Introduction
Medulloblastoma is the most common type of brain
tumor in children and arises in the cerebellum (1). Current therapeutic approaches include
surgical resection, craniospinal irradiation and chemotherapy, all
of which are associated with severe long-term side effects,
including intellectual damage and endocrine dysfunction (2–6).
Identification of molecular markers that can be used to monitor the
therapeutic outcomes of patients with medulloblastoma and
decreasing the long-term sequelae with improved molecular-targeted
therapies is required. Despite progress from recent studies
regarding prognostic molecular markers for medulloblastoma,
numerous patients still require an appropriate clinical prognostic
marker (7–10).
The Hippo signaling pathway is a major regulator of
tissue growth and organ size (11).
The major function of this signaling pathway is largely determined
by the downstream transcriptional regulator tafazzin (TAZ), which
is a transcriptional co-activator with a PDZ-binding domain
(11,12). TAZ, also termed WW domain-containing
transcription regulator 1, serves pivotal roles in organ
development (13,14). TAZ functions by cooperating with
several transcriptional factors, including T-box 5, paired box-8
and TEA domain family members (15–17).
Given its biological effects in development and tissue homeostasis,
TAZ has been a point of interest in cancer development and
progression studies. The dysregulation of TAZ leads to concurrent
uncontrolled cellular proliferation, inhibition of apoptosis and
enhanced migration and invasion (18–20). In
addition, the activity of TAZ is required to sustain the
self-renewal and tumor-initiation capacities of cancer stem cells
(CSCs). Cordenonsi et al (21) reported that the levels of TAZ protein
are elevated in breast cancer CSCs and in poorly differentiated
human malignant tumors. Additionally, a gain of TAZ was identified
to induce a self-renewal capacity in non-CSCs.
Despite this interest in elucidating the role of TAZ
in cancer biology, to the best of our knowledge, the impact of TAZ
expression on the prognosis and biological behavior of human
medulloblastoma has not been investigated. The present study aimed
to evaluate the expression of TAZ protein in medulloblastoma by
immunohistochemistry and analyzed the association of TAZ expression
with clinical outcomes by multivariate analysis. Furthermore, the
functional role of TAZ in the proliferation of medulloblastoma
cells was investigated.
Materials and methods
Patient tumor tissues
The use of tumor tissues from patients with
medulloblastoma in the current study was approved by the Ethics
Committee of Daping Hospital (Chongqing, China). A total of 72
patients with medulloblastoma, who initially underwent excision of
their tumor without any other prior treatment, were enrolled
between June 2005 and June 2012 at Daping Hospital or the Second
Affiliated Hospital of Dalian Medical University (Dalian, China).
These patients included 50 males and 22 females, (range, 5–43
years, mean 14.2±7.4 years). From these 72 patients with
medulloblastoma, 55 patients were between the ages of 5 and 16
years, with a mean average age of 10.9±3.4 years, and the remaining
17 patients ranged between 17 and 43 years, with an mean average
age of 24.6±7.4 years. All patients were followed up until May
2014. The detailed characteristics of the patients are presented in
Table I. The present study was
performed according to the Declaration of Helsinki and written
informed consent was obtained from all patients with tumors and
donors of normal brain tissue samples.
 | Table I.Demographics of the 72 patients with
medulloblastoma. |
Table I.
Demographics of the 72 patients with
medulloblastoma.
| Characteristic | n (%) |
|---|
| Sex |
|
| Male | 50 (69.44) |
|
Female | 22 (30.56) |
| Age, years |
|
| ≤16 | 55 (76.39) |
|
>16 | 17 (23.61) |
| Symptoms |
|
|
Headache | 57 (79.17) |
|
Vomiting | 15 (20.83) |
|
Ataxia | 5 (6.94) |
|
Dizziness | 8 (11.11) |
| Leg
weakness | 2 (2.78) |
| Visual
symptoms | 2 (2.78) |
| Tumor maximal
diameter, cm |
|
| ≤2 | 12 (16.67) |
| >3,
≤5 | 53 (73.61) |
|
>5 | 7 (9.72) |
| Tumor location |
|
|
Hemisphere | 27 (37.50) |
|
Vermis | 45 (62.50) |
| Metastases at
diagnosis | 23 (31.94) |
| Extent of
resection |
|
| Gross
total | 46 (63.89) |
|
Subtotala | 26 (36.11) |
| Recurrence | 53 (73.61) |
| Tumor bed | 19 (26.39) |
| Metastatic | 26 (36.11) |
| Mixed | 8 (11.11) |
| Postoperative
treatment |
|
| Surgery
only | 17 (23.61) |
| Surgery
and irradiation | 34 (47.22) |
|
Irradiation and
chemotherapy | 21 (29.17) |
| Histology |
|
|
Classical | 51 (70.83) |
|
Nodular-desmoplastic | 9 (12.50) |
| Large
cell/anaplastic | 12 (16.67) |
| Patient
survival | 27 (37.50) |
| Patient
mortality | 45 (62.50) |
Immunohistochemistry
For immunohistochemistry, preserved 10%
formalin-fixed (4°C, 6 h) and paraffin-embedded tissue samples,
including human and mouse medulloblastoma tissue and normal tissue
samples were cut into 5-mm-thick sections. Following
deparaffinization in xylene, the sections were rehydrated in a
graded alcohol series at room temperature (100, 95, 85 and 75%
ethanol). Endogenous peroxidase activity was blocked 15 min by
incubation with 3% hydrogen peroxide in methanol at room
temperature, after which the sections were microwaved (100°C) in
0.01 M sodium citrate buffer to retrieve the epitopes. The sections
were incubated sequentially with primary antibodies against TAZ
(dilution, 1:200; BD Biosciences; cat. no., 560235), and Ki-67
(dilution, 1:100; BD Biosciences; cat. no., 550609) overnight at
4°C and goat anti-mouse antibodies (dilution, 1:100; Santa Cruz
Biotechnology, Inc; cat. no., sc-3738) for 30 min at room
temperature. The sections were then incubated with
3,3′-diaminobenzidine for chromogen detection. A total of 10
randomly selected microscopy fields were observed under a light
microscope (magnification, ×20; Olympus-CKX41; Olympus Corporation,
Tokyo, Japan) and the mean number of positive cells was calculated.
The immunohistochemistry images were obtained under identical
microscope conditions and magnification (×20).
Hematoxylin and eosin (H&E)
staining
For immunohistochemistry, preserved 10%
formalin-fixed (4°C, 6 h) and paraffin-embedded tissue samples,
including human and mouse medulloblastoma tissue and normal tissue
samples were cut into 5-mm-thick sections, and stained with H&E
staining using the Hematoxylin and Eosin Staining Kit (Beyotime
Institute of Biotechnology; cat. no., C0105), according to the
manufacturer's protocols.
Semi-quantitative scoring of
immunoreactivity
All tumor sections analyzed in the present study
were reviewed by two pathologists (Chenyi Mao and Hualiang Xiao;
Daping Hospital, Third Military Medical University). Tumor cell
immunoreactivity was scored according to intensity and the extent
of staining. The extent of immunopositivity was scored as follows:
1, <30% positive cells; 2, 30–50% positive cells; and 3, >50%
positive cells. For statistical analysis, sections with scores of 1
were considered to exhibit low expression and those with scores of
2 and 3 were considered to exhibit high expression.
Cell culture and lentivirus-mediated
stable knockdown of TAZ expression
The medulloblastoma cell line Daoy was obtained from
the American Type Culture Collection (Manassas, VA, USA) and
cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F-12;
Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine
serum (Thermo Fisher Scientific, Inc.), 1% penicillin and
streptomycin. The GFP plasmid and TAZ-targeting short hairpin RNA
(shRNA) in pLKO.1-Puro lentiviral vectors were kindly provided by
Dr Heng Xiao (Department of Hepatobiliary and Pancreatic Surgery,
First Affiliated Hospital of Zhejiang University, Zhejiang, China).
Before transfection, 5×106 293FT cells (American Type
Culture Collection) were plated on 100-mm plate with lentiviral
medium and incubated at 37°C overnight, Then 0.5 µg pLKO.1 vector
were mixed with 0.5 µg of pLP1, pLP2 and pLP/VSVG (packing)
plasmids, and diluted in 6 µl Lipofectamine™ 2000 (Thermo Fisher
Scientific, Inc.) with 250 µl OPTI medium (no serum, penicillin and
streptomycin; Thermo Fisher Scientific, Inc.). After 20 min at room
temperature, the mixture was added dropwise into each 6 well plate,
and 850 µl of the 293FT cell suspension (1×106 cells)
was added to the plate containing OPTI medium and
DNA-Lipofectamine™ 2000 complexes. It was mixed gently by rocking
the plate back and forth, and incubated at 37°C with 5%
CO2. The medium was replaced with lentiviral medium
after 6–8 h transfection. After 48 h, virus-containing supernatants
were harvested, passed through a 0.45-µm filter, and then infected
with target cells. Cells stably expressing GFP and TAZ shRNA were
generated after 3 days of culture in the presence of puromycin (3
µg/ml).
MTT assay
Cell viability was examined using the MTT assay.
Cells (1×103 cells/well) were seeded in 96-well plates
with three replicates, and then were detected between day 0 and day
5. Purple formazan was dissolved by dimethylsulfoxide, and the
absorbance was measured at a wavelength of 490 nm. using a
microplate reader. Each experiment was performed independently at
least three times.
5-Bromo-2-deoxyuridine (BrdU)
staining
For BrdU staining, 2×104 cells were
seeded on coverslips in 24-well plates. Then cells were incubated
with 10 µg/ml BrdU (Sigma-Aldrich; Merck KGaA) for 30 min at 37°C
with 5% CO2 and fixed in 4% paraformaldehyde for 15 min
at room temperature. The cells were then incubated with BrdU
Detection Antibody kit (dilution, 1:300; Biovision, Inc.; cat. no.,
K306-1000) overnight at 4°C, followed by the anti-mouse HRP-linked
Antibody from the above BrdU Detection Antibody kit (dilution,
1:2,000; Biovision, Inc.) for 2 h, according to the manufacturer's
protocols. A total of 300 nM DAPI was then added for
counterstaining, according to the BrdU Detection Antibody kit. The
images were captured using a light microscope (magnification, ×20)
with Image-Pro Plus software (X-Ray, Celina; http://www.xrayscan.com/software-image-pro-plus/) for
image analysis and green staining indicated BrdU-positive
cells.
Western blot analysis
Cellular proteins were extracted with RIPA lysis
buffer (Thermo Fisher Scientific, Inc.). The protein concentration
of the proteins was determined by the BCA Protein Assay kit
(Beijing Solarbio Science & Technology Co., Ltd.; cat. no.,
PC0020). Equal amounts of protein (50 µg) were subjected to 10%
SDS-PAGE and electrotransferred onto a hydrophobic PVDF membrane
(Roche Diagnostics). Membranes were subsequently blocked with 5%
nonfat milk in TBST (0.1% Tween) for 2 h at room temperature and
subsequently incubated with primary antibodies against TAZ
(dilution, 1:2,000; BD Biosciences; cat. no., 560235), tubulin
(dilution, 1:3,000; Beyotime Institute of Biotechnology; cat.no.,
A01080HRP), cyclin-dependent kinase 4 (CDK4, dilution, 1:200; Santa
Cruz Biotechnology, Inc.; cat. no., sc-70831), cyclin D1 (CCND1,
dilution, 1:200; Santa Cruz Biotechnology, Inc.; cat. no., sc-8396)
and cyclin E2 (CCNE2, dilution, 1:200; Santa Cruz Biotechnology,
Inc.; cat. no., sc-28351) under slow rotation overnight at 4°C.
Subsequently, the membranes were incubated with the corresponding
secondary antibodies HRP-labeled goat anti-mouse IgG (H+L) (cat.
no., A0216; dilution, 1:5,000; Beyotime Institute of Biotechnology)
and goat anti-rabbit IgG (H+L) (cat. no., A0216; dilution, 1:5,000;
Beyotime Institute of Biotechnology) for 2 h at 4°C. They were
subsequently captured using the ECL Reagent (Cell Signaling
Technology, Inc.) and visualized by iBright Imaging Systems
(iBright FL 1000; Thermo Fisher Scientific, Inc.).
Xenograft assay
A total of 12 female SCID mice (4 weeks old; 18–20
g) were purchased from Beijing Laboratory Animal Research Center
and housed in an SPF room, which maintained standard housing
conditions with 12 h light–dark cycle with food and water available
ad libitum and at a constant temperature (22°C-25°C) and
humidity (40–50%). The human medulloblastoma Daoy cells
(1×106) stably transfected with sh-GFP or sh-TAZ were
injected slowly into the subcutis on the dorsum of each mouse. The
tumor size was monitored every week using a vernier caliper and
tumor volume (V) was calculated using the formula: V = (length ×
width2)/2. At the termination of the experiment (day
45), the tumor mass was harvested, weighed and stored for
immunohistochemical analysis. All studies were approved by the
Ethics Committee of Daping Hospital (Chongqing, China).
Statistical analysis
All statistical analyses were performed using SPSS
11.5 software (SPSS, Inc., Chicago, IL, USA). Differences in the
levels of TAZ expression among various subgroups were analyzed
using a χ2 test. The overall survival and tumor-free
survival rates were evaluated using Kaplan-Meier analysis and the
survival of groups of patients was compared using a log-rank test.
All experiments were performed in triplicate and the quantitative
data are presented as the mean ± standard deviation. Two-tailed
Student's t-tests were performed to calculate significance with a
95% confidence level in data with a normal distribution and
different but similar standard deviations. P<0.05 was considered
to indicate a statistically significant difference.
Results
Immunostaining of TAZ expression in
medulloblastoma tissues
Immunohistochemistry was performed to detect TAZ
expression in 72 medulloblastoma and three normal brain tissue
samples. The results revealed low expression of TAZ in the three
normal brain tissues (Fig. 1A). By
contrast, TAZ expression, which was predominantly observed in the
nuclei, was elevated in a high proportion of medulloblastoma
tissues. In total, 25 of the tumors (34.72%) demonstrated low
expression of TAZ (Fig. 1B-D) and 47
of the tumors (65.28%) demonstrated high expression (Fig. 1E and F; Table II). These results suggest TAZ may
serve an important role in the progression of medulloblastoma.
| Table II.Association of TAZ expression with
clinicopathological features of patients with medulloblastoma. |
Table II.
Association of TAZ expression with
clinicopathological features of patients with medulloblastoma.
| Characteristic | TAZ low expression
(n=25) | TAZ high expression
(n=47) | P-value |
|---|
| Sex |
|
| 0.465 |
|
Male | 16 | 34 |
|
|
Female | 9 | 13 |
|
| Age, years | 25 | 47 | 0.522 |
|
≤16 | 18 | 37 |
|
|
>16 | 7 | 10 |
|
| Tumor maximal
diameter, cm |
|
| 0.912 |
| ≤3 | 4 | 8 |
|
|
>3 | 21 | 39 |
|
| Tumor location |
|
| 0.848 |
|
Hemisphere | 9 | 18 |
|
|
Vermis | 16 | 29 |
|
| Metastases at
diagnosis | 6 | 17 | 0.292 |
| Recurrence | 14 | 38 | 0.025a |
| Histology |
|
| 0.212 |
|
Classic | 20 | 31 |
|
|
Non-classic | 5 | 16 |
|
Association of TAZ expression and
clinicopathological features of patients with medulloblastoma
The TAZ immunohistochemistry results were analyzed
for potential associations with clinicopathological characteristics
of patients with medulloblastoma. As presented in Table II, no significant associations were
identified between TAZ expression and sex (P=0.465), age (P=0.522),
tumor location (P=0.848), tumor maximal diameter (P=0.912),
histological type (P=0.212) or tumor metastases at diagnosis
(P=0.292). However, the expression of TAZ was significantly
associated with tumor recurrence. Among the 52 patients who had
recurrence during follow-up, 38 patients exhibited a high
expression of TAZ and the others had low expression. The recurrence
rate in patients with tumors with high TAZ expression was 80.9%,
whereas the rate in patients with tumors with low TAZ-expressing
tumors was only 56% (P=0.025).
Association of TAZ expression with
overall survival and tumor-free survival
As presented in Fig.
2, the association of TAZ expression with overall survival and
tumor-free survival rate was evaluated by log-rank test. Patients
with high TAZ expression demonstrated significantly shorter overall
survival and tumor-free survival times compared with patients with
low TAZ expression (Fig. 2).
Univariate and multivariate Cox regression analyses were performed
to determine the independent prognostic factors for overall
survival of patients with medulloblastoma. Univariate Cox
regression analysis first revealed TAZ expression (P=0.047),
metastasis at diagnosis (P<0.001) and postoperative therapy
(P=0.011) as significant prognostic factors for patients with
medulloblastoma (Table III).
However, histological type and resection extent were not
significant prognostic factors. Furthermore, multivariate analysis
identified TAZ expression (P=0.046), metastasis at diagnosis
(P<0.001) and postoperative therapy strategies (P=0.014) as
independent prognostic factors for the overall survival of patients
with medulloblastoma (Table
IV).
| Table III.Univariate Cox regression analysis of
overall survival. |
Table III.
Univariate Cox regression analysis of
overall survival.
| Factor | Wald-value | Exp(B) | 95% CI for
Exp(B) | P-value |
|---|
| TAZ expression | 3.952 | 2.042 | 1.010–4.130 | 0.047a |
| Sex | 0.002 | 1.015 | 0.509–2.023 | 0.967 |
| Age | 1.236 | 0.638 | 0.289–1.408 | 0.266 |
| Tumor location | 0.023 | 0.945 | 0.458–1.950 | 0.879 |
| Metastasis at
diagnosis | 23.805 | 6.569 | 3.084–13.992 |
<0.001a |
| Tumor diameter | 0.238 | 1.263 | 0.494–3.232 | 0.626 |
| Resection
extent | 2.862 | 1.714 | 0.918–3.198 | 0.091 |
| Tumor
histology | 1.050 | 0.795 | 0.514–1.232 | 0.305 |
| Postoperative
therapy | 6.426 | 0.557 | 0.355–0.876 | 0.011a |
| Table IV.Multivariate Cox regression analysis
of overall survival. |
Table IV.
Multivariate Cox regression analysis
of overall survival.
| Factors | Wald-value | Exp(B) | 95% CI for
Exp(B) | P-value |
|---|
| TAZ expression | 3.965 | 2.008 | 1.011–3.986 | 0.046a |
| Metastasis at
diagnosis | 25.008 | 6.132 | 3.012–12.482 |
<0.001a |
| Postoperative
therapy | 5.987 | 0.587 | 0.383–0.899 | 0.014a |
Downregulation of TAZ inhibits cell
proliferation of Daoy cells
Enhanced expression of TAZ was identified to be
associated with short overall survival and tumor-free survival
times of patients with medulloblastoma. However, the functional
role of TAZ in medulloblastoma progression remains unknown. To
address this issue, the present study established Daoy cells with
stable expression of shRNA targeting TAZ (sh-TAZ) and control shRNA
targeting GFP (sh-GFP). As presented in Fig. 3A, the protein level of TAZ was
significantly downregulated in sh-TAZ cells compared with sh-GFP
cells. Subsequently, the role of TAZ in the regulation of the
proliferation of Daoy cells was examined. MTT assay demonstrated
that knockdown of TAZ expression significantly inhibited the
proliferation of Daoy cells (Fig.
3B). This result was further confirmed using a BrdU
incorporation assay. The percentage of BrdU-positive cells was
significantly decreased in sh-TAZ cells compared with sh-GFP cells
(Fig. 3C and D). Finally, it was
identified that the expression levels of CCND1 and CCNE2 were
decreased in the sh-TAZ group compared with the sh-GFP group, while
no obvious changes were observed in CDK4 expression (Fig. 3E). In summary, these results
suggested that TAZ may regulate the proliferation and expression of
cell-cycle-associated proteins in medulloblastoma cells, which
indicates a role for TAZ in the progression of medulloblastoma.
Downregulation of TAZ inhibits the
tumor formation of Daoy cells in vivo
To investigate the effects of TAZ expression on
medulloblastoma tumor formation, a nude mouse xenograft experiment
was performed. In the nude mouse xenograft experiment, tumor growth
was significantly inhibited in nude mice injected with
TAZ-knockdown Daoy cells compared with control mice (Fig. 4A and B). Immunohistochemical staining
revealed that the expression of Ki-67, a cell proliferation marker,
was significantly reduced in the tissues samples of mice injected
with sh-TAZ cells compared with those injected with sh-GFP cells
(Fig. 4C and D).
Discussion
Medulloblastoma, which arises in the cerebellum, is
the most common type of malignant brain tumor in children (22). Despite progress in the treatment of
medulloblastoma, ~30% of patients will succumb to the disease and
40% of children diagnosed with the disease will experience tumor
recurrence (23). Although
conventional therapies can treat a large proportion of patients
with medulloblastoma, the majority of survivors experience a
reduced quality of life.
Current risk stratification strategies are largely
based on clinical parameters, including age at diagnosis,
metastatic status, extent of surgical resection and histological
features (24). Molecular
stratification and the use of molecular targeted therapy for
patients with medulloblastoma is still in its infancy and has yet
to be routinely implemented in the clinical practice (25). An improved understanding of the
molecular and genetic basis of medulloblastoma will contribute to
appropriate stratification and the treatment of patients based on
biological markers, leading to improved patient outcomes with
reduced sequelae.
Medulloblastoma is a heterogeneous tumor type
consisting of at least four distinct subgroups, including the WNT
subgroup, sonic hedgehog subgroup, subgroup C and subgroup D
(26). This classification is based
on immunohistochemistry data and has been widely used in
medulloblastoma research, directing numerous studies toward
elucidating the mechanisms of activation of WNT and SHH signaling
pathways and the development of new strategies to inhibit them,
thus leading to the improvement of patient stratification and
prognosis (27–29). Additionally, the outcomes of patients
with medulloblastoma have been reported to be associated with c-MYC
copy number and the expression of microRNA-183/96/182 and
cyclin-dependent kinase 6 (30,31).
However, additional markers that can be utilized for monitoring
medulloblastoma progression and therapeutic outcome are
required.
The present study examined the expression of TAZ in
72 patients with medulloblastoma. High TAZ expression was observed
in 65.28% of patients. Low or undetectable immunostaining for TAZ
was observed in normal brain tissues. Subsequently, it was
demonstrated that expression of TAZ is a negative prognostic factor
for the overall survival rate of patients with medulloblastoma. To
the best of our knowledge, this is a new finding, as no previous
studies have assessed the impact of TAZ expression on the prognosis
of patients with medulloblastoma. The present results are
consistent with studies of adenocarcinoma of the esophagogastric
junction and colorectal cancer (32,33).
However, in the current study, a lower rate of patients with
long-time survival was observed compared with a previous study
(23). This may be due to the fact
that fewer patients in the present study had received combined
therapy (23.61 and 47.22% received surgery alone and combined
surgery and irradiation, respectively). In addition, it was
identified that TAZ expression was significantly associated with
medulloblastoma recurrence. However, the current results are
limited by the amount of clinical data and different patient
follow-up periods. Therefore the sample size should be increased
and the median follow-up time should be analyzed in future
studies.
Additional functional experiments in the present
study revealed that the expression of TAZ promoted tumor
proliferation. Additionally, silencing TAZ expression decreased the
proliferation and tumor formation of medulloblastoma cells. The
present study highlights the important role of TAZ in
medulloblastoma progression and its association with patient
survival.
In summary, high expression of TAZ was detected in a
majority of medulloblastoma tissue samples and was identified to
predict a poor outcome for patients. As a result, the expression of
TAZ may serve as a prognostic marker and a potential therapeutic
target for patients with medulloblastoma. However, this conclusion
is limited by the small number of cases analyzed in the present
study. Further studies with a large number of cases are required to
validate the clinical significance of TAZ in patients with
medulloblastoma and elucidate the precise mechanism by which TAZ
regulates the malignant behavior of medulloblastoma.
Acknowledgements
The authors would like to thank Dr Heng Xiao
(Zhejiang University School of Medicine, Hangzhou, China) for
technical support.
Funding
The present study was supported by the Natural
Science Foundation of China (grant no. 81300965). The Natural
Science Foundation of Chongqing (grant no. cstc2017jcyjAX0337) and
the Chongqing Basic Science Frontier Technology Special Key Project
(grant nos. cstc2017jcyjB0164, cstc2017jcyjBX0021 and
cstc2018jcyjAX0600).
Availability of data and materials
The datasets used and/or analyzed during the present
study are available from the corresponding author on reasonable
request.
Authors' contributions
HW and JZ collected, analyzed and interpreted the
data, and wrote the manuscript. DY, LY, XW, YO and DHY collected
and analyzed the data. LX and MX designed the study, provided
financial support, analyzed and interpreted the data, and revised
the manuscript. All authors read and approved the final
manuscript.
Ethics approval and consent to
participate
All animal studies were approved by the Ethics
Committee of Daping Hospital (Chongqing, China). The use of tissue
samples from patients was approved by the Ethics Committee of
Daping Hospital. The current study was performed according to the
Declaration of Helsinki and written informed consent was obtained
from all patients.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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