with Giemsa stain for
8 min, washed, and finally observed under a microscope.
Flow cytometric cell cycle analysis
Cells were trypsinized, collected, and washed with cold PBS
before being fixed with 70% ethanol at −20°C overnight. The
fixed BGC-823 cells were washed in cold PBS, and then
incubated at 37°C with 100 ng/mL ribonuclease and 10 ng/
mL propidium iodide (PI) for 30 min. After filtering through
a nylon mesh, samples were analyzed with a FACScan flow
cytometer, and data were analyzed with ModFit software
(Verity Software House, Topsham, ME, USA).
Methylation-specific PCR (MSP)
We detected the methylation state of ECRG4 by MSP.
DNA was isolated from GC cell lines and tissues by using
phenol–chloroform–isoamyl alcohol. Then, sodium hydrogen
sulfite was removed from DNA by using the Wizard DNA
cleanup system kit. The purified DNA was immediately
used to perform MSP. The primers for methylated sequences
were 5′-TGGCGTTTTTATGGTGTTC-3′ (upstream) and
generating a 137-bp PCR product. The primers for unmethylated
sequences were 5′-ATGTGGTGTTTTTATGGTGTTT-3′
(upstream) and 5′-AAACACCACTTCACACTTATACA-3′
(downstream), generating another 137-bp PCR product. The
total reaction volume was 50 μL, and the mixture contained
DNA, sense and antisense primers, 10× Dream Taq buffer,
dNTP mix, and Dream Taq DNA polymerase. The reaction
conditions were as follows: pre-denaturation at 95°C for
5 min, followed by 34 cycles at 95°C for 30 s, 62°C for 30 s,
and 72°C for 30 s, with a final extension at 72°C for 10 min.
The PCR products were subjected to agarose gel
electrophoresis at 120 V for 40 min. The gels were photographed by
using an electrophoretic gel imaging analysis system.
cell treatment with 5-aza-2
We first maintained BGC-823 cells under starvation
conditions in RPMI-1640 medium containing 1% fetal bovine
serum and then treated the cells with 1 μmol/L, 5 μmol/L,
or 10 μmol/L 5-Aza-dC. The cells were transferred daily
into a fresh culture medium containing 5-Aza-dC at the three
experimental concentrations. On the third day, the cells were
harvested for further analysis. Simultaneously, BGC-823
cells were cultivated without drug treatment as a control.
Continuous variables are presented as the mean values and
standard deviation. Differences between groups were
determined by using an independent sample t-test and χ2 test. Each
assay was repeated three times, and p,0.05 was considered
statistically significant. All analyses were performed by using
the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).
expression of ecrg4 is downregulated
and closely related to lymph node
metastasis in gc
A heatmap of the 100 most downregulated genes in the
GSE63089 public data set, which includes expression data
from 45 paired GC and normal tissues, is presented in
Figure 1A. The expression level of ECRG4 in normal gastric
tissues was about 2.46-fold higher than that in GC tissues
(log fold change [log FC] = 2.46, p,0.01). A qRT-PCR assay
on tissues from 35 patients showed that ECRG4 mRNA was
downregulated in GC tissues ( p,0.05; Figure 1B). Using
an IHC assay, we also observed a lower expression
intensity of the ECRG4 protein in GC tissues from 102 patients
(Figure 1C). Among the 102 GC patients, 35.3% (38/102)
showed a lower expression of ECRG4 in GC tissues, while
3.9% (4/102) showed a lower expression of ECRG4 in normal
tissues ( p,0.01). The ECRG4 protein was also related to the
0128 differentiation state of GC. As shown in Figure 1C, lower
l--Ju ECRG4 expression was always associated with poorer
dif13 ferentiation. To further confirm the ECRG4 expression in
no7 cells, we performed WB and qRT-PCR using four GC cell
.211 lines. Both the assays demonstrated that protein and mRNA
.823 expression of ECRG4 was downregulated in all GC cell lines
45 compared to that in a normal cell line (Figure 1D and E).
/yb Based on the levels of ECRG4 protein and mRNA
expres.com sion, the 102 GC patients were divided into high and low
rsse . expression groups. The t-test showed that the ECRG4 protein
.vdoep lsyeon level in GC was associated with lymph node metastasis
( p,0.05) and histological grade ( p,0.05) (Table 1).
Similarly, the ECRG4 mRNA level in GC was also related to
lymph node metastasis ( p,0.05), as shown in Table 2. Thus,
the expression level of ECRG4 is positively related to lymph
inhibition of ecrg4 promotes
proliferation and invasion of gc cells
An ECRG4-specific siRNA was transfected into BGC-823
cells, which had a relatively high level of ECRG4 expression.
The expression level of ECRG4 in the siRNA-transfected
ECRG4 group was significantly reduced compared to that
in the non-transfected group or in the group transfected with
a negative plasmid (Figure 2A). The CCK-8 assay showed
that the proliferation rate of the siRNA-ECRG4 group was
higher than that of the other groups (Figure 2B). In addition,
the migration ability of BGC-823 cells transfected with
siRNA-ECRG4 significantly increased in the Transwell
assay, as shown in Figure 2C. These results indicated that the
inhibition of ECRG4 expression could enhance the growth
and invasion of GC cells.
effect of ecrg4 expression on the cell
cycle of gc cells
To determine the effect of ECRG4 expression in altering the
cell cycle in GC cells, BGC-823 cells were transfected with
pcDNA3.1, pcDNA3.1-ECRG4, siRNA-control, or
siRNAECRG4. The cell cycle was analyzed by flow cytometry
following PI staining. Compared with the control groups,
the percentage of G1 phase cells was significantly increased
in the pcDNA3.1-ECRG4 group (70.72% vs 61.95%) and
decreased in the siRNA-ECRG4 group (50.89% vs 61.95%)
(Figure 3). Our results revealed that ECRG4 expression
was associated with variation in the cell cycle in GC cells.
In addition, overexpression of ECRG4 induced cycle arrest
in the G1 phase and inhibited the proliferation of GC cells,
consistent with the results of the CCK-8 assay.
The ecrg4 promoter is highly methylated in gc
We performed the MSP analysis to investigate the
methylation status of ECRG4 in GC cells. As shown in Figure 4A,
the ECRG4 promoter was highly methylated in four GC
cell lines but partially methylated in GES1. In addition, we
analyzed the ECRG4 methylation status in 74 GC tissues
(Figure 4B) and confirmed that methylation of the ECRG4
promoter was also higher in GC tissues than that in non-tumor
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tissues ( p,0.05). ECRG4 was hypermethylated in 66.2%
(49/74) of the carcinoma tissues but only in 18.9% (14/74) of
the paracarcinoma tissues. However, there was no correlation
between the methylation level of the ECRG4 promoter and
clinicopathological characteristics ( p.0.05; Table 3).
The demethylation agent 5-aza-dc could
upregulate ecrg4 expression in gc
5-Aza-dC is an effective suppressor of DNA methylation.
After treatment with 5-Aza-dC for 72 h, the expression of
ECRG4 mRNA and protein was markedly upregulated in
BGC-823 cells ( p,0.05; Figure 4C and D). Furthermore,
the elevation in ECRG4 expression was positively
associated with the drug concentration of 5-Aza-dC. These results
indicated that 5-Aza-dC increases the expression of ECRG4
by suppressing its methylation.
ECRG4 is a highly conserved gene that plays an important
role in tumorigenesis in vertebrates. Studies have revealed
the potential TSG role of ECRG4 in different cancers. In our
study, we confirmed the association between GC
tumorigenesis and ECRG4 expression, particularly, the role of ECRG4
High-throughput analysis showed that ECRG4 might act
as a TSG of GC. Using IHC, WB, and qRT-PCR assays, we
showed that the expression of ECRG4 mRNA and protein was
downregulated in GC, consistent with a tumor-suppressing
role of ECRG4. The relationship between clinicopathological
characteristics and ECRG4 expression has been reported in
several cancers. Studies have revealed that ECRG4
expression is associated with lymph node metastasis, predicting a
poor prognosis in patients with tumors. In our study, we also
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demonstrated the relationship between ECRG4 expression
and lymph node metastasis in GC patients. However, a
longterm and complete follow-up is needed to confirm the role
of ECRG4 as a marker for GC prognosis.
Furthermore, we investigated the tumor-suppressing
function of ECRG4 at the cellular level. As previously
reported, ECRG4 may be associated with the inhibition of
some malignant phenotypes of tumor cells. In our study, we
showed that downregulation of ECRG4 promoted
proliferation and invasion of GC cells, as demonstrated by CCK-8
and Transwell assays. Notably, Wen et al have found that
ECRG4 expression in esophageal cancer is closely
associated with the invasive depth and tumor node metastasis
stage, also confirming the inhibitory effect of ECRG4 on
malignant phenotypes.13 However, the mechanism of effects
of ECRG4 effects in tumorigenesis is unclear. Lu et al have
reported that ECRG4 may suppress the proliferation and
invasion of breast cancer cells by regulating M-phase cell
cycle genes.16 The results of our analysis showed that gain
or loss of ECRG4 function led to changes in cell cycle in
GC cells. Overexpression of ECRG4 induced cell cycle
arrest in G1 phase and then suppressed the proliferation of
GC cells, which is consistent with previous studies.17,18 In
addition, Jia et al have revealed that ECRG4 is associated
not only with proliferation but also with apoptosis.17 In
laryngeal cancer, overexpression of ECRG4 induced apoptosis
by regulating the expression of apoptosis-related factors,
such as Bax, Bcl-2, and caspase-3. ECRG4 could also play
a role in anti-inflammatory activities by interacting with an
immunity receptor complex, which might be important for
Promoter DNA methylation plays an important role in
the tumorigenesis of various human cancers via long-term
silencing of TSGs.5 In esophageal, colorectal, and renal
cancers, promoter methylation is the main mechanism of ECRG4
silencing, and treatment with demethylating agents can
restore gene expression.20–22 In GC, Huang et al reported that
DNA methylation is common in gastric intestinal metaplasia
(IM), and hypermethylated regions of IM were recapitulated
in GC.23 Moreover, Qu et al reported that methylation levels
of ECRG4 were significantly higher in GC tissue (69.4%)
than in normal tissue (6.7%).4 In the present study, we
confirmed that the ECRG4 promoter was highly methylated,
which could explain the downregulation of ECRG4 in GC
patients. Although we did not find an association between
ECRG4 methylation and clinicopathological characteristics
of GC patients, Wang et al22 have reported that aberrant
ECRG4 promoter methylation could be a predictor of GC
pathological stage. In their study, methylation levels of
ECRG4 were higher in stage III/IV (24/30) than in stage I/II
In our study, we found that the demethylating agent
5-Aza-dC enhanced ECRG4 expression by effectively
suppressing DNA methylation, and this suppressive effect
was positively associated with the drug concentration of
5-Aza-dC. Therefore, reversing the hypermethylation of
ECRG4 with 5-Aza-dC might be a new approach for GC
treatment. Based on a literature review, 5-Aza-dC, combined
with adjuvant chemotherapy, might be an effective measure
against GC. For example, Jiang et al have reported that
overexpression of ECRG4 may improve the chemosensitivity
of the GC-derived SGC-7901 cell line to 5-fluorouracil
(5-FU).24 Therefore, we hypothesized that 5-Aza-dC may
improve the curative effect of 5-FU to some extent, which
would have important clinical significance.
Our results suggested that ECRG4 is a novel TSG for GC,
and DNA methylation of ECRG4 is a potential inducer
of tumorigenesis. Inhibition of ECRG4 expression may
promote the growth and migration of GC cells. Conversely,
demethylation of ECRG4 with 5-Aza-dC could suppress the
tumorigenesis of GC cells. Thus, restoring the expression of
the ECRG4 in the tumor, either by epigenetic therapy or by
using a recombinant protein, may offer a promising novel
therapeutic approach for GC treatment.
This study was funded by the Liaoning Province Science and
Technology Plan Project (No. 2013225021) and the Natural
Science Foundation of Liaoning Province (No. 201602817).
The funders had no role in study design, data collection and
analysis, preparation, or publication of the manuscript.
The authors report no conflicts of interest in this work.
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OncoTargets and Therapy is an international, peer-reviewed, open
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