Introduction
Wound repair is the main form of adult skin wound
healing and is a complex, multistep process, which includes the
interaction of various cells, growth factors and cytokines
(1,2). Proliferation and migration of
keratinocytes are considered to be important processes of wound
repair (3). Connective tissue
growth factor cellular communication network factor 2 promotes the
re-epithelialization at the wound site, thereby completing the
process of wound repair (4).
Therefore, the better understanding of the factors that affect
keratinocyte proliferation and migration may provide novel
therapeutic strategies for wound repair.
Previous studies have indicated that the process of
skin wound healing was associated with the expression of microRNAs
(miRNAs/miRs), including miR-105, miR-29a and miR-125b (5-7).
miRNAs are small non-coding RNAs, which bind to mRNA resulting in
its transcriptional inhibition or degradation, thereby regulating
gene expression (8,9). The abnormal regulation of certain
miRNAs has been indicated to serve a vital role in the aberrant
healing of wounds, such as miR-140 and miR-126 (5,7). Yang
and Yee (10) demonstrated that
miR-185 could bind to the 3'-untranslated region (3'-UTR) of
versican and participate in wound healing in transgenic mice.
However, the specific mechanism of miR-185 function in wound
healing remains unclear.
Peroxisome proliferator-activated receptors (PPARs)
are ligand-activated receptors that belong to the nuclear hormone
receptor family, including three different subtypes: PPARα, PPARβ/δ
and PPARγ (11). Previous studies
have suggested that PPARβ is associated with inflammation and tumor
progression (12-14).
In addition, PPARβ has also been demonstrated to participate in
wound healing. For instance, it has been reported that the
anti-apoptotic function of PPARβ was important in maintaining the
proliferation and migration of keratinocytes (15). Moreover, PPARβ has been indicated to
alleviate the inflammatory response of macrophages and inhibit the
apoptosis of keratinocytes, therefore indicating that it may be
used as a target in the development of wound healing drugs
(16). Tan et al (17) revealed that the upregulation of
PPARβ, which was induced by the inflammatory response, was critical
for skin wound healing. The aforementioned studies have suggested
that PPARβ may be a key molecule involved in wound healing.
The present study investigated the effects of
miR-185 and PPARβ on the proliferation and migration of HaCaT
keratinocytes. In addition, the mechanism by which miR-185
regulated these processes in HaCaT keratinocytes was explored. The
current study aimed to provide a novel basis for elucidating the
mechanism of wound healing.
Materials and methods
Cell culture
HaCaT keratinocytes were donated by Dr Petra Boukamp
(German Cancer Research Center, Heidelberg, Germany). The cells
were cultured in DMEM (Beijing Solarbio Science & Technology
Co., Ltd.) containing 10% FBS (Gibco; Thermo Fisher Scientific,
Inc.) and 1% antibiotics (100 U/ml penicillin and 100 µg/ml
streptomycin) at 37˚C in an incubator with 5% CO2. HaCaT
keratinocytes were treated with 50 µmol/l PI3K inhibitor LY294002
(Genomeditech Biotechnology) for 24 h at 37˚C. According to the
amount of LY294002 added, the equal amounts of DMSO (Genomeditech
Biotechnology) were added to the cells as control.
Cell transfection
miR-185 agomir (5'-UGGAGAGAAAGGCAGUUCCUGA-3') or
antagomir (5'-UCAGGAACUGCCUUUCUCUCCA-3') and their negative
controls (NCs), miR-NC (5'-CUAGUCAUCGAUGUCGUAGCA-3') or anti-miR-NC
(5'-CAGUACAUUGGUUCUGCAA-3'), were synthesized by Vigene
Biosciences. PPARβ overexpression plasmid (PPARβ forward,
5'-GCTCTAGAGCGGAGCGTGTGACGCTGCG-3' and reverse,
5'-GGGGTACCTTAAATATTTAATTCCCATT-3') or small interfering (si)RNA
(si-PPARβ forward, 5'-GCAAGCCCUUCAGUGACAUTT-3' and reverse,
5'-AUGUCACUGAAGGGCUUGCTT-3') and their negative controls (vector
forward, 5'-CTAGAGAACCCACTGCTTAC-3' and reverse
5'-TAGAAGGCACAGTCGAGG-3'; or si-NC forward
5'-UUCUCCGAACGUGUCACGUTT-3' and reverse
5'-ACGUGACACGUUCGGAGAATT-3') were purchased from Shanghai GeneChem
Co., Ltd. Transfection was performed using
Lipofectamine® 2000 (Invitrogen; Thermo Fisher
Scientific, Inc.) at 37˚C for 48 h. HaCaT keratinocytes were seeded
into six-well-plates at a density of 1x105 cells/m and
incubated at 37˚C for 48 h. A total of 50 nM oligonucleotides and 2
µg plasmid were transfected into HaCaT keratinocytes at a
confluence of 60%. After transfection for 48 h, the cells were
collected for subsequent experiments.
Reverse transcription-quantitative PCR
(RT-qPCR)
Total RNA was extracted from HaCaT keratinocytes
using TRIzol® reagent (Invitrogen; Thermo Fisher
Scientific, Inc.) according to the manufacturer's protocol.
PrimeScript™ RT Reagent kit (Takara Biotechnology Co., Ltd.) was
used to synthesize cDNA according to the manufacturer's
instructions with the following temperature protocol: 37˚C for 30
min and 95˚C for 10 min. mirVana qRT-PCR miRNA Detection kit
(Thermo Fisher Scientific, Inc.) was used to quantify the
expression of miR-185 using the following thermocycling conditions:
95˚C for 3 min, followed by 40 cycles at 95˚C for 15 sec and 60˚C
for 30 sec. SYBR Premix Ex Taq™ (Tli RNaseH plus; Takara
Biotechnology Co., Ltd.) was used for qPCR. The thermocycling
conditions were as follows: 95˚C for 5 min, followed by
denaturation at 94˚C for 30 sec, annealing at 60˚C for 30 sec, for
45 cycles. The primer sequences used were as follows: miR-185
forward, 5'-GTGCAGGGTCCGAGGTATG-3' and reverse,
5'-TGGAGAGAAAGGCAGTTCCTGA-3'; U6 forward,
5'-GCAGGAGGTCTTCACAGAGT-3' and reverse, 5'-TCTAGAGGAGAAGCTGGGGT-3';
PPARβ forward, 5'-TGAGCCTAAGTTTGAATTTGC-3' and reverse,
5'-TCTCGGTTTCGGTCTTCTTG-3'; GAPDH forward,
5'-GCACCGTCAAGCTGAGAAC-3' and reverse, 5'-TGGTGAAGACGCCAGTGGA-3'.
Relative expression was determined with the 2-ΔΔCq
method (18) using U6 or GAPDH as
internal controls.
Cell Counting Kit-8 (CCK-8) assay
HaCaT keratinocytes (~5,000 cells) were seeded into
96-well plates. Following transfection, HaCaT keratinocytes were
cultured for 24, 48 or 72 h at 37˚C. A CCK-8 kit (Beijing Solarbio
Science & Technology Co., Ltd.) was used to detect the
proliferative ability of HaCaT keratinocytes. In brief, 10 µl CCK-8
reagent was added to each well at a certain time point (24, 48 or
72 h) and cultured for 4 h at 37˚C. The absorbance at 450 nm was
measured using an immunoassay analyzer (Bio-Rad Laboratories,
Inc.).
Colony formation assay
Following transfection for 48 h, HaCaT keratinocytes
were seeded into six-well plates (~200 cells/well). After 2 weeks,
HaCaT keratinocytes were washed with PBS, fixed with 4%
paraformaldehyde for 20 min and stained with 0.5% crystal violet
for 15 min at room temperature. The number of cell colonies (>50
cells) was measured using ImageJ software (Version 1.8.0; National
Institutes of Health), and at least three independent repeats were
conducted for each treatment.
Western blotting
Total protein was obtained from HaCaT keratinocytes
using RIPA lysis buffer (Wuhan Boster Biological Technology, Ltd.)
and quantified using BCA Protein Assay kit (Pierce; Thermo Fisher
Scientific, Inc.). Subsequently, proteins were separated with 10%
SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories,
Inc.). After blocking with 5% non-fat milk at room temperature for
2 h, the membranes were incubated with the following primary
antibodies: Cyclin D1 (1:200; cat. no. ab16663), CDK6 (1:3,000;
cat. no. ab151247), CDK4 (1:1,000; cat. no. ab95255), p-AKT-T308
(1:1,000; cat. no. ab8933), AKT (1:500; cat. no. ab8805) (all from
Abcam), MMP-2 (1:2,000; cat. no. sc-10736), MMP-9 (1:2,000; cat.
no. sc-10737), PPARβ (1:5,000; cat. no. sc-74440), integrin-linked
kinase (ILK; 1:2,000; cat. no. sc-20019) (all from Santa Cruz
Biotechnology, Inc.), phosphoinositide-dependent protein kinase 1
(PDK1, 1:2,000; cat. no. BA4499), p-AKT-S473 (1:1,000; cat. no.
P00024-6), (all from Boster Biological Technology Co., Ltd.) or
GAPDH (1:2,500; cat. no. ab9485; Abcam) at 4˚C overnight.
Subsequently, the membranes were incubated with horseradish
peroxidase-conjugated secondary antibody Goat Anti-Rabbit IgG
H&L HRP (1:50,000; cat. no. ab205718; Abcam) or Goat Anti-Mouse
IgG H&L HRP (1:20,000; cat. no. ab205719; Abcam) at room
temperature for 1 h. The protein signals were visualized using a
Western Blotting Luminol Reagent (cat. no. sc-2048; Santa Cruz
Biotechnology, Inc.). Image-Pro Plus software v6.0 (Media
Cybernetics, Inc.) was used to analyze the protein signals.
Transwell assay
HaCaT keratinocytes were digested with trypsin at
37˚C for 2 min and resuspended in DMEM after transfection for 48 h.
Subsequently, cell suspensions (2x105 cells/ml) were
added to the upper chambers of Transwell equipment with 8-µm
polycarbonate membrane filter (Corning, Inc.). The lower chambers
were filled with DMEM containing 10% FBS. After incubation for 24 h
at 37˚C, the migrated HaCaT keratinocytes were stained with 0.5%
crystal violet for 15 min at room temperature and counted using an
optical inverted microscope (magnification, x100; Leica
Microsystems GmbH).
Dual-luciferase reporter assay
The binding sites between the PPARβ 3'-UTR and
miR-185 was predicted using the DIANA tools website (v5.0;
http://diana.imis.athena-innovation.gr/DianaTools/index.php).
PPARβ 3'-UTRs containing wild-type (WT) and mutant (Mut) miR-185
binding sites were cloned into pMIR-REPORT miRNA expression
reporter vector (Thermo Fisher Scientific, Inc.) to generate
PPARβ-WT and PPARβ-Mut reporter vectors. HaCaT keratinocytes were
co-transfected with the aforementioned reporter vectors and
miR-185, anti-miR-185, miR-NC or anti-miR-NC using
Lipofectamine® 2000 (Invitrogen; Thermo Fisher
Scientific, Inc.). A Dual-Luciferase Reporter Assay kit
(Genomeditech Biotechnology) was used to detect the relative
luciferase activity, which was normalized to Renilla
luciferase activity, after transfection for 48 h.
Statistical analysis
Data are presented as the mean ± SD of three
independent experimental repeats, and were analyzed using an
unpaired Student's t-test or one-way ANOVA followed by Tukey's post
hoc test. GraphPad Prism v5 software (GraphPad Software, Inc.) was
used to perform statistical analysis. P<0.05 was considered to
indicate a statistically significant difference.
Results
miR-185 inhibits the proliferation of
HaCaT keratinocytes
To investigate the effect of miR-185 on HaCaT
keratinocytes, miR-185 or anti-miR-185 were transfected into HaCaT
keratinocytes. RT-qPCR results indicated that miR-185 significantly
upregulated, while anti-miR-185 inhibited the expression level of
miR-185, validating their transfection efficiency (Fig. 1A). Subsequently, the effect of
miR-185 on the proliferative ability of HaCaT keratinocytes was
investigated. CCK-8 assay results demonstrated that miR-185
overexpression significantly suppressed cell proliferation, while
miR-185 inhibition exhibited the opposite effect compared with
their respective controls (Fig.
1B). Furthermore, the colony formation assay revealed that
overexpression of miR-185 decreased, while knockdown of miR-185
increased the number of colonies in HaCaT keratinocytes (Fig. 1C). Moreover, detection of the
expression level of proliferation-related proteins demonstrated
that overexpression of miR-185 reduced, while knockdown of miR-185
increased the protein expression level of cyclin D1, CDK6 and CDK4
(Fig. 1D). Taken together, the
results indicated that miR-185 suppressed proliferation in HaCaT
keratinocytes.
miR-185 suppresses migration in HaCaT
keratinocytes
The migratory ability of HaCaT keratinocytes was
examined using Transwell and western blot assays. The results
indicated that miR-185 significantly inhibited the number of
migrated HaCaT keratinocytes. By contrast, anti-miR-185 markedly
increased the number of migrated HaCaT keratinocytes (Fig. 2A). Moreover, detection of the
protein expression levels of MMP-2 and MMP-9, which have been
associated with cell migration (19), revealed that miR-185 significantly
reduced the expression level of MMP-2 and MMP-9, while
anti-miR-185exhibited the adverse effect (Fig. 2B). Therefore, the results indicated
that miR-185 reduced the migratory ability of HaCaT
keratinocytes.
miR-185 directly targets PPARβ in
HaCaT keratinocytes
The main role of miRNAs is the recognition of target
mRNAs via base complementary pairing, thereby degrading or
inhibiting the translation of the target mRNAs (20). To determine the molecular mechanism
by which miR-185 affects HaCaT keratinocyte proliferation and
migration, the DIANA tools website (v5.0; http://diana.imis.athena-innovation.gr/DianaTools/index.php)
was used to predict the potential target genes of miR-185, the
results of which indicated that PPARβ 3'-UTR presented a
complementary binding sequence with miR-185 (Fig. 3A). To further validate this
association, a dual-luciferase reporter assay was performed. The
results indicated that miR-185 significantly inhibited, and
anti-miR-185 enhanced the luciferase activity of PPARβ-WT 3'-UTR,
but neither exhibited any effect on the luciferase activity of
PPARβ-Mut 3'-UTR (Fig. 3B and
C). In addition, the effect of
miR-185 on PPARβ expression level was investigated, the results of
which indicated that miR-185 overexpression suppressed the mRNA and
protein level of PPARβ, whereas miR-185 knockdown resulted in the
adverse effect (Fig. 3D and
E). Moreover, the levels of
PI3K/AKT signaling pathway-related proteins were examined, and it
was revealed that overexpression of miR-185 reduced the protein
level of ILK and PDK1, the phosphorylation level of AKT at S473 and
T308 and the p-AKT/AKT ratio. Conversely, inhibition of miR-185
increased the protein level of ILK and PDK1, the phosphorylation
level of AKT at S473 and T308 and the p-AKT/AKT ratio (Fig. 3E). These data indicated that miR-185
regulated PPARβ expression and inhibited activation of the PI3K/AKT
signaling pathway.
 | Figure 3miR-185 directly targets PPARβ in
HaCaT keratinocytes. (A) The WT and mutant binding sites between
miR-185 and PPARβ are presented. The matched sequences of miR-185
and PPARβ 3'-UTR are expressed in uppercase, and those that cannot
be matched are expressed in lowercase. (B and C) A dual-luciferase
reporter assay was performed to verify the binding of miR-185 to
PPARβ 3'-UTR. (D) The expression level of PPARβ was examined by
reverse transcription-quantitative PCR. (E) Western blot analysis
was performed to determine the protein level of PPARβ, ILK, PDK1,
p-AKT and AKT. *P<0.05. PPARβ, peroxisome
proliferator-activated receptor β; ILK, integrin-linked kinase;
PDK1, phosphoinositide-dependent protein kinase 1; miR, microRNA;
NC, negative control; WT, wild-type; Mut, mutant; p,
phosphorylated. |
PPARβ promotes the proliferation and
migration of HaCaT keratinocytes
To investigate the effect of PPARβ on proliferation
and migration in HaCaT keratinocytes, si-PPARβ or PPARβ
overexpression plasmids were transfected into HaCaT keratinocytes.
Detection of the protein level of PPARβ via western blotting
demonstrated that si-PPARβ significantly decreased, and the PPARβ
overexpression plasmid increased the expression level of PPARβ
compared with their respective controls, indicating that
transfection was successful (Fig.
4A). CCK-8 and clone formation assays indicated that PPARβ
silencing inhibited proliferation in HaCaT keratinocytes, whereas
its overexpression exhibited the opposite effect (Fig. 4B and C). Moreover, PPARβ knockdown significantly
suppressed, while its overexpression promoted the migration of
HaCaT keratinocytes (Fig. 4D). In
addition, western blot analysis revealed that knockdown of PPARβ
decreased the protein level of cyclin D1, CDK6, CDK4, MMP-2 and
MMP-9, while PPARβ overexpression resulted in the opposite effect
(Fig. 4E). The results indicated
that PPARβ promoted proliferation and migration in HaCaT
keratinocytes.
Inhibition of the PI3K/AKT pathway
represses proliferation and migration in HaCaT keratinocytes
To verify the role of the PI3K/AKT signaling pathway
in HaCaT keratinocytes, cells were treated with 50 µmol/l PI3K
inhibitor LY294002, and the results revealed that LY294002
significantly inhibited the phosphorylation level of AKT at S473
and T308 and the p-AKT/AKT ratio (Fig.
5A). CCK-8 and colony formation assays indicated that
inhibition of PI3K significantly reduced cell proliferation
(Fig. 5B and C), and Transwell assay demonstrated that
LY294002 suppressed the migration of HaCaT keratinocytes (Fig. 5D). In addition, protein detection
results revealed that PI3K inhibitor LY294002 significantly reduced
the expression level of proliferation and migration-related
proteins (Fig. 5E). The results
suggested that activation of the PI3K/AKT signaling pathway was
required to maintain the normal functions of HaCaT
keratinocytes.
miR-185 suppresses the proliferation
and migration of HaCaT keratinocytes by targeting PPARβ to inhibit
the PI3K/AKT signaling pathway
In order to confirm whether the effects of miR-185
and PPARβ on HaCaT keratinocytes were mediated by the PI3K/AKT
signaling pathway, miR-185 and PPARβ overexpression plasmid were
co-transfected into HaCaT keratinocytes. The results indicated that
PPARβ overexpression restored the suppressive effect of miR-185
overexpression on the protein level of ILK and PDK1, the
phosphorylation level of AKT at S473 and T308 and the p-AKT/AKT
ratio, indicating that PPARβ promoted activation of the PI3K/AKT
signaling pathway (Fig. 6A). In
addition, PPARβ overexpression reversed the inhibitory effect of
miR-185 overexpression on the proliferation and migration of HaCaT
keratinocytes (Fig. 6B-D).
Moreover, PPARβ overexpression reversed the suppressive effect of
miR-185 overexpression on the protein expression level of cyclin
D1, CDK6, CDK4, MMP-2 and MMP-9, further suggesting that PPARβ and
miR-185 were implicated in regulating proliferation and migration
in HaCaT keratinocytes (Fig. 6E).
The results indicated that miR-185 inhibited the activation of the
PI3K/AKT signaling pathway by targeting PPARβ in HaCaT
keratinocytes.
 | Figure 6Effect of miR-185 and PPARβ
overexpression on the proliferation and migration of HaCaT
keratinocytes. HaCaT keratinocytes were transfected with miR-185
and PPARβ overexpression plasmid or their NCs. (A) The protein
level of PPARβ, ILK, PDK1, p-AKT-S473, p-AKT-T308 and AKT in HaCaT
keratinocytes was determined via western blot analysis. (B)
Proliferation of HaCaT keratinocytes was assessed using Cell
Counting Kit-8 assay. (C) The number of colonies in HaCaT
keratinocytes was determined using colony formation assay. (D)
Transwell assay was used to assess the number of migrated HaCaT
keratinocytes. (E) The protein level of cyclin D1, CDK6, CDK4,
MMP-2 and MMP-9 in HaCaT keratinocytes was assessed via western
blot analysis. *P<0.05. PPARβ, peroxisome
proliferator-activated receptor β; ILK, integrin-linked kinase;
PDK1, phosphoinositide-dependent protein kinase 1; miR, microRNA;
NC, negative control; p, phosphorylated. |
Discussion
Skin damage initiates wound healing, which is a
complex process involving numerous cell types, growth factors,
cytokines and extracellular matrix components (21,22).
Wound repair comprises three main stages, inflammation, new tissue
formation and remodeling, where the formation of new tissue
involves the proliferation and migration of various cell types,
including keratinocytes (3). HaCaT
is the first normal differentiated permanent epithelial cell line
from adult skin, and its function is similar to that of normal
keratinocytes. Its discovery provides a convenient cell model for
the study of human keratinocytes (23,24).
miR-185 has been revealed to be a cancer-related
miRNA participating in various cellular processes, including
proliferation, invasion and migration (25,26).
In the present study, the function of miR-185 in keratinocytes was
explored, and it was demonstrated that miR-185 suppressed the
proliferation and migration of HaCaT keratinocytes, indicating that
miR-185 may be an effective target for wound repair.
Previous studies have revealed that the level of
PPARβ was increased rapidly in the early phase after skin injury,
and it was expressed at a high level during the wound healing
process (27,28). Lack of PPARβ has been reported to
promote the apoptosis of keratinocytes and reduce the migratory
ability of cells in wound healing (29). In the present study, PPARβ was
indicated to be a target gene of miR-185, and its expression was
revealed to be regulated by miR-185. In subsequent experiments,
knockdown of PPARβ was demonstrated to decrease keratinocyte
proliferation and migration, and it was indicated that
overexpression of PPARβ may present a positive effect on wound
repair.
The PI3K/AKT signaling pathway is a classical
signaling pathway regulating cell proliferation, differentiation
and apoptosis (30,31). AKT has been indicated to be
recruited to the cell membrane along with PDK1, and amino acid
residues T308 and S473 of AKT have been demonstrated to be
phosphorylated by PDK1, thereby activating AKT (32). Activated AKT can phosphorylate
multiple target proteins, including E-cadherin, β-catenin and
Vimentin, serving an important regulatory role in tumor
aggressiveness (33,34). Decreased phosphorylation of AKT has
been reported to reduce the proliferation and migration of human
primitive skin keratinocytes, leading to impaired wound healing
(35). The present study revealed
that miR-185 decreased the protein level of ILK, PDK1, p-AKT-S473
and p-AKT-T308, therefore it was hypothesized that the PI3K/AKT
signaling pathway participated in the regulation of keratinocyte
functions. To further verify this hypothesis, LY294002 was used to
suppress the activation of PI3K/AKT signaling pathway. The results
indicated that inhibition of the PI3K/AKT signaling pathway
significantly decreased the proliferation and migration of HaCaT
keratinocytes. Moreover, western blot results demonstrated that
inhibition of the PI3K/AKT signaling pathway reduced the protein
level of cyclin D1, CDK6, CDK4, MMP-2 and MMP-9. Previous studies
have revealed that PPARβ served a vital role in regulating the
PI3K/AKT signaling pathway (15,36).
In the present study, the overexpression results of PPARβ indicated
that PPARβ restored the inhibitory effect of miR-185 overexpression
on the PI3K/AKT signaling pathway. These results verified that the
PI3K/AKT signaling pathway was associated with the proliferation
and migration of keratinocytes.
The present study presents certain limitations.
Firstly, the effect of miR-185 was only examined on HaCaT
keratinocytes, but it is unclear whether it exhibits the same
effect on other types of keratinocytes, therefore additional
confirmation is required. Secondly, only the proliferation and
migration of keratinocytes were examined, without considering the
density, stratification and differentiation of keratinocytes. On
the other hand, in addition to affecting the proliferation and
migration of keratinocytes, whether miR-185 exhibits the same
effect on fibroblasts, endothelial, perivascular and inflammatory
cells remains unknown. Lastly, only in vitro experiments
were conducted without additional in vivo experimental data.
Therefore, these issues should be further explored in future
studies.
In conclusion, the results of the present study
demonstrated that miR-185 inhibited proliferation and migration in
HaCaT keratinocytes by targeting PPARβ to modulate the PI3K/AKT
signaling pathway. The elucidation of the effect of miR-185 on
keratinocyte proliferation and migration may provide a theoretical
basis for the study of factors affecting wound repair. In addition,
the understanding of miR-185 function also provides novel insights
for improving the process of wound repair.
Acknowledgements
Not applicable.
Funding
Funding: No funding was received.
Availability of data and materials
All data generated or analyzed during the present
study are included in this published article.
Authors' contributions
JY and FC conceived and designed the present study.
JY, PD, YQ, XF, HW and FC performed the experiments and acquired
the data. JY performed data analysis and interpretation. JY and PD
were involved in the preparation of the manuscript. JY and PD
confirm the authenticity of all the raw data. All authors have read
and approved the final manuscript.
Ethics approval and consent to
participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
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