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\begin{document}

\title{Over-expression of EBP50 supresses the invasion and migration of CIA-FLS
in vitro by downregulating MMP-9}



\author[1]{Jie Zhou}%
\author[2]{yu gao}%
\author[3]{Chaofei Tu}%
\author[4]{HuaPing Zhou}%
\author[2]{ri su}%
\author[3]{zi huang}%
\author[5]{Junming Li}%
\author[6]{qing luo}%
\author[3]{yang guo}%
\affil[1]{Maternal and Child Health Hospital of Jiangxi Province}%
\affil[2]{Nanchang University}%
\affil[3]{The First Affiliated Hospital of Nanchang University}%
\affil[4]{People’s Hospital of Jiangxi Province}%
\affil[5]{the First Affiliated Hospital of Nanchang University}%
\affil[6]{The First Affiliated Hospital Of Nanchang University}%


\vspace{-1em}



  
  \date{June 05, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Studies had confirmed that the abnormal proliferation and activation of
FLS play a key role in the process of arthropathy. Inhibiting the
abnormal proliferation and activation of FLS may be an effective method
to control the development of RA. Many studies showed that EBP50 was a
powerful factor in inhibiting the abnormal tumor cell proliferation and
activation, but the expression characteristics and function of EBP50 in
FLS had not yet been reported. In this study we showed that
overexpression of EBP50 in CIA-FLS can inhibit the proliferation, which
had no effect on the autophagy, and promoted the cell apoptosis by
activating caspase-3. At the same time, relative~results revealed that
EBP50 overexpression markedly down-regulated the expression of MMP-9,
but had no effect on MMP-2, E-cadherin and \selectlanguage{greek}β-\selectlanguage{english}catenin, suggesting that
one of the mechanisms for EBP50-regulated aggressive behavior of FLS is
reducing MMP-9 production.%
\end{abstract}%



\sloppy


Title Page

Title

\textbf{Over-expression of EBP50 supresses the} \textbf{invasion and
migration of CIA-FLS in vitro by downregulating MMP-9}

Full names

Jie Zhou\textsuperscript{1,2*}, Yujie Gao\textsuperscript{3*}, Chaofei
Tu\textsuperscript{1}, Huaping Zhou\textsuperscript{4},~Rigu
Su\textsuperscript{1},Zikun Huang\textsuperscript{1},~Junming
Li\textsuperscript{1\#},~Qing Luo\textsuperscript{1\#} and Yang
Guo\textsuperscript{1\#}

Institutional addresses

1 Department of Clinical Laboratory, The First Affiliated Hospital of
Nanchang University, Nanchang, Jiangxi 330006, China.

2 Department of Clinical Laboratory, Maternal and Child Health Hospital
of Jiangxi Province,Nanchang, Jiangxi 330006, China.

3 Department of medical college, Nanchang University, Nanchang, Jiangxi
330006,China.

4 Department of Clinical Laboratory, People's Hospital of Jiangxi
Province, Nanchang, Jiangxi 330006, China.

Email addresses for all authors

Jie Zhou: 352346483@qq.com

Yujie Gao: 1024608270@qq.com

Chaofei Tu: 247930460@qq.com

Huaping Zhou: 879123144@qq.com

Rigu Su:~851242097@qq.com

Zikun Huang: 491353062@qq.com

Junming Li: lisir361@163.com

Qing Luo: lxc042@163.com

Yang Guo: 839444295@qq.com

*equally contributed to this study

\#Corresponding Author

Dr. Junming Li. Department of Clinical Laboratory, First Affiliated
Hospital of Nanchang University, Nanchang, Jiangxi 330006, China. Tel:
(+86) 0791-88692794. Fax: (+86) 0791-88623153. E-mail: lisir361@163.com

Qing Luo. Department of Clinical Laboratory, First Affiliated Hospital
of Nanchang University, Nanchang, Jiangxi 330006, China. Tel: (+86)
0791-88692794. Fax: (+86) 0791-88623153. E-mail: lxc042@163.com

Yang Guo. Department of Clinical Laboratory, First Affiliated Hospital
of Nanchang University, Nanchang, Jiangxi 330006, China. Tel: (+86)
0791-88697032. Fax: (+86) 0791-88623153. E-mail: 839444295@qq.com

\textbf{Absract}

Studies had confirmed that the abnormal proliferation and activation of
FLS play a key role in the process of arthropathy. Inhibiting the
abnormal proliferation and activation of FLS may be an effective method
to control the development of RA. Many studies showed that EBP50 was a
powerful factor in inhibiting the abnormal tumor cell proliferation and
activation, but the expression characteristics and function of EBP50 in
FLS had not yet been reported. In this study we showed that
overexpression of EBP50 in CIA-FLS can inhibit the proliferation, which
had no effect on the autophagy, and promoted the cell apoptosis by
activating caspase-3. At the same time, relative~results revealed that
EBP50 overexpression markedly down-regulated the expression of MMP-9,
but had no effect on MMP-2, E-cadherin and \selectlanguage{greek}β-\selectlanguage{english}catenin, suggesting that
one of the mechanisms for EBP50-regulated aggressive behavior of FLS is
reducing MMP-9 production.

\textbf{Keywords:} EBP50, FLS, Rheumatoid arthritis

\textbf{Introduction}

Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease
characterized by aggressive and symmetric
polyarthritis\textsuperscript{{[}1,2{]}}. The principal pathological
changes in RA include chronic persistent synovial inflammation, abnormal
proliferation of synovial cells, extensive infiltration of inflammatory
cells, formation of synovial membrane pannus, gradually eroded cartilage
and subchondral bone, then resulting in destruction of joint tissue
structure and loss of function\textsuperscript{{[}3,4{]}}. RA has been
reported to occured in people aged 40 to 60, while showing predominance
in women more than men. Although current clinical treatment can
alleviate some symptoms, it cannot reverse the damage caused to
cartilage and related bone tissue, and the high disability rate of RA
seriously affects the quality of patients life. The~latest~research have
shown that the occurrence and development of RA is due to a combination
of genetic, environmental and immune factors, but the specific mechanism
is still unclear\textsuperscript{{[}5{]}}.

Fibroblast-like synoviocytes (FLS) are predominant component of joint
synovial membrane\textsuperscript{{[}6{]}}, and plays an pivotal
physiological role in controlling the volume of synovial fluid and
maintaining the stability of joint cavity\textsuperscript{{[}7{]}}. At
the same time, FLS are also important factor leading to joint
pathological damage\textsuperscript{{[}8{]}}. During the occurrence and
development of RA, FLS can occur three major abnormal changes: such as
releasing large amounts of proinflammatory
cytokines\textsuperscript{{[}9{]}}, FLS can promote the occurrence and
maintenance of inflammatory response by involving in the activation of
macrophages, T lymphocytes and NK cells\textsuperscript{{[}10{]}}; such
as dysplasia and apoptosis defect, both proliferation and invasion of
FLS were significantly enhanced\textsuperscript{{[}11{]}}, exhibiting
tumor-like behavior, accompanied by significant inhibition of
apoptosis\textsuperscript{{[}12{]}}; such as secreting matrix degrading
enzymes, including matrix apoptosis-deficient metalloproteinases and
cathepsin,leading to destruction of bone and
cartilage\textsuperscript{{[}13,14{]}}. A large number of studies have
confirmed that abnormal proliferation and activation of FLS play a key
role in the process of joint lesions in RA
patients\textsuperscript{{[}15,16{]}}. Therefore, inhibiting abnormal
proliferation and activation of FLS may be a potentially therapeutic
strategy to block or delay the progression of RA.

EBP50, also known as Na+/H+~exchange regulatory factor (NHERF1), is a
PSD-95/Dlg-1, Drosophila disk large/ZO-1 (PDZ)-containing scaffolding
protein that regulates a variety of physiological functions, such as
controlling the localization, delivery, surface stability and function
of transporters, integral membrane proteins, and ion channel proteins,
as well as clustering of proteins to specific cellular domains to
facilitate signaling. As a novel tumor suppressor gene. Previous studies
have demonstrated that EBP50 expression was down-regulated in breast
cancer\textsuperscript{{[}17{]}}, gastric
cancer\textsuperscript{{[}18{]}} and prostate
cancer\textsuperscript{{[}19{]}}, and overexpression of EBP50 could
effectively inhibit the proliferation of tumor cells and promote the
apoptosis of tumor cells, at the same time, Related researchs also show
that EBP50 can inhibit the migration and invasion of breast cancer cells
by down-regulating MMPs and interacting with
\selectlanguage{greek}β-\selectlanguage{english}catenin\textsuperscript{{[}20,21{]}}.

However, most studies of EBP50 have focused on tumor cells, and it is
not clear whether EBP50 can perform the same function in other cells. In
this study, we investigated the effects and mechanisms of EBP50
overexpression on the Proliferation and Migration of Fibroblast-Like
Synoviocytes in vitro.

\textbf{Materials and Methods}

Materials. Antibodies specific for EBP50 (ab3452), MMP-2 (ab37150),
MMP-9 (ab38898) were purchased from Abcam (San Francisco, CA); Caspase-3
(9662), LC-3 (4108), \selectlanguage{greek}β-\selectlanguage{english}catenin (8480) and E-cadherin(14472) were
purchased from CST (Cell Signaling Technology); \selectlanguage{greek}β-\selectlanguage{english}actin mAb (AC062),HRP
Goat Anti-Rabbit IgG (AS014) and HRP Goat Anti-Mouse IgG (AS003) was
purchased from ABclonal Technology; The CIA-FLS were kindly gifted by
professor Yingyuan Fu from the Department of Immunology, Medical College
of Nanchang University, Nanchang, China, The lentiviral expression
plasmid pLVX-IRES-ZsGreen1 was kindly gifted by ZhangLin Zhang from the
Department of Transfusion, First Affiliated Hospital of Nanchang
University, Nanchang, Jiangxi, China.

{\label{materials.-antibodies-specific-for-ebp50-ab3452-mmp-2-ab37150-mmp-9-ab38898-were-purchased-from-abcam-san-francisco-ca-caspase-3-9662-lc-3-4108-ux3b2-catenin-8480-and-e-cadherin14472-were-purchased-from-cst-cell-signaling-technology-ux3b2-actin-mab-ac062hrp-goat-anti-rabbit-igg-as014-and-hrp-goat-anti-mouse-igg-as003-was-purchased-from-abclonal-technology-the-cia-fls-were-kindly-gifted-by-professor-yingyuan-fu-from-the-department-of-immunology-medical-college-of-nanchang-university-nanchang-china-the-lentiviral-expression-plasmid-plvx-ires-zsgreen1-was-kindly-gifted-by-zhanglin-zhang-from-the-department-of-transfusion-first-affiliated-hospital-of-nanchang-university-nanchang-jiangxi-china.}}

Cell culture. The CIA-FLS were maintained in Dulbecco's modified Eagle
medium (DMEM, Sigma-Aldrich) supplemented with 10\% fetal bovine serum
(FBS). 293T cells (ATCC, TIB-202) were maintained in RPMI 1640 medium
(GIBCO, Grand Island, NY) supplemented with 10\% FBS. The cells were
cultured at 37 with an atmosphere of 5\% CO\textsubscript{2} and 95\%
air.

{\label{cell-culture.-the-cia-fls-were-maintained-in-dulbeccos-modified-eagle-medium-dmem-sigma-aldrich-supplemented-with-10-fetal-bovine-serum-fbs.-293t-cells-atcc-tib-202-were-maintained-in-rpmi-1640-medium-gibco-grand-island-ny-supplemented-with-10-fbs.-the-cells-were-cultured-at-37-with-an-atmosphere-of-5-co2-and-95-air.}}

Lentivirus vector construction. The EBP50 encoding gene was amplified
from RAW264.7 cells by reverse-transcription-polymerase chain reaction
(RT-PCR). The primers containing EcoRI and BamHI sites for PCR were as
follows, forward primer: 5'-AGAGAATTC-ATGAGCGCGGACG-3'; reverse primer:
5'-CGCGGATCC-TCAGAGGTTGCTGAAGA-3'. The thermal cycling condition for PCR
was 95 for 5 min, 30 cycles at 94 for 40 s, 54 for 30 s and 72 for
90 s, followed by a final round of 72 for 10 min. The PCR product was
digested by EcoRI and BamHI, and ligated with linearized
Plvx-Ires-ZsGreen1 by T4 DNA ligase to form recombinant lentiviral
expression vector Plvx-EBP50.

{\label{lentivirus-vector-construction.-the-ebp50-encoding-gene-was-amplified-from-raw264.7-cells-by-reverse-transcription-polymerase-chain-reaction-rt-pcr.-the-primers-containing-ecori-and-bamhi-sites-for-pcr-were-as-follows-forward-primer-5-agagaattc-atgagcgcggacg-3-reverse-primer-5-cgcggatcc-tcagaggttgctgaaga-3.-the-thermal-cycling-condition-for-pcr-was-95-for-5-min-30-cycles-at-94-for-40-s-54-for-30-s-and-72-for-90-s-followed-by-a-final-round-of-72-for-10-min.-the-pcr-product-was-digested-by-ecori-and-bamhi-and-ligated-with-linearized-plvx-ires-zsgreen1-by-t4-dna-ligase-to-form-recombinant-lentiviral-expression-vector-plvx-ebp50.}}

\subsubsection*{}

{\label{section}}

Lentiviral vectors package. 293T cells were cultured into 6-well plates
at 37 \selectlanguage{ngerman}°C with 5\% CO2 in humidified atmosphere. After reaching
70\%\textasciitilde{}80\% confluence, 293T cells were triple transfected
with the lentiviral expression vector Plvx-EBP50 and two packaging
vectors pSPAX2 and pMD2.G as a group named LV-EBP50, and
Plvx-Ires-ZsGreen1, pSPAX2 with pMD2.G as a group named LV-Plvx by using
FuGene 6 transfect regent following the manufacturers protocol. Culture
supernatants were collected every 24 h for 3 days, filtered through a
0.45-\selectlanguage{greek}μ\selectlanguage{english}m poresize filter, and concentrated two times with
ultracentrifugation at 50,000\selectlanguage{ngerman}× g for 120min. Each group was diluted
1:10, 1:100, and 1:1000 times to determine virus titers. The virus
particles were resuspended in sterile PBS, and stored at --80 °C until
use.

{\label{lentiviral-vectors-package.-293t-cells-were-cultured-into-6-well-plates-at-37-c-with-5-co2-in-humidified-atmosphere.-after-reaching-7080-confluence-293t-cells-were-triple-transfected-with-the-lentiviral-expression-vector-plvx-ebp50-and-two-packaging-vectors-pspax2-and-pmd2.g-as-a-group-named-lv-ebp50-and-plvx-ires-zsgreen1-pspax2-with-pmd2.g-as-a-group-named-lv-plvx-by-using-fugene-6-transfect-regent-following-the-manufacturers-protocol.-culture-supernatants-were-collected-every-24-h-for-3-days-filtered-through-a-0.45-ux3bcm-poresize-filter-and-concentrated-two-times-with-ultracentrifugation-at-50000-g-for-120min.-each-group-was-diluted-110-1100-and-11000-times-to-determine-virus-titers.-the-virus-particles-were-resuspended-in-sterile-pbs-and-stored-at-80-c-until-use.}}

Real-time PCR. Total cellular RNA was extracted from cells using TRIzol
reagent (Invitrogen, Carlsbad, CA), following the manufacturer's
instruction, then reverse-transcribed into complementary DNA using the
Prime Script RT reagent kit (Takara, Shiga, Japan) according to the
manufacturer's instructions. The expression of EBP50 was determined by
real-time PCR using SYBR Premix Ex Taq (Takara, Shiga, Japan) and Step
One Plus Real-Time PCR System (Applied Biosystems, CA). Primers used for
PCR amplification were: GAPDH: 5-GCACCGTCAAGGCTGAGAAC-3\selectlanguage{english}' (forward),
5\selectlanguage{english}'-TGGTGAAGACGCCAGTGGA-3\selectlanguage{english}' (reverse); EBP50:
5'-AGGCCAGTTCATCCGAGCAG-3'(forward), 5'-CAGCAGCTTGGCCTCATCAC-3'
(reverse). The mRNA levels of EBP50 relative to the control was
calculated by 2\selectlanguage{english}\textsuperscript{-^^CT}.

{\label{real-time-pcr.-total-cellular-rna-was-extracted-from-cells-using-trizol-reagent-invitrogen-carlsbad-ca-following-the-manufacturers-instruction-then-reverse-transcribed-into-complementary-dna-using-the-prime-script-rt-reagent-kit-takara-shiga-japan-according-to-the-manufacturers-instructions.-the-expression-of-ebp50-was-determined-by-real-time-pcr-using-sybr-premix-ex-taq-takara-shiga-japan-and-step-one-plus-real-time-pcr-system-applied-biosystems-ca.-primers-used-for-pcr-amplification-were-gapdh-5-gcaccgtcaaggctgagaac-3-forward-5-tggtgaagacgccagtgga-3-reverse-ebp50-5-aggccagttcatccgagcag-3forward-5-cagcagcttggcctcatcac-3-reverse.-the-mrna-levels-of-ebp50-relative-to-the-control-was-calculated-by-2ct.}}

Western blotting. The cellular protein was collected and lysed using
RIPA lysis buffer containing 1 mM phen-ylmethylsulfonyl fluoride and 1\%
(vol/vol) protease inhibitor cocktail (Beyotime, Haimen, Jiangsu,
China).Equal amounts of proteins were separated on sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and
transferred to polyvinylidene fluoride membranes at 4\selectlanguage{english}for 1 h. Membranes
were blocked in 5\% BSA and incubated with primary antibodies against
EBP50, LC3, Caspase-3, \selectlanguage{greek}β-\selectlanguage{english}catenin, E-cadherin, Akt, MMP-2 and MMP-9 at 4
overnight. Then membranes were incubated at room temperature for 1 h
with relevant secondary antibodies, and blots were visualized using
enhanced chemiluminescence (ECL; Thermo Pierce, Rockford, Illinois, USA)
according to the manufacturer's instructions and quantified by
densitometry using Quantity One image software with \selectlanguage{greek}β-\selectlanguage{english}actin used as an
internal control.

{\label{western-blotting.-the-cellular-protein-was-collected-and-lysed-using-ripa-lysis-buffer-containing-1-mm-phen-ylmethylsulfonyl-fluoride-and-1-volvol-protease-inhibitor-cocktail-beyotime-haimen-jiangsu-china.equal-amounts-of-proteins-were-separated-on-sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis-sds-page-gels-and-transferred-to-polyvinylidene-fluoride-membranes-at-4for-1-h.-membranes-were-blocked-in-5-bsa-and-incubated-with-primary-antibodies-against-ebp50-lc3-caspase-3-ux3b2-catenin-e-cadherin-akt-mmp-2-and-mmp-9-at-4-overnight.-then-membranes-were-incubated-at-room-temperature-for-1-h-with-relevant-secondary-antibodies-and-blots-were-visualized-using-enhanced-chemiluminescence-ecl-thermo-pierce-rockford-illinois-usa-according-to-the-manufacturers-instructions-and-quantified-by-densitometry-using-quantity-one-image-software-with-ux3b2-actin-used-as-an-internal-control.}}

\textbf{MTT cytotoxicity assay.} FLS were cultured in DMEM with 10\%
heat-inactivated FBS and antibiotics at 37 \selectlanguage{ngerman}°C in a humidifified
atmosphere with 5\% CO2. 5×10\textsuperscript{3} cells/well were plated
in a 96-well plate. After 12 h, 20 \selectlanguage{greek}μ\selectlanguage{english}L MTT solution (0.5 mg/mL) was added
in FLS culture mediumand incubated for 4 h. The culture mediumand in
each well was discarded and added 150 \selectlanguage{greek}μ\selectlanguage{english}L~DMSO. The absorption of each
well was measured at 490 nm.

\textbf{Phagocytosis assay by flow cytometry (FCM).} FLS transduced with
or without recombinant lentivirus for 48 h,72 h,96 h. Then cells were
washed thoroughly with D-Hanks buffer to remove extracellular
recombinant lentivirus and depleted media, at the same time, fls were
transfered seedind in 96-well plates 5\selectlanguage{ngerman}×10\textsuperscript{3}/cells with
annexin V binding buffer resuspended. Then were incubated at 2-8\selectlanguage{english} for 15
min with 5\selectlanguage{greek}μ\selectlanguage{english}l Annexin V-FITC, and were incubated at 2-8 for 5 min with
5\selectlanguage{greek}μ\selectlanguage{english}l PI, finally cells were collected and analyzed using Cytomics FC500
flow cytometer (Beckman Coulter Inc., Brea, CA, USA).

\textbf{In Vitro Migration and Invasion Assay of FLS} . In vitro cell
migration assays were performed as described previously using Trans-well
chambers (8 \selectlanguage{greek}μ\selectlanguage{english}M pore size,Corning, NY, USA). When cells reached
subconfluency (75\%--80\%), cells were serum-starved for 24 h. After
detachment with trypsin, cells were washed with PBS and resuspended in
serum-free medium. Next, 100 \selectlanguage{greek}μ\selectlanguage{english}L of cell suspension
(2\selectlanguage{ngerman}×10\textsuperscript{5} cells/mL) was added to the upper chamber while
complete medium was added to the bottom wells. 24 h later, cells that
had not migrated were removed from the upper surface of the filters
using cotton swabs. Cells that had migrated were fixed with 5\%
glutaraldehyde solution to determine the number of migratory cells. The
lower surface of the filters was stained with crystal violet. Images of
three fields were captured from each membrane, and the number of
migratory cells were counted. The mean of triplicate assays for each
experimental condition was used.

\textbf{Statistical analysis.} All the presented data and results were
confirmed in at least three independent experiments. Unpaired Student's
t test or one-way analysis of variance was used to determine the
significance of theresults. Data were considered statistically
significant at P\textless{} 0.05.

\textbf{Results}

\textbf{Overexpression of EBP50 Suppresses the proliferation of FLS}

Inhibiting abnormal proliferation and activation of FLS may be a
potentially therapeutic strategy to block or delay the progression of
RA. To investigate the potential effects of EBP50 on the proliferation
of FLS, fls were transfected with LV-EBP50 or LV-Plvx for 72 h, then
analyzed for the EBP50 expression and the proliferation of FLS. The
results showed that LV-EBP50 transfection significantly increased the
expression of EBP50 in both mRNA levels (Fig. 1A) and protein levels
(Fig. 1B-1C), and MTT assay demonstrated that overexpression of EBP50
can suppresse the proliferation of FLS significantly, when compared with
untransfected cells and LV-Plvx transfected cells (Fig. 1D). It has been
reported that EBP50 can inhibit the proliferation of breast cancer cells
by stimulating the autophagic lysosomal degradation, so we speculate
whether EBP50 inhibits FLS cell proliferation by inhibiting autophagy.
Then, the expression of LC3 was measured by western blotting in LV-EBP50
and LV-plvx transfected cells and untreated cells.As shown in (Fig.
1E-1F), the expressions of LC3 in FLS remain unchanged after transfected
with LV-EBP50.Collectively, these results suggest that overexpression of
EBP50 suppresses the proliferation of FLS and has no influence on
autophagy.

\textbf{Overexpression of EBP50 promote apotosis of FLS By activating
caspase-3}

FLS can exhibit tumor-like behavior, such as significant inhibition of
apoptosis. As the major tumor suppressor genes, related literature
reports have indicated that overexpression of EBP50 promoted
significantly the tumor cells apotosis, and our previous results have
showed that the overexpression of EBP50 upregulates the apoptosis ratio
of macrophage by increasing the expression of Bax and caspase-3 in an
NO-dependent mechanism. To investigated whether EBP50 can perform the
same function in FLS, FLS were transfected with LV-EBP50 and LV-Plvx at
the MOIs of 10 for 48 h, 72 h, 96 h, and the cell apoptosis of FLS was
analyzed by FCM. Results showed that EBP50 overexpression increased
distinctly the apoptosis of FLS when compared with LV-Plvx transduced
FLS (P\textless{} 0.001) (Fig. 2A); The expression of apoptosis-related
protein caspase-3 in FLS were measured by western blotting. As shown in
(Fig. 2B), when FLS were transduced with LV-EBP50 and LV-Plvx for 96h
,the protein expressions level of cleaved caspase-3 were significantly
increased .

\textbf{overexpression of EBP50Suppresses the} \textbf{Migration and
Invasion of FLS}

\textbf{By downregulating MMP-9}

Related researchs have showed that EBP50 could inhibit the migration and
invasion of breast cancer cells by down-regulating MMPs and interacting
with \selectlanguage{greek}β-\selectlanguage{english}catenin(30,33). Since FLS migration and invasion are essential
steps leading to joint destruction of RA, we evaluated the effect of
EBP50 on these aggressive phenotypes of FLS~in vitro. The primary FLS
were left untreated (CON) or transfected with LV-EBP50 or LV-Plvx for 72
h, then the migration and invasion of FLS were evaluated using the
transwell assay. FLS overexpressing EBP50 displayed dramatic reduction
in the number of migrating and invading cells across transwell assay
(Fig. 3A-3B). As we known, the FLS could express MMPs and a variety of
surface adhesion molecules to influence the migration and invasion. To
study the potential mechanisms underlying the biological effect of EBP50
in FLS, the expression of \selectlanguage{greek}β-\selectlanguage{english}catenin , E-cadherin , MMP2 and MMP9 were
analyzed in LV-EBP50 and LV-Plvx transfected cells and untreated cells.
The results have showed that protein levels of \selectlanguage{greek}β-\selectlanguage{english}catenin were unchanged
and E-cadherin were not expressed (Fig. 3C-3D), and the expression of
MMP-9 in the EBP50-overexpressing cells were significantly decreased,
whereas MMP-2 remained the same (Fig. 3E-3F-3G).These results have
indicated that overexpression of EBP50 impaired both the migratory and
invasive ability of FLS by downregulating MMP-9.

\textbf{DISCUSSION}

So far, the pathogenesis of RA has not been completely clarified,
therefore no therapeutic specific has been found clinically. The current
treatment mainly focuses on drug therapy and surgical treatment. Current
clinically used drugs include nonsteroidal anti-inflammatory drugs,
glucocorticoid, disease modifying antirheumatic drugs, biologics,
traditional~Chinese~medicine, etc. However, various therapies have their
own limitations in the clinical treatment of RA. Recent studies have
suggested genetic approach may offer new therapeutic methods, several
genetics have been identified as genetic factors that contribute to RA,
or have potential role in regulating the pathogenesis of RA, such as
microRNAs\textsuperscript{{[}22,23{]}},
lncRNAs\textsuperscript{{[}24{]},} etc. There is growing evidence that
FLS of active RA play a major role in the onset and progression of RA.
In addition, FLS obtained from synovia of patients with RA exhibit
tumour-like behaviours, including aggressive proliferation, increased
migration, invasion, and reduced apoptosis. Emerging evidence indicate
that inhibition of RA-FLS effector molecules might be beneficial for RA
therapy. In this study, we expolred the role of overexpression of EBP50
in FLS proliferation, migration/invasion, and cell apoptosis.

There have many studies reported that EBP50 expression was
down-regulated in breast cancer, gastric cancer and prostate cancer, and
overexpression of EBP50 could effectively inhibit the proliferation of
tumor cells and promote the apoptosis of tumor cells. Here in our study,
to explore the potential effects of EBP50 on the proliferation of FLS,
FLS were left untreated (CON) or transfected with control lentivirus
(LV-Plvx) or LV-EBP50 for 72 h, the expression of EBP50 were detected by
WB and qRT-PCR firstly. The results indicated that the expression of
EBP50 was significantly up-regulated in LV-EBP50 transfected cells
compared with untransfected cells and LV-Plvx transfected cells. Then,
we found that EBP50 overexpression in FLS significantly inhibited cell
proliferation by MTT assay, consistent with previous reports showing
EBP50 as an inhibitor against cell proliferation in certain
proliferative diseases such as cancer\textsuperscript{{[}25-27{]}}. At
the same time, EBP50 can stimulate autophagy and silencing EBP50
restrains it in cytoplasm\textsuperscript{{[}28{]}}. EBP50, an activator
of autophagy, causes lysosomal degradation of c-Myc to suppress the
growth of MCF-7 breast cancer cells
overexpressing\textsuperscript{{[}29{]}}. Recent studies also have shown
that in RA-FLS from RA patients or from animal models of RA, the
autophagy pathway is persistently stimulated and apoptosis is
downregulated, leading to synovial fibroblast proliferation and
activation. So, we investigated the effect of EBP50 overexpression on
autophagy in FLS. We observed that the autophagy-related proteins LC3
remain unchanged. Our results thus suggest that EBP50 overexpression had
no effect on the autophagy of FLS, which might inhibit FLS proliferation
through other pathways.

Apoptosis is another important target for anticancer therapy. Related
literature reports that overexpression of EBP50 significantly promoted
the tumor cells apotosis, and we found that overexpression of EBP50
upregulates the apoptosis ratio of macrophage by increasing the
expression of Bax and caspase-3 in an NO-dependent mechanism in a
previous study\textsuperscript{{[}30{]}}. Thus, we investigated whether
EBP50 can perform the same function in FLS. It is well known that the
Bcl-2 and caspase families play a vital role in cell survival/death. The
Bcl-2 family can be functionally divided into antiapoptotic members
(Bcl-2, Bcl-XL, Bcl-w, and CED 9), and proapoptotic members (Bax, Bad,
and Bid)\textsuperscript{{[}31,32{]}}. Among the members of the caspase
family, caspases-3, -6, and -7 are the effector caspases involved in the
execution of cells in response to apoptotic stimuli, and caspase-3, the
most important terminal executive molecule in the process of apoptosis,
activation will lead to irreversible
apoptosis\textsuperscript{{[}33{]}}. In this study, after transduced
with LV-EBP50, the apoptosis of FLS were increased when compared with
LV-Plvx transduced FLS (Fig. 4B). Furthermore, we evaluated the
expression of the apoptosis-related proteins Caspase3 (Fig. 4B). The
results showed that cleaved caspase-3 were significantly increased in
LV-EBP50-transduced FLS, which revealed that EBP50 overexpression
promote apotosis of FLS through activating caspase-3. Our results
demonstrated EBP50 transient overexpression significantly increased
RA-FLS apoptosis, and therefore, may offer novel approaches against
synovial hyperplasia.

FLS is the major cell population involved in the development of RA in
synovial tissues. Previous studies have shown that FLS migrate and
invade the cartilage and bone, contributing to pannus formation and
tissue damage during RA progression. Regulation of FLS migration and
invasion may be a new therapeutic strategy to prevent the destructive
progress of RA. Prior studies indicated that the suppression of FLS
migration and invasion might protect against joint destruction in RA.
Therefore, we determined the effect of overexpression of EBP50 on the
migration and invasion of FLS in this study. As a result, transfection
of FLS with the LV-EBP50 over-expression vector had a profound
inhibitory effect on their migration and invasion. It is well documented
that membrane-associated proteins formed by the combination of
E-cadherin and \selectlanguage{greek}β-\selectlanguage{english}catenin participate in the regulation of cell adhesion,
and cell movement, including metastasis and invasion, is also regulated
by membrane-associated adhesion proteins. EBP50 stabilizes \selectlanguage{greek}β-\selectlanguage{english}catenin on
the cell surface by interacting with \selectlanguage{greek}β-\selectlanguage{english}catenin and promotes the binding
of \selectlanguage{greek}β-\selectlanguage{english}catenin to E-cadherin complex, inhibiting cell migration and thus
tumor cell metastasis. In addition, Matrix metalloproteinases (MMPs) are
the main proteases responsible for the invasion and degradation of
basement membranes and extracellular matrix. MMP-1, MMP-3, MMP-9, and
MMP-13 are markedly elevated in synovial fluid from RA patients and play
a significant role in collagen degradation, causing disease progression
and bone erosion\textsuperscript{{[}34{]}}. Among these proteins,
endogenous MMP2 and MMP9 contribute to synovial fibroblast survival,
proliferation, migration, and invasion in RA \textsuperscript{{[}35{]}}.
Studies have shown that EBP50 can down-regulate the expression of MMPs
in breast cancer cells, especially MMP-2 and MMP-9, thus inhibiting the
invasion ability of breast cancer cells, indicating that EBP50 can
inhibit the invasion of tumor cells by inhibiting the expression of
MMPs. To determine the effect of EBP50 on E-cadherin, \selectlanguage{greek}β-\selectlanguage{english}catenin, MMP2
and MMP9 expression of FLS, we used western blot to measure the
expression of E-cadherin, \selectlanguage{greek}β-\selectlanguage{english}catenin, MMP2 and MMP9. In the present
study, we observed that EBP50 overexpression markedly down-regulated the
expression of MMP-9, but had no effect on MMP-2, E-cadherin and
\selectlanguage{greek}β-\selectlanguage{english}catenin, suggesting that one of the mechanisms for EBP50-regulated
aggressive behavior of FLS is reducing MMP-9 production.

\textbf{Conclusions}

To summarize, EBP50 suppressed FLS proliferation, migration and
invasion, and induced apoptosis. These findings have therapeutic
significance and indicate the potential of EBP50 as a therapeutic target
for RA, because the EBP50-mediated effects on FLS involve targeting
synovial hyperplasia. Further research is needed to investigate the
pathways involved in these protective effects and clarify if EBP50 could
reduce proinflammatory cytokine secretion from RA-FLS.

\textbf{Acknowledgements}

This work was supported by the National Natural Science Foundation of
China (81660277), Jiangxi Provincial Natural Science Foundation of China
(20202BAB216001), the Science and Technology Project of Health and
Family Planning Commission of Jiangxi Provinceof China (20203091).

\textbf{Disclosure}

The authors declare no conflicts of interest.

\textbf{References}

{[}1{]} Charlotte Hua, Claire I Daien, Bernard Combe, et al. Diagnosis,
prognosis and classification of early arthritis: results of a systematic
review informing the 2016 update of the EULAR recommendations for the
management of early arthritis{[}J{]}. RMD Open. 2017;3(1):e000406.

{[}2{]} Josef S Smolen, Robert B M Landew\selectlanguage{ngerman}é, Johannes W J Bijlsma, et al.
EULAR recommendations for the management of rheumatoid arthritis with
synthetic and biological disease-modifying antirheumatic drugs: 2019
update{[}J{]}.Ann Rheum Dis. 2020;79(6):685-699.

{[}3{]} Buch MH, Emery P. New therapies in the management of rheumatoid
arthritis{[}J{]}. Curr Opin Rheumatol.2011;23(3):245-251.

{[}4{]} Chan AC, Carter PJ. Therapeutic antibodies for autoimmunity and
inflammation{[}J{]}. Nat Rev Immunol.2010;10(5):301-316.

{[}5{]} Gary Firestein, MD, Iain B McInnes FRCP PhD2. Immunopathogenesis
of rheumatoid arthritis{[}J{]}. Immunity. 2017;46(2):183-196.

{[}6{]} Bartok B, Firestein GS. Fibroblast-like synoviocytes: key
effector cells in rheumatoid arthritis{[}J{]}.Immunol
Rev.2010;233(1):233-255.

{[}7{]} Iwanaga T, Shikichi M, Kitamura H, et al. Morphology and
functional roles of synoviocytes in the joint{[}J{]}. Arch Histol
Cytol,2000; 63(1):17-31.

{[}8{]} Chang SK, Gu Z,Brenner MB. Fibroblast-like synoviocytes in
inflammatory arthritis pathology: the emerging role of
cadherin-11{[}J{]}. Immunol Rev.2010;233(1):256-266.

{[}9{]} McInnes IB, Schett G. Cytokines in the pathogenesis of
rheumatoid arthritis{[}J{]}. Nat Rev Immunol.2007;7(6):429-442.

{[}10{]} Hu F, Liu H, Xu L, et al. Hypoxia-inducible
factor-1\selectlanguage{greek}α\selectlanguage{english}perpetuates synovial fibroblast interactions with T cells and B
cells in rheumatoid arthritis{[}J{]}. Eur J Immunol.2016;46(3):742-751.

{[}11{]} Garc\selectlanguage{ngerman}ía S, Liz M, Gómez-Reino JJ, et al. Akt activity protects
rheumatoid synovial fibroblasts from Fas-induced apoptosis by inhibition
of Bid cleavage{[}J{]}. Arthritis Res Ther.2010;12(1):R33.

{[}12{]} Kim SK, Park KY, Yoon WC, et al. Melittin enhances apoptosis
through suppression of IL-6/sIL-6R complex-induced NF- \selectlanguage{greek}κ \selectlanguage{english}B and STAT3
activation and Bcl-2 expression for human fibroblast-like synoviocytes
in rheumatoid arthritis{[}J{]}. Joint Bone Spine.2011;78(5):471-477.

{[}13{]} Hsiao HB, Hsieh CC, Wu JB, et al. Kinsenoside inhibits the
inflammatory mediator release in a type-II collagen induced arthritis
mouse model by regulating the T cells responses{[}J{]}. BMC Complement
Altern Med.2016;16(1):80.

{[}14{]} Mu N, Gu J, Huang T, et al. A novel NF- \selectlanguage{greek}κ \selectlanguage{english}B/YY1/microRNA-10a
regulatory circuit in fibroblast-like synoviocytes regulates
inflammation in rheumatoid arthritis{[}J{]}. Sci Rep.2016;6:20059.

{[}15{]} Shang CH,Zhang QQ,Zhou JH.Oridonin Inhibits Cell Proliferation
and Induces Apoptosis in Rheumatoid Arthritis Fibroblast-Like
Synoviocytes{[}J{]}. Inflammation.~2016 ;39(2):873-80.

{[}16{]} Zou S, Wang C, Cui Z, et al. \selectlanguage{greek}β -\selectlanguage{english}Elemene induces apoptosis of
human rheumatoid arthritis fibroblast-like synoviocytes via reactive
oxygen species-dependent activation of p38 mitogen-activated protein
kinase{[}J{]}. Pharmacol Rep.2016;68(1):7-11.

{[}17{]} Yao W, Feng D, Bian W, et al. EBP50 inhibits EGF-induced breast
cancer cell proliferation by blocking EGFR phosphorylation{[}J{]}. Amino
Acids.2012;43(5):2027-2035.

{[}18{]} Lv XG, Ji MY, Dong WG, et al. EBP50 gene transfection promotes
5-fluorouracil-induced apoptosis in gastric cancer cells through Bax-
and Bcl-2-triggered mitochondrial pathways{[}J{]}. Mol Med
Rep.2012;5(5):1220-1226.

{[}19{]} Bartholow TL, Becich MJ, Chandran UR, et al.
Immunohistochemical analysis of ezrin-radixin-moesin-binding
phosphoprotein 50 in prostatic adenocarcinoma{[}J{]}. BMC
Urol.2011;11:12.

{[}20{]} Peng XL, Ji MY, Yang ZR, et al. Tumor suppressor function of
ezrin-radixin-moesin-binding phosphoprotein-50 through \selectlanguage{greek}β
-\selectlanguage{english}catenin/E-cadherin pathway in human hepatocellular cancer{[}J{]}. World
J Gastroenterol.2013;19(8):1306-1313.

{[}21{]} Li H, Zhang B, Liu Y, et al. EBP50 inhibits the migration and
invasion of human breast cancer cells via LIMK/cofilin and the
PI3K/Akt/mTOR/MMP signaling pathway{[}J{]}. Med Oncol.2014;31(9):162.

{[}22{]} Jie Wu,~Wenqiang Fan,~Ling Ma, et al. miR-708-5p promotes
fibroblast-like synoviocytes' cell apoptosis and ameliorates rheumatoid
arthritis by the inhibition of Wnt3a/\selectlanguage{greek}β-\selectlanguage{english}catenin pathway{[}J{]}. Drug Des
Devel Ther. 2018;12:3439-3447.

{[}23{]} Jie Gao, Ruina Kong, Xiaoli Zhou , et al. MiRNA-126 expression
inhibits IL-23R mediated TNF-\selectlanguage{greek}α \selectlanguage{english}or IFN-\selectlanguage{greek}γ\selectlanguage{english}production in fibroblast-like
synoviocytes in a mice model of collageninduced rheumatoid
arthritis{[}J{]}.Apoptosis. 2018;23(11-12):607-615.

{[}24{]} Chun-Wang Zhang, Xia Wu, Dan Liu, et al. Long non-coding RNA
PVT1 knockdown suppresses fibroblast-like synoviocyte inflammation and
induces apoptosis in rheumatoid arthritis through demethylation of
sirt6{[}J{]}. J Biol Eng. 2019;13:60.

{[}25{]} Yao W, Feng D, Bian W, et al. EBP50 inhibits EGF-induced breast
cancer cell proliferation by blocking EGFR phosphorylation{[}J{]}. Amino
Acids. 2012;43(5):2027-2035.

{[}26{]} Lv XG, Ji MY, Dong WG, et al. EBP50 gene transfection promotes
5-fluorouracil-induced apoptosis in gastric cancer cells through Bax-
and Bcl-2-triggered mitochondrial pathways{[}J{]}. Mol Med
Rep.2012;5(5):1220-1226.

{[}27{]} Bartholow TL, Becich MJ, Chandran UR, et al.
Immunohistochemical analysis of ezrin-radixin-moesin-binding
phosphoprotein 50 in prostatic adenocarcinoma{[}J{]}. BMC
Urol.2011;11:12.

{[}28{]} Tang MK, Zhang JT. Caspase and its effects on apoptosis. Bull
Med Res. 2000;

{[}29{]} Minxi Lao, Maohua Shi, Yaoyao Zou, et al. Protein inhibitor of
activated STAT3 regulates migration, invasion, and activation of
fibroblastlike synoviocytes in rheumatoid arthritis{[}J{]}. J Immunol.
2016;196(2):596-606.

{[}30{]} Guo Y, Deng Y, Huang Z, et al. EBP50 induces apoptosis in
macrophages by upregulating nitric

oxide production to eliminate intracellular Mycobacterium
tuberculosis{[}J{]}. Sci Rep.2016;6:18961.

{[}31{]} A. Pena-Blanco, A.J. Garcia-Saez. Bax, Bak and beyond -
mitochondrial performance in apoptosis{[}J{]}, FEBS J. 285 (3) (2018)
416--431.

{[}32{]} M.A. Elhinnawi, R.M. Mohareb, H.M. Rady, et al. Novel
pregnenolone derivatives modulate apoptosis via Bcl-2 family genes in
hepatocellular carcinoma in vitro{[}J{]}. Steroid Biochem. Mol. Biol.
183 (2018) 125--136.

{[}33{]} Bao J, Wang S, Gunther LK, et al. The actin-bundling protein
TRIOBP-4 and -5 promotes the motility of pancreatic cancer cells{[}J{]}.
Cancer Lett.~2015;356(2 Pt B):367-73.

{[}34{]} Shan Zeng, Kefeng Wang , Mingcheng Huang, et al. Halofuginone
inhibits TNF-\selectlanguage{greek}α-\selectlanguage{english}induced the migration and proliferation of
fibroblast-like synoviocytes from rheumatoid arthritis patients{[}J{]}.
Int Immunopharmacol. 2017;43:187-194.

{[}35{]} P Pratheeshkumar, Girija Kuttan. Andrographolide inhibits human
umbilical vein endothelial cell invasion and migration by regulating
MMP-2 and MMP-9 during angiogenesis{[}J{]}. J Environ Pathol Toxicol
Oncol. 2011;30(1):33-41.

\textbf{Figure legends}

\textbf{Figure 1. Overexpression of EBP50 Suppresses the proliferation
of FLS.}

FLS were left untreated (CON) or transfected with control lentivirus
(LV-Plvx) or LV-EBP50 for 72 h.The mRNA levels of EBP50 were measured by
real-time RT-PCR (A). The protein levels of EBP50 were detected by
Western blotting (B).Band density of the specific protein was analyzed
with Quantity One image software, and the results were expressed as
average density to \selectlanguage{greek}β-\selectlanguage{english}actin (C).FLS were left untreated (CON) or
transfected with control lentivirus (LV-Plvx) or LV-EBP50 for 48 h, Then
were transfered seedind in 96-well plates
5\selectlanguage{ngerman}×10\textsuperscript{3}/cells,after night,20 \selectlanguage{greek}μ\selectlanguage{english}L MTT solution (0.5 mg/mL)
was added in FLS culture mediumand incubated for 4 h in 5\% CO2
atmosphere at 37. The culture mediumand in each well was discarded and
added 150 \selectlanguage{greek}μ\selectlanguage{english}L~DMSO. The absorption of each well was measured at 490
nm.(D). The protein levels of LC3 were detected by Western blotting
(E).Band density of the specific protein was analyzed with Quantity One
image software, and the results were expressed as average density to
\selectlanguage{greek}β-\selectlanguage{english}actin (F).Data are represented as mean\selectlanguage{ngerman}±SEM of at least three
independent experiments.*P \textless{} 0.05, **P \textless{} 0.01,***P
\textless{} 0.001 compared with the uninfected group.

\textbf{Figure 2. Overexpression of EBP50 promote apotosis of FLS By
activating caspase-3}

FLS were left untreated (CON) or transfected with control lentivirus
(LV-Plvx) or LV-EBP50 for 48 h,72 h,96 h.the apoptosis incidence was
measured by FCM(A).The expression levels of caspase-3 were determined by
Western blotting(B). Data are represented as mean±SEM of at least three
independent experiments.*P \textless{} 0.05, **P \textless{} 0.01,***P
\textless{} 0.001 compared with the uninfected group.

\textbf{Figure 3. overexpression of EBP50 Suppresses the Migration and
Invasion of FLS}

\textbf{By downregulating MMP-9}

FLS were left untreated (CON) or transfected with control lentivirus
(LV-Plvx) or LV-EBP50 for 72 h. Then the effect of EBP50 on migration
was detected with transwell Boyden chamber,The images are representative
of migration through the membrane after staining. Original
magnifification 200×(A,C). The effect of EBP50 on migration was detected
with transwell Boyden chamber,The images are representative of invasion
through the membrane after staining. Original magnifification
200×(B,D).The expression levels of \selectlanguage{greek}β-\selectlanguage{english}catenin and E-cadherin were
determined by Western blotting(E). Band density of the specific protein
was analyzed with Quantity One image software (F). The expression levels
of MMP-2 and MMP-9 were determined by Western blotting(G). Band density
of the specific protein was analyzed with Quantity One image software
(H-I). Data are represented as mean\selectlanguage{ngerman}±SEM of at least three independent
experiments.*P \textless{} 0.05, **P \textless{} 0.01,***P \textless{}
0.001 compared with the uninfected group.

\textbf{Figure 1. Overexpression of EBP50 Suppresses the proliferation
of FLS.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 2. Overexpression of EBP50 promote apotosis of FLS By
activating caspase-3}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 3. overexpression of EBP50 Suppresses the Migration and
Invasion of FLS}

\textbf{By downregulating MMP-9}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& CON & LV-Plvx & LV-EBP50\tabularnewline
A & & &\tabularnewline
B & & &\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image17/image17}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image18/image18}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image19/image19}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image20/image20}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image21/image21}

\end{center}
\end{figure}

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