The new 6q27 tumor suppressor DACT2, frequently silenced by CpG methylation, sensitizes nasopharyngeal cancer cells to paclitaxel and 5-FU toxicity via β-catenin/Cdc25c signaling and G2/M arrest
Zhang et al. Clinical Epigenetics
The new 6q27 tumor suppressor DACT2, frequently silenced by CpG methylation, sensitizes nasopharyngeal cancer cells to paclitaxel and 5-FU toxicity via β-catenin/ Cdc25c signaling and G2/M arrest
Yan Zhang 0 1
Jiangxia Fan 0 1
Yichao Fan 3
Lili Li 3
Xiaoqian He 1
Qin Xiang 1
Junhao Mu 1
Danfeng Zhou 1
Xuejuan Sun 1
Yucheng Yang 2
Guosheng Ren 1
Qian Tao 1 3
Tingxiu Xiang 1
0 Equal contributors
1 Chongqing Key Laboratory of Molecular Oncology and Epigenetics, the First Affiliated Hospital of Chongqing Medical University , Chongqing , China
2 Department of Otolaryngology, the First Affiliated Hospital of Chongqing Medical University , Chongqing , China
3 Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, CUHK-Shenzhen Research Institute, The Chinese University of Hong Kong , Hong Kong , China
Background: Nasopharyngeal carcinoma (NPC) is prevalent in South China, including Hong Kong and Southeast Asia, constantly associated with Epstein-Barr virus (EBV) infection. Epigenetic etiology attributed to EBV plays a critical role in NPC pathogenesis. Through previous CpG methylome study, we identified Disheveled-associated binding antagonist of beta-catenin 2 (DACT2) as a methylated target in NPC. Although DACT2 was shown to regulate Wnt signaling in some carcinomas, its functions in NPC pathogenesis remain unclear. Methods: RT-PCR, qPCR, MSP, and BGS were applied to measure expression levels and promoter methylation of DACT2 in NPC. Transwell, flow cytometric analysis, colony formation, and BrdU-ELISA assay were used to assess different biological functions affected by DACT2. Immunofluorescence, Western blot, and dual-luciferase reporter assay were used to explore the mechanisms of DACT2 functions. Chemosensitivity assay was used to measure the impact of DACT2 on chemotherapy drugs. Results: We found that DACT2 is readily expressed in multiple normal adult tissues including upper respiratory tissues. However, it is frequently downregulated in NPC and correlated with promoter methylation. DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored its expression in NPC cells. DACT2 methylation was further detected in 29/32 (91%) NPC tumors but not in any (0/8) normal nasopharyngeal tissue samples. Ectopic expression of DACT2 in NPC cells suppressed their proliferation, migration, and invasion through downregulating matrix metalloproteinases. DACT2 expression also induced G2/M arrest in NPC cells through directly suppressing β-catenin/Cdc25c signaling, which sensitized NPC cells to paclitaxel and 5-FU, but not cisplatin. Conclusion: Our results demonstrate that DACT2 is frequently inactivated epigenetically by CpG methylation in NPC, while it inhibits NPC cell proliferation and metastasis via suppressing β-catenin/Cdc25c signaling. Our study suggests that DACT2 promoter methylation is a potential epigenetic biomarker for the detection and chemotherapy guidance of NPC.
DACT2; Nasopharyngeal cancer; Cdc25c; Paclitaxel; 5-FU
Unlike other malignancies, the incidence of
nasopharyngeal carcinoma (NPC) has great ethnic and geographic
differences. Its incidence is high in Chinese and Malay
populations in Southeast Asia and North Africa [
Specific biomarkers would be helpful in populations with a
high incidence of NPC, but few are available. Study of
NPC pathogenesis should aim to identify diagnostic
]. The Disheveled-associated binding
antagonist of β-catenin (DACT) family, also known as Dapper/
Frodo, are small intracellular scaffold proteins. There are
three family members, DACT1, 2, and 3 [
]. DACT2 is
repressed by promoter methylation in various cancers,
including breast [
], colon , lung [
], and gastric
], but the mechanisms differ. In breast cancer,
our findings demonstrated that DACT2 antagonizes
Akt/GSK-3 and Wnt/β-catenin signaling to suppress
epithelial-to-mesenchymal transition (EMT) [
glioma cells, DACT2 interacts with Wnt/β-catenin
signaling to prevent Yes-associated protein translocation to
the nucleus, resulting in its sequestration and
degradation in the cytoplasm [
]. In esophageal squamous cell
cancer, DACT2 suppresses TGFβ/SMAD2/3 activity via
both the proteasome and lysosomal degradation
]. Zebrafish DACT2 was reported to inhibit
TGF-β/Nodal signaling during mesoderm induction by
interacting with type 1 receptors ALK5 and ALK4 and
further promoting lysosomal degradation [
In our recent study, DACT2 gene was identified to be
a methylated target in NPC [
], but its molecular
functions and mechanism were not determined. Here, we
intend to investigate the expression and methylation of
DACT2 in NPC cells and tissues. The effect of DACT2
on the cell cycle was evaluated to explore the influence
of DACT2 overexpression on drug treatment.
DACT2 was downregulated in NPC by promoter methylation
Reverse transcription (RT)-PCR confirmed that DACT2
was expressed in the majority of normal adult tissues
(Fig. 1a). To investigate the expression of DACT2 in
NPC, we analyzed the gene expression data of DACT2
in Oncomine online database
(https://www.oncomine.org/), and it clearly shows that its expression is
suppressed in the T1 and N0 stage NPC, which means
DACT2 has potential to be an early diagnosed
biomarker (Fig. 1b). DACT2 expression was downregulated
in HNE1 and HONE1 NPC cells and was restored by
5aza-2′-deoxycytidine (Aza) without or with trichostatin
A (TSA). Following treatment, quantitative
methylationspecific PCR (qMSP) showed a decrease of methylated
level and an increase in un-methylated level (Fig. 1c).
Thus, DACT2 expression was downregulated in these
NPC cell lines by promoter methylation.
The methylation status of eight normal nasopharyngeal
tissues and 32 NPC tissues was assayed by
methylationspecific polymerase chain reaction (MSP), which found
that the DACT2 promoter was not methylated in any of
the normal nasopharyngeal tissues but was methylated in
29 of 32 (91%) NPC tissues (Fig. 1d, e). Bisulfite genomic
sequencing (BGS) was used to assay methylated DACT2
promoter alleles in two normal nasopharyngeal tissue and
two NPC tissue samples to confirm the result of MSP and
found that DACT2 methylation was more frequent in
NPC than in normal nasopharyngeal tissues (Fig. 1f ).
Overexpression of DACT2 inhibited NPC cell proliferation, viability, and colony formation
The overexpression of DACT2 after DACT2 plasmid
transfection was confirmed using RT-PCR and Western
blot by comparing to empty control (Fig. 2a, b). The
MTS assay (Fig. 2c) showed that cell viability was
significantly reduced in DACT2-expressing cells. Colony
formation (Fig. 2d) was also significantly suppressed
compared with the control cells. These results indicated
that DACT2 suppressed both viability and growth of
DACT2 induced G2/M cell cycle arrest and apoptosis in
The influence of DACT2 on tumor cell proliferation
might be mediated by its effects on the cell cycle and
apoptosis. Flow cytometry of HONE1 and HNE1 cells
found that the percentage of cells in the G2/M phase
was increased in those that overexpressed DACT2
compared with controls transfected with an empty
vector (Fig. 3a, b), accompanied by the increased cell
population of S phase. Furthermore, the BrdU-ELISA
assay, which reflects active DNA synthesis, revealed that
the cell proliferation rate was decreased in
DACT2expressing cells (Fig. 3c). DACT2 overexpression also
promoted cell apoptosis compared with controls (Fig. 3d, e).
These results indicated that DACT2 inhibited cell
proliferation by blocking the cell cycle in G2/M and by inducing
DACT2 inhibited NPC cell migration and invasion
Wound healing and Transwell assays were used to
investigate the influence of DACT2 expression on NPC cell
migration and invasiveness. In the Transwell assay,
significantly fewer DACT2-overexpressing cells passed
through the membrane than control cells (p < 0.001)
(Fig. 4a). Further wound healing assay revealed that
scratches made in confluent layers of
DACT2-overexpressing cells healed significantly slower than control
cell layers over 24 h for HNE1 (p < 0.001) or 33 h for
HONE1 (p < 0.01, Fig. 4b), which showed that DACT2
inhibited NPC cell migration. In the Transwell assay
including a Matrigel barrier, DACT2 overexpression was
associated with significant inhibition of NPC cancer cell
invasion through the Matrigel before traversing the
Transwell chamber membrane (p < 0.01, p < 0.001 at
24 h, Fig. 4c). qPCR and Western blot assays assessed
the effect of DACT2 on expression of matrix
metalloproteinases (MMPs) 2 and 9 (Fig. 4d, e), which are
essential for cell migration and invasion [
the results indicate that DACT2 suppressed cell
migration and invasion in NPC cells.
DACT2 induced G2/M cell cycle arrest through the
β-catenin/Cdc25c signaling pathway in nasopharyngeal
Investigation of the mechanism of G2/M cell cycle arrest
was based on a previous report that DACT2 suppressed
β-catenin activity in colon cancer by competition for
LEF1 binding [
]. In addition, the gene coding for cell
division control protein 25C (Cdc25c), a regulator of
G2/M cell cycle progression, has a LEF1 binding site on
its promoter region. The function of that protein may
thus be responsive to Wnt/β-catenin signaling activity
]. It is thus reasonable that DACT2 inhibits the
activity of β-catenin/LEF1 complex by competitively
binding with β-catenin followed by downregulation of
Cdc25c, which ultimately blocks cell division in the G2/M
phase. DACT2 regulation of the β-catenin/Cdc25c
pathway to produce G2/M cell cycle arrest was assessed
by immunofluorescent staining of β-catenin. As shown in
Fig. 5a, the morphology of NHE1 cells overexpressing
DACT2 differed from that of control cells (Fig. 5a), and
the expression of total β-catenin did not significantly
change but active β-catenin decreased in the cell nuclear
location (Fig. 5b, c). qRT-PCR showed that DACT2
overexpression was associated with decreased expression of
Cdc25c and cyclin B1 (Fig. 6a). Western blots revealed
that DACT2 suppressed the expression of active
β-catenin, Cdc25c, and the downstream target genes of
β-catenin/Cdc25c signaling (Fig. 6b). A dual-luciferase
reporter assay was performed to confirm whether
DACT2 inhibited the activity of the β-catenin/LEF1
complex. It showed that DACT2 downregulated the
induced TOPflash luciferase activities (Fig. 6c).
Overall, the results indicate that DACT2 led to G2/M cell
cycle arrest by inhibiting the β-catenin/Cdc25c
DACT2 induced sensitivity of NPC cells to paclitaxel and
5-FU but not cisplatin
As DACT2 expression led to G2/M cell cycle arrest, the
impact of DACT2 overexpression on the sensitivity of
NPC cells to cell cycle phase-specific and
phasenonspecific chemotherapy drugs was tested. Paclitaxel,
5-fluorouracil (5-FU), and cisplatin were selected.
Paclitaxel acts by arrest of the cell cycle in G2/M [
5-FU is an atypical periodic chemotherapy drug
targeting on the S and other phases [
]. Cisplatin is cell cycle
phase-nonspecific chemotherapy drug [
]. As shown in
Fig. 7, DACT2 expression increased the sensitivity of
HNE1 and HONE1 cells to paclitaxel and 5-FU
compared with controls, but had no effect on cisplatin
sensitivity (Fig. 7).
Discussion and conclusion
Promoter CpG methylation, which downregulates the
expression of tumor suppressor genes, is essential to the
pathogenesis of malignancies including NPC [
Specific epigenetic therapy may increase the
effectiveness of NPC treatment . In papillary thyroid cancer
] and hepatocellular cancer [
], the expression of
DACT2 is downregulated by promoter methylation. In
this study, DACT2 was strongly expressed in normal
adult tissues but weakly expressed and hyper-methylated
in NPC cell lines. DACT2 expression was restored in
NPC cell lines by Aza and TSA demethylation. Promoter
methylation was detected in 29 of 32 (91%) NPC tissue
samples but was not detected in any of the normal
nasopharyngeal tissue samples. The results indicated
that the low expression of DACT2 in NPC was caused
by promoter CpG methylation. The function of DACT2
was investigated in HONE1 and HNE1 cells transfected
with DACT2 gene. The restoration of DACT2
expression inhibited NPC cell proliferation, migration, and
invasiveness and induced G2/M cell cycle arrest.
The Wnt signaling pathway is active in tumorigenesis,
cell differentiation, and cell proliferation [
is a transcription cofactor that induces target gene
expression by binding to T cell factor/lymphoid enhancer
factor (TCF/LEF) in the activated Wnt pathway [
The DACT family members are inhibitors of Disheveled,
an important Wnt pathway component that suppresses
c-Jun N-terminal kinase (JNK) signaling and the
βcatenin cascades [
]. DACT2 has been reported to
decrease LEF1-β-catenin binding in colon cancer by
competing with β-catenin . In this study, DACT2
decreased the expression of active β-catenin and its
downstream genes in NPC cells and suppressed the
activity of the β-catenin/LEF1 complex. Cdc25c, which
has been shown to regulate the G2/M checkpoint, has
also been reported to have a functional LEF binding site
14, 15, 29, 30
]. In NPC cells, DACT2 was found to
suppress the expression of Cdc25c and its downstream
genes related to G2/M arrest. We conclude that DACT2
downregulated the activity of the β-catenin/LEF1
complex by binding to β-catenin and that the decreased
expression of Cdc25c induced G2/M arrest (Fig. 8).
DACT2 overexpression increased the sensitivity of NPC
cells to the cell cycle phase-specific chemotherapy drugs,
paclitaxel and 5-FU.
In summary, DACT2 was silenced by promoter
methylation as a tumor suppressor in NPC cells and induced
G2/M phase arrest by the regulating β-catenin/Cdc25c
signaling pathway. The results show that DACT2 is a
tumor suppressor in nasopharyngeal cancer and support
continuing evaluation of its value for early diagnosis and
for targeted therapy of nasopharyngeal cancer.
Tumor cell lines and tissues
HNE1 and HONE1, two poorly differentiated
nasopharyngeal squamous carcinoma cell lines, were used [
Cells were maintained in RPMI 1640 (Gibco BRL, MD,
USA) with 10% fetal bovine serum (Gibco, CA, USA) and
1% penicillin and streptomycin (Gibco BRL). Thirty-two
nasopharyngeal cancer and eight normal nasopharyngeal
tissue samples were obtained from Department of
Otolaryngology of the First Affiliated Hospital of
Chongqing Medical University between December 2010 and July
]. All samples were verified by histology. All
patients gave written informed consent.
RNA isolation, reverse transcription-PCR, and quantitative real-time PCR
Total RNA was extracted from cell lines and tissues
using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA)
following the manufacturer’s instructions, and aliquots
containing 1 μg of total RNA were reverse-transcribed
to 20 μl cDNA. PCR was performed using Go-Taq
(Promega, Madison, WI, USA) with initial denaturation at
95 °C for 2 min, followed by 32 cycles (95 °C for 30 s,
55 °C for 30 s, and 72 °C for 30 s) of amplification, with
a final extension at 72 °C for 3 min [
] with β-actin
used as a control. Twenty-three cycles of amplification
were performed. The primer sequences are shown in
Table 1. qPCR was performed using SYBR Green
(Thermo Fisher) following the manufacturer’s
instructions (7500 Real-Time PCR System, Applied Biosystems,
Foster City, CA, USA). Each sample was tested in
triplicate. Gene expression level was calculated by the 2−ΔΔCt
5-Aza-2′-deoxycytidine and trichostatin A treatment
Cell lines were treated with 10 μmol/L
5-aza-2′-deoxycytidine (Aza, Sigma-Aldrich, Steinheim, Germany), a
DNA methyltransferase (DNMT) inhibitor, for 3 days
and then without or with 100 nmol/L trichostatin A
(TSA, Sigma-Aldrich) for 24 h as previously
DNA isolation, bisulfite modification of DNA, methylationspecific PCR, and quantitative methylation-specific PCR
Genome DNA was extracted from tissues using a
QIAamp DNA Mini kit (Qiagen, Hilden, Germany)
following the manufacturer’s instructions. Bisulfite
modification of DNA was performed as previously described
]. The MSP primers (annealing temperature of
60 °C, 40 cycles) are shown in Table 1, and have been
confirmed not amplify any nonbisulfited DNA . MSP
was performed using AmpliTaq-Gold DNA Polymerase
(Applied Biosystems). The PCR products were identified
on 2% agarose gels. qMSP was performed with the 7500
Real-Time PCR System (Applied Biosystems, Foster City,
CA, USA) [
Bisulfite genomic sequencing
BGS primers (Table 1) were used to amplify
bisulfitetreated DNA, and the PCR products were cloned into a
pCR4-Top vector (Invitrogen). Eight to 12 colonies were
randomly chosen and sequenced.
Construction of vector- and DACT2-expressed stable cell lines
Stable cell lines were constructed by transfecting cell lines
with plasmids using Lipofectamine 2000 (Invitrogen)
following the manufacturer’s instructions. pcDNA3.1 and
pcDNA3.1–DACT2 plasmids were transfected at a
concentration of 4 μg and selection by geneticin at 48 h
after transfection [
]. Ectopic expression of DACT2 was
assayed by RT-PCR and Western blotting prior to the
other experimental procedures.
Cell viability assay
Cell viability was evaluated with a CellTiter 96 AQueous
One Solution Cell Proliferation Assay (MTS, Promega)
following the manufacturer’s instructions. HONE1 and
HNE1 cells were cultured in 96-well plates after
transient transfection with DACT2 and vector (pcDNA3.1)
plasmids. Cell viability was measured at 0, 24, 48, and
72 h. Absorbance was read in a microplate reader at
490 nm. All experiments were performed in triplicate.
Colony formation assay
Cell proliferation was assayed by colony formation assay
]. DACT2- and vector-expressing cells were plated in
6-well plates at densities of 200, 400, or 600 cells/well
with geneticin. Surviving colonies (≥ 50 cells/colony)
were counted on day 10 of culture after fixation and
staining with Gentian violet. All experiments were
performed in triplicate.
BrdU cell proliferation enzyme-linked immunosorbent assay
Cells were seeded in 96-well plates at 1 × 104 cells per
well after transfection with DACT2 and vector
(pcDNA3.1) plasmids for 48 h. After 24-h culture, BrdU
(bromodeoxyuridine) was added to the wells to
incorporate into proliferating cells for 4 h. The BrdU-ELISA
assay was performed by BrdU Cell Proliferation ELISA
Kit (colorimetric) (Abcam, Cambridge, UK) following
Fig. 8 Proposed models how DACT2 affects the β-catenin/Cdc25c pathway in nasopharyngeal carcinoma. When β-catenin/LEF1 complex is
activated, it promotes the development of nasopharyngeal carcinoma by targeting its downstream genes including Cdc25c, which plays a
crucial role in regulating cell cycle G2/M stage. DACT2 inhibits the activity of β-catenin/LEF1 complex by competitively binding to β-catenin
and downregulates the expression of Cdc25c and its downstream genes, which can suppress nasopharyngeal carcinoma growth
Product size (bp)
Annealing temperature (°C)
the kit manufacturer’s instructions. The results were
read at 450 nm using a microplate reader.
Flow cytometry was used for cell cycle analysis and to
assay apoptosis [
]. To assess cell cycle status, cells
were stained with propidium iodide (PI) following
transfection and fixation. For apoptosis, cells were
doublestained with annexin V-fluorescein isothiocyanate and
PI. The flow cytometry results were evaluated using a
Cell Quest kit (BD Biosciences, CA, USA) and were
performed in triplicate.
Wound healing, Transwell, and Matrigel assays
Cell migration was evaluated by wound healing and
Transwell assays [
]. Stably transfected DACT2- and
vector-(pcDNA3.1) HONE1 and HNE1 cells were plated
in 6-well plates and were wounded when confluent by
scratching with a sterile pipette tip. Migration was
measured on phase-contrast micrographs (Leica DMI4000B,
Milton Keynes, Buckinghamshire, UK) at 0, 12, and 24 h
for HNE1 and 0, 11, 24, and 33 h for HONE1. Transwell
chambers (Corning Life Sciences, Corning, NY, USA)
with a pore size of 8 μm were used to evaluate cell
migration and cell invasion. To assay cell invasiveness, a
Matrigel (BD Biosciences) barrier was added on top of
the Transwell membrane. Cells on the lower surface of
the chamber at 24 h were photographed using a
phasecontrast microscope (Leica) after fixation and staining
and were then counted. All experiments were performed
The effect of DACT2 on the cytotoxicity of paclitaxel,
cisplatin, and 5-fluorouracil (5-FU) was assayed using a
Cell Counting Kit-8 (CCK-8). Briefly, HONE1 and
HNE1 cells transfected with DACT2 or vector
(pcDNA3.1) plasmids were plated at 5000/well in
96well plates. After cell attachment, culture media
containing different concentrations of the tested drugs
were added. Cells were then counted at 24 or 48 h
with CCK-8 (Dojindo, Shanghai, China) following the
manufacturer’s instructions. Absorbance was read with
a microplate reader at 450 nm. The half-maximal
inhibitory concentration (IC50) was calculated for each
drug concentration. All experiments were performed in
Dual-luciferase reporter assay
The effect of DACT2 on TCF/LEF transcriptional
activities was investigated by a dual-luciferase reporter assay.
pTopflash and pFopflash were used in our previous
]. pTopflash was constructed with TCF/LEF
binding sites but pFopflash containing a mutant TCF/
LEF binding sites as a control. HONE1 and HNE1 cells
were transiently co-transfected with a pTOPflash or
pFOPflash and DACT2 or vector (pcDNA3.1) with a
Renilla luciferase reporter pRL-TK (Promega) as an
internal control. Luciferase activity was measured after
48 h transfection using a dual-luciferase reporter assay
kit (Promega) following the manufacturer’s instructions.
All experiments were performed in triplicate.
Cells were seeded in 24-well plates containing glass
coverslips and then transfected with pcDNA3.1-DACT2
or pcDNA3.1 plasmid for 48 h. After transfection, cells
were fixed with 4% paraformaldehyde in pH 7.4 PBS for
10 min, permeabilized for 10 min in 0.5% Triton X-100,
and blocked with blocking buffer for 1 h at room
temperature. Cells were incubated with primary antibodies
against DACT2 (TA306668, Origene) and β-catenin
(#2677, Cell Signaling Technology, Danvers, MA, USA) or
β-actin (sc-8432, Santa Cruz Biotechnology, CA, USA),
Flag-M2 (F3165, Sigma-Aldrich, Darmstadt, Germany)
and non-p-β-catenin (#19807, Cell Signaling Technology)
overnight at 4 °C. After primary antibody binding, cells
were incubated with Alexa Fluor 594- or 488-conjugated
goat anti-rabbit or anti-mouse secondary antibody (Jackson
ImmunoResearch, West Grove, PA, USA) for an additional
30 min. Nuclei were counterstained with 4,
6-diamidino-2phenylindole (DAPI, Roche, Palo Alto, CA, USA). The
slides were observed with a confocal laser scanning
microscope and photographed. Phalloidin staining was
performed as our previous work [
]. Stable cells were used.
Phalloidin-iFluor™ 594 Conjugate (23122, AAT Bioquest,
CA, USA) at room temperature for 1 h.
Western blot assay
Western blotting was performed as previously described
]. Aliquots of 40 μg of protein lysate were separated
by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and then transferred onto
polyvinylidene difluoride (PVDF) membranes (Bio-Rad,
Hercules, CA, USA). Membranes were incubated with
DACT2 (TA306668, Origene), active β-catenin (#4270;
Cell Signaling Technology), total β-catenin (#9562; Cell
Signaling Technology), MMP9 (ab76003, Abcam),
MMP2 (ab86607, Abcam), c-Myc (#13987, Cell Signaling
Technology), Cyclin D1(sc-450), p-GSK3β (sc-373800),
Cdc25c (sc-13138), Cdc2 (sc-54), p-Cdc2 (pY15.44)
(sc136014), β-actin (sc-8432) (all from Santa Cruz
Biotechnology, CA, USA), or CyclinB1 (ab32053, Abcam)
primary antibodies. Proteins were visualized using an
Immobilon Western Chemiluminescent HRP Substrate
kit (Millipore Corporation, Billerica, MA, USA).
SPSS16 (SPSS, Chicago, IL, USA) was used to perform
the statistical analysis. Differences were evaluated for
significance with the χ2 test and Fisher’s exact test. p
values < 0.05 were considered statistically significant.
5-FU: 5-Fluorouracil; Aza: 5-Aza-2-deoxycytidine; BGS: Bisulfite genomic
sequencing; BrdU: Bromodeoxyuridine; CCK-8: Cell Counting Kit-8; Cdc25c: Cell
division control protein 25C; DACT2: Disheveled-associated binding antagonist
of beta-catenin 2; DAPI: 4, 6-diamidino-2-phenylindole; DNMT: DNA
methyltransferase; EBV: Epstein-Barr virus; ELISA: Enzyme-linked immunosorbent
assay; EMT: Epithelial-to-mesenchymal transition; IC50: Half-maximal inhibitory
concentration; JNK: c-Jun N-terminal kinase; MMPs: Matrix metalloproteinases;
MSP: Methylation-specific polymerase chain reaction; NPC: Nasopharyngeal
carcinoma; PI: Propidium iodide; PVDF: Polyvinylidene difluoride;
qMSP: Quantitative methylation-specific polymerase chain reaction;
qPCR: Quantitative real-time polymerase chain reaction; RT-PCR: Reverse
transcription polymerase chain reaction; SDS-PAGE: Sodium dodecyl sulfate
polyacrylamide gel electrophoresis; TCF/LEF: T Cell factor/lymphoid enhancer
factor; TSA: Trichostatin A
This study was supported by the National Natural Science Foundation of China
(#81572769, #31420103915), Natural Science Foundation of Chongqing.
Municipal Commission of Health and Family Planning (#2016ZDXM006) and
VC special research fund from The Chinese University of Hong Kong.
Availability of data and materials
The datasets analyzed during the current study are available in the
Oncomine repository (https://www.oncomine.org/).
YZ, TX, and QT contributed to the conception and design of the study. YZ, JF,
DZ, XS, and JM performed the experiments and analyzed the data. XH and QX
contributed to the RNA and DNA extraction. YY collected the samples. YZ and
TX prepared the figures and drafted the manuscript. GR, LL, and YF reviewed
the manuscript. TX and QT finalized the manuscript. All authors reviewed and
approved the final manuscript.
Ethics approval and consent to participate
This research was approved by the Institutional Ethics Committees of the
First Affiliated Hospital of Chongqing Medical University (#20130306) and
conformed to the tenets of the Declaration of Helsinki.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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