Expression and methylation levels of suppressor of cytokine signaling 3 in rheumatic arthritis synovial fibroblasts

Journal Pre-proof Expression and methylation levels of suppressor of cytokine signaling 3 in rheumatic arthritis synovial fibroblasts

Chunqing Meng, Hong Qi, Xiaohong Wang, Xinghuo Wu, Wei Huang, Hong Wang, Yu He PII:

S0014-4800(18)30436-2

DOI:

https://doi.org/10.1016/j.yexmp.2019.104361

Reference:

YEXMP 104361

To appear in:

Experimental and Molecular Pathology

Received date:

21 September 2018

Revised date:

11 November 2019

Accepted date:

14 December 2019

Please cite this article as: C. Meng, H. Qi, X. Wang, et al., Expression and methylation levels of suppressor of cytokine signaling 3 in rheumatic arthritis synovial fibroblasts, Experimental and Molecular Pathology(2019), https://doi.org/10.1016/ j.yexmp.2019.104361

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof

Expression and methylation levels of suppressor of cytokine signaling 3 in rheumatic arthritis synovial fibroblasts Running title: SOCS3 in RA synovial fibroblasts Chunqing Meng1# , MD; Hong Qi2# , MD; Xiaohong Wang1 , PhD; Xinghuo Wu1 , PhD; Wei Huang1 ,PhD; Hong Wang1 *, MM; Yu He1 *, PhD Orthopedic Department, Union Hospital, Tongji Medical College, HuaZhong

oo

f

1

University of Science and Technology, Wuhan, 430022, China

Anesthesia Department, Union Hospital, Tongji Medical College, HuaZhong

pr

2

e-

University of Science and Technology, Wuhan, 430022, China

Pr

# Chunqing Meng and Hong Qi should be regarded as co-first authors. *Corresponding authors: Hong Wang and Yu He

al

Tel and fax: +86-027 85726489

rn

E-mail: [email protected]

Jo u

Address: Orthopedic Department of Union Hospital, Jiefang Road 1277#, Wuhan, Hubei, 430022, China

1

Journal Pre-proof

Highlights: 1. The RA model was successfully demonstrated. 2. SOCS3 level was significantly increased in the RA rat.

rn

al

Pr

e-

pr

oo

f

There was no difference of SOCS3 methylation between two groups.

Jo u

3.

2

Journal Pre-proof

Abstract Background: In the present study, we aimed to understand the expression and methylation levels of the suppressor of cytokine signaling 3 (SOCS3) in rheumatoid arthritis (RA) synovial fibroblasts. Method: The RA model was established using Freund’s complete adjuvant, and then

oo

f

the synovial fibroblasts were isolated and cultured. Next, RNA extraction and reverse transcription were performed. The SOCS3 transcription level was detected using qPCR,

pr

and SOCS3 protein expression was detected using western blotting (WB). Lastly,

e-

methylation-specific PCR (MSP) was performed.

Pr

Results: The RA model was successfully demonstrated. SOCS3 gene (p < 0.01) and protein expression levels were significantly increased in the RA rat group compared to

al

in the wild type (WT) group. However, no significant difference was observed in the

rn

MSP products between the RA and WT groups.

Jo u

Conclusion: The increased expression of the SOCS3 can be correlated with the development of RA.

Keywords: SOCS3; rheumatoid arthritis; synovial fibroblasts; expression; methylation

3

Journal Pre-proof

1. Introduction Rheumatoid arthritis (RA), which is characterized by the presence of persistent synovitis, systemic inflammation, and joint destructio n, is a chronic and debilitating autoimmune disease (Scott, 2010). Patients may present with lower energy levels, in addition to pain and stiffness in the wrist and hands (Scott, 2010). Furthermore, the

oo

f

symptoms persist from several weeks to months (Majithia and Geraci, 2007). In the developed world, RA is estimated to affect 0.5% to 1% of adults each year. The etiology

pr

of RA remains unclear, and requires urgent investigation to provide a theoretical basis

e-

for RA therapy.

Pr

Suppressor of cytokine signaling 3 (SOCS3) plays an important role in the attenuation of inflammatory cytokine signaling, and is strongly induced by interleukin

al

(IL)-1, IL-6, IL-10, and IFN-γ (Guimarães et al., 2013). O'Shea et al. reported that

rn

SOCS negatively regulates the JAK-STAT pathway in the T cells, neutrophils, and

Jo u

macrophages involved in inflammatory responses (O'Shea and Murray, 2008). Besides, SOCS3 plays a critical role in regulating the chondrocyte responses during inflammatory arthritis (Liu et al., 2014). SOCS-1 was involved in the mechanism of non- immunologically mediated zymosan- induced arthritis in the STAT-1−/− mice (de Hooge et al., 2004). In addition, Shouda et al. stated that SOCS3/CIS3 could be considered a potential therapeutic target in the treatment of inflammatory arthritis. (Shouda et al., 2001). Moreover, SOCS3 mediates the regulation of the cholinergic pathway in the synovitis in RA (Li et al., 2018). Here, we speculated that there might be

4

Journal Pre-proof

a strong correlation between SOCS3 and RA. It is believed that synovial fibroblasts derived from patients with RA are critically involved in the process of joint destruction (Müllerladner et al., 1996; Stanczyk et al., 2008). In this study, we first established the RA model. Next, the synovial fibroblasts were isolated and cultured. After RNA extraction and reverse transcription, the

oo

f

transcription and translation levels of SOCS3 were detected using qPCR and western blotting (WB), respectively. Lastly, methylation-specific analysis (MSP) was performed.

pr

Our objective was to understand the expression and methylation levels of SOCS3 in RA

e-

synovial fibroblasts.

Pr

2. Methods

2.1. Sprague-Dawley (SD) rats and RA model

al

Six Sprague-Dawley (SD) rats (8 weeks old, 220 ± 25 g) were obtained from the

rn

Shanghai SLAC Laboratory Animal Co. LTD. All rats were housed under a normal

Jo u

photoperiod and were maintained on a standard laboratory diet. After adaptive feeding for one week, the RA model was established using Freund’s complete adjuvant (FCA, F-5881, Sigma-Aldrich Chemical Co., Milwaukee, USA,) (Dai et al., 2003). First, hairs surrounding the bilateral hind leg knee joint were shaved, and the skin was disinfected with 75% ethanol. In the RA model group, the knee was slightly bent, and FCA (0.5 ml/kg) was injected into the knee joint cavity of the rats, with the knee patella margin lateral to the patellar ligament sag used as the puncture point. The control group (WT group) was injected with phosphate-buffered saline (PBS). Approximately 12-14 days

5

Journal Pre-proof

later, rats with an arthritis index (AI) close to 4 were used for the following experiments. For the RA model, AI = 0 indicated no arthritis, AI = 1 indicated red spots or mild swelling of joints, AI = 2 indicated moderate swelling of joints, AI = 3 indicated partial swelling in the phalanges and swelling in the limb, and intact or impeded mobility; AI = 4 indicated swelling in the phalanges and limb and inability to move.

oo

f

2.2 Hematoxylin and eosin (HE) staining Using the standard HE staining method, each specimen was evaluated to observe

pr

the difference between the control and RA model, and to assess the degree of

e-

destruction in the RA model. Briefly, tissues from the lower extremity of the knee joint

Pr

from the rats were fixed in 10% formalin. Next, the tissues were sectioned and were deparaffinized with xylene and ethanol. Finally, the sections were stained with HE, and

rn

Italy).

al

observed under an inverted fluorescence microscope (Optika XDS-3FL4, Ponteranica,

Jo u

2.3 Isolation and culture of synovial fibroblasts The synovial fibroblasts were isolated and cultured as follows: The rats were sacrificed via CO 2 asphyxiation and the synovial tissues were surgically isolated from the knee joint. Next, the synovial tissues were placed in a culture dish and were cut into pieces, followed by filtration with Collagenase IV (Sigma Chemical Co., St. Louis, MO USA). The synovial tissues were mixed with DMEM (Dulbecco’s modified Eagle’s medium; Gibco-BRL, Grand Island, New York, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL, Grand Island, New York, USA), and incubated at 37°C for

6

Journal Pre-proof

50-60 min. The mixture was centrifuged at 1100 rpm for 10 min, and the precipitates were collected. Subsequently, the cells were collected and suspended in DMEM (10% FBS, 1% L-glutamine, 1% pen/strep). The medium was changed once every three days, and when the cells grew to 90%-100%, cell passage was performed with trypsin digestion.

oo

f

2.4. Identification of synovial fibroblasts After the isolated cells were cultured, immunofluorescence was performed to

pr

observe the synovial fibroblasts. Cells were fixed with 4% paraformaldehyde for 30 min

e-

at room temperature, and blocked with 10% goat serum at 37°C for 30 min. Next, the

Pr

cells were incubated with the Vimentin primary antibody (1:50, ab8069, Abcam, Cambridge, UK) at 4°C overnight, followed by incubation with the secondary antibody

al

(1:50, Anti- goat IgG, A21447, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at

rn

room temperature. The section was stained with DAPI (H-1200, Vector Laboratories,

Jo u

Burlingame, CA, USA) and observed under a scanning confocal microscope (Leica Microsystems Inc., Wetzlar, Germany) at 647 nm. 2.5 RNA extraction

The cells were extracted using RNAiso Plus (Dalian Takara Co., Ltd., Dalian, Liaoning, China) and maintained at room temperature for 2-3 min. After centrifugation at 12,000 ×g for 15 min at 4°C, the colorless upper layer was transferred into another Eppendorf tubes. Isopropanol (1/2 volume) was added into the Eppendorf tube and maintained at room temperature for 10 min. Following centrifugation, the precipitate

7

Journal Pre-proof

was washed with 1 ml of 75% ethanol. Next, centrifugation was performed again, and the precipitate was dissolved using 20 μL DEPC (diethylpyrocarbonate; Dalian Takara Co., Ltd., Dalian, Liaoning, China). Finally, the RNA was placed on ice or stored at –80°C until further experimentation. 2.6 Reverse transcription

oo

f

The reaction system of reverse transcription (Dalian Takara Co., Ltd., Dalian, Liaoning, China) was prepared using the following components: 2 μL 5× PrimeScript

pr

Master Mix (Perfect Real Time) (Takara, Bio lnc., Shiga, Osaka, Japan), 500 ng total

e-

RNA, and Rnase Free dH2 O up to 10 μL. The transcription was performed at 37°C for

Pr

15 min, 85°C for 5 min, and cooling at 4°C. Next, the reaction solution was used for the real-time PCR.

al

2.7 Detection of the SOCS3 expression using qPCR

were

listed

Jo u

sequences

rn

The primers for SOCS3 were designed by the Primer 5.0 software, and the as

follows:

β-actin

Forward

Primer:

5ʹ-CCCATCTATGAGGGTTACGC-3ʹ,

β-actin

Reverse

Primer:

5ʹ-TTTAATGTCACGCACGATTTC-3ʹ,

SOCS3

Forward

Primer:

5ʹ-CCGCCGCCGGGATGAGCC-3ʹ, 5ʹ-CGGTTACGGCACTCCAGTAGA-3ʹ.

and The

SOCS3 reaction

Reverse system

is

Primer: shown

in

Supplementary Table 1. Real- time PCR was performed under an ABI ViiA7 fluorescent quantitation PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA) according to the following process: ViiA7 fluorescent quantitative PCR cycle, 95°C for 15 sec,

8

Journal Pre-proof

60°C for 60 sec, then 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. 2.8 Detection of SOCS3 protein expression using western blotting (WB) The cells were collected and protein was extracted by the RIPA lysis solution (P0013, Beyotime Institute of Biotechnology, Shanghai, China). The concentration of protein was determined by the Bicinchoninic Acid protein assay (BCA) kit (Wanleibio,

oo

f

Shenyang, Liaoning, China). The protein concentration was calculated based on the standard curve. Subsequently, the protein was separated by SDS-PAGE (sodium

pr

dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred onto the PVDF

e-

(polyvinylidene difluoride, Beyotime Institute of Biotechnology, Shanghai, China)

Pr

membrane.

The PVDF membrane was soaked with carbinol for 2 min, followed by a washing

al

with ddH2 O and the transfer buffer. The protein was transferred to the membrane on ice

rn

under the constant current condition of 160 mA for 90 min. Next, the membrane was

Jo u

rinsed in ddH2 O. After blocking with 5% non-fat dry milk for 1.5 h at room temperature, the bands were incubated overnight with the primary antibody (SOCS3 Antibody: 14025-1-AP, Proteintech,Wuhan, Hubei, China ) in a table concentrator at 4°C. The bands were incubated with horse radish peroxidase (HRP)- labeled secondary antibody for 1.5 h at 37°C. Imaging was performed using the ChemiDoc XRS System (Bio-Rad Laboratories, lnc., Hercules, CA, USA). 2.9 Methylation-specific PCR (MSP) Based on Liu et al. (Liu et al., 2010), the primer sequences were designed as

9

Journal Pre-proof

follows:

SOCS3-P-M-F,

GGATTTTATTGGAGTGTCGTAATC;

SOCS3-P-M-R,

AATACGTAAATTCTTAATCCCCGAC;

SOCS3-P-U-F,

GGATTTTATTGGAGTGTTGTAATTG;

SOCS3-P-U-R,

AATACATAAATTCTTAATCCCCAAC. DNA was extracted using the genome DNA extraction kit (Tiangen Biotech Co., Ltd, Beijing, China) according to the

oo

f

manufacturer’s instructions. The DNA was treated with hydrogen sulfite using the EpiTect Fast DNA Bisulfite Kit (Catalogue No. 59824, Qiagen, CA, USA) according to

pr

the manufacturer’s instructions. The PCR procedure was performed in a reaction

e-

volume containing a 2 μ L template (DNA treated with hydrogen sulfite), 4 μL 10×

Pr

buffer, 2 μL primer F, 2 μL primer R, 1 μL 10 mM dNTP, 0.5 μL Mg2+, 0.15 Taq polymerase (NEB), and ddH2 O up to 40 μL. PCR reactions were performed using the

al

following cycling conditions: 95°C for 2 min, 40 cycles of 94°C for 30 sec, 60°C for 30

rn

sec, 68°C for 30 sec, and a final extension at 68°C for 5 min. Following PCR,

Jo u

electrophoresis (3% agarose gel, 1× TAE buffer, 130 V, 30 min) was performed, and the bands were imaged with the Furi FR-980 image analysis system (Shanghai Furi Co, Shanghai, China). 2.10

Statistical analysis All results are presented as the mean ± SEM (standard error of the mean). The

differences between groups were compared using the Student’s t-test. One-way analysis of variance (ANOVA) was used for comparison between the groups. P value<0.05 was regarded as statistically significant.

10

Journal Pre-proof

3

Results

3.1. RA model After the establishment of the animal model, the physical state and pathological condition of the rats were observed. The changes in the physical and mental state of rats were assessed following the FCA injection. In the control group, the appearance of the

oo

f

knee joint was smooth and bright (Figure 1A). The internal synovium of the joint was smooth and opaque, without vegetation (Figure 1B). In the RA model group, the knee

pr

joint appeared swollen and dim with vegetation (Figure 1C). The internal synovium of

e-

the joint was not smooth or opaque, and presented with hyperplasia and cartilage

Pr

infiltration (Figure 1D). In addition, the HE staining demonstrated significant differences between the WT and RA model (Figure 1 E and F), with more cells

al

congregated in the knee joint of the RA rat, compared to the control. In the control

rn

group, HE staining showed no obvious changes in chondrocytes, and the structure was

Jo u

normal. Besides, the cells were round and scattered (Figure. 1E). In the model group, irregular fissures appeared on the surface of the local cartilage, and the number of chondrocytes was abnormally reduced or increased, and the structure was disordered. There was inflammatory cell infiltration, clustering- like cell cluster formation phenomenon (Figure. 1F). The AI for the rats in the RA model group was closer to 4, which indicated that RA model had been developed successfully. 3.2. Identification of synovial fibroblasts As shown in Figure 2 (A-C), vimentin was positively expressed in the isolated

11

Journal Pre-proof

cells, which suggested that the cells were synovial fibroblasts. 3.3. Detection of SOCS3 transcription level using qPCR As shown in Figure 3, the transcriptional level of the SOCS3 gene was significantly increased in the RA group compared to the WT group (P < 0.01). 3.4 Detection of SOCS3 protein expression using WB

oo

f

In order to analyze the protein expression of SOCS3, we performed a WB analysis. The relative expressions of SOCS3 in WT and RA groups were 0.45 and 1.04,

pr

respectively. The expression of SOCS3 was significantly higher in the RA group than

e-

the WT group (P < 0.01) (Figure 4).

Pr

3.5 MSP

According to the U primer, the gray value of the PCR amplification in the RA

al

group was 16396±1818, and 16880±1885 in the WT group. Additionally, after

rn

amplification by M primer, the gray value was 10100±832 in the RA group, and

Jo u

10870±2172 in the WT group (Figure 5). No significant differences were observed in the MSP products between the RA and WT group (P>0.05), which may be attributed to individual differences. 4

Discussion RA is a heterogeneous chronic disease, with presently no identified therapeutic

agent that universally and persistently is effective in all RA patients (Burmester et al., 2013). In the present study, the RA model was successfully established. The transcription and expression levels of the SOCS3 were significantly increased in rats in

12

Journal Pre-proof

the RA group compared with the WT group (P< 0.01). However, no significant difference was observed in the MSP products between the RA and WT group. In the RA model group, the knee joint appeared swollen, dim, presented with vegetation, and the internal synovium of the joint was not smooth or opaque, demonstrating hyperplasia and cartilage infiltration. Thus, the AI in the RA model group was close to 4, and indicated

oo

f

that the RA model was successful. Cytokines have been suggested to play a critical role in the mechanism of RA

pr

development. Cytokines such as IL-1, IL-6, IL-10, and IFN-γ act as proinflammatory

e-

cytokines and are found to be accumulated in the rheumatoid synovium (Feldmann et al.,

Pr

1996; Firestein, 2003). It has been claimed that SOCS3 is strongly induced by IL-1, IL-6, IL-10, and IFN-γ (Guimarães et al., 2013). Cytokines such as IL-6, IL-10, and

al

IL-12 were activated in the JAK-STAT pathway in RA, which is negatively regulated by

rn

the SOCS proteins(Tamiya et al., 2011; Walker and Smith, 2005). SOCS proteins play

Jo u

key roles in the immune and inflammatory response, and may be important regulators in the development of arthritis (Warren S, 2002). It is reported that the expression of SOCS3 was significantly increased in mononuclear cells in the peripheral blood of RA patients (Isomaki et al., 2007), which was consistent with our findings. Thus, we speculated that the increased expression of SOCS3 might play a key role in the pathogenesis of RA. In previous reports, He et al. suggested that SOCS3 was frequently silenced by hyper- methylation in lung cancer (He et al., 2003). Niwa et al. indicated that silencing

13

Journal Pre-proof

of SOCS3 by methylation promotes cell growth and migration in hepatocellular carcinoma (Niwa et al., 2005). In addition, SOCS3 is also frequently methylated in head and neck squamous cell carcinoma (Weber et al., 2005). Fourouclas et al. suggested a role of SOCS3 methylation in myeloproliferative disorders (Fourouclas et al., 2008). The above reports demonstrated that SOCS3 is frequently silenced by methylation in

oo

f

certain diseases. However, in the present study, no significant difference was noted in the MSP products between the RA and WT group, which could have resulted due to

e-

detection of RA was not adequately reliable.

pr

individual differences. Thus, we speculated that direct methylation sequencing for the

Pr

5 Conclusion

In conclusion, the expression level of SOCS3 was significantly increased in the RA

al

synovial fibroblasts compared with those of the WT, but no significant difference was

rn

observed in the MSP products. We concluded that the increased expression of SOCS3

Jo u

could potentially be correlated with the pathogenesis of RA. Moreover, in order to further understand the roles of SOCS3 in RA, a gene knockdown experiment will be performed in our further research. Acknowledgements This work was supported by Hubei provincial health and family planning research project. (Project No. Wj2015mb072). Conflict of interest The authors declare that there are no conflicts of interest.

14

Journal Pre-proof

References Burmester, G. R., Blanco, R., Charles-Schoeman, C., et al., 2013. Tofacitinib (CP-690,550) in combination with methotrexate in patients with active rheumatoid arthritis with an inadequate response to tumour necrosis factor inhibitors: a randomised phase 3 trial. Lancet. 381, 451-460.

oo

f

Dai, M., Wei, W., Wang, N., Chen, Q., 2003. Effects of papain on adjuvant arthritis in rats. Chinese Pharmacological Bulletin. 19, 340-344.

pr

de Hooge, A. S., van de Loo, F. A., Koenders, M. I., et al., 2004. Local activation of

e-

STAT‐1 and STAT‐3 in the inflamed synovium during zymosan‐induced

Pr

arthritis: Exacerbation of joint inflammation in STAT ‐ 1 gened arthritis: Exacerbation of joint inflammation in STAT2004. Local activation of STAT.

al

Chinese Pharmacological B

rn

Feldmann, M., Brennan, F. M., Maini, R. N., 1996. Role of cytokines in rheumatoid

Jo u

arthritis. Annual Review of Immunology. 14, 397-440. Firestein, G. S., 2003. Evolving concepts of rheumatoid arthritis. Nature. 423, 356-361. Fourouclas, N., Li, J., Gilby, D. C., et al., 2008. Methylation of the suppressor of cytokine

signaling

3

gene

(SOCS3)

in

myeloproliferative

disorders.

haematologica. 93, 1635-1644. Guimarães, M. R., Leite, F. R. M., Spolidorio, L. C., Kirkwood, K. L., Rossa, C., 2013. Curcumin abrogates LPS-induced pro- inflammatory cytokines in RAW 264.7 macrophages. Evidence for novel mechanisms involving SOCS-1,-3 and p38

15

Journal Pre-proof

MAPK. Archives of oral biology. 58, 1309-1317. He, B., You, L., Uematsu, K., et al., 2003. SOCS3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proceedings of the National Academy of Sciences. 100, 14133-14138. Isomaki, P., Alanara, T., P, Lagerstedt, A., et al., 2007. The expression of SOCS is

oo

f

altered in rheumatoid arthritis. Rheumatology. 46, 1538. Li, T., Wu, S., Li, S., Bai, X., Luo, H., Zuo, X., 2018. SOCS3 participate s in cholinergic

pr

pathway regulation of synovitis in rheumatoid arthritis. Connective tissue

e-

research. 59, 287-294.

Pr

Liu, W. B., Ao, L., Zhou, Z. Y., et al., 2010. CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during

al

rat lung carcinogenesis induced by 3- methylcholanthrene and diethylnitrosamine.

rn

Biochemical & Biophysical Research Communications. 402, 507-514.

Jo u

Liu, X., †, B. A. C., ‡, I. K. C., et al., 2014. Key Role of Suppressor of Cytokine Signaling 3 in Regulating gp130 Cytokine–Induced Signaling and Limiting Chondrocyte Responses During Murine Inflammatory Arthritis. Arthritis & Rheumatology. 66, 2391–2402. Majithia, V., Geraci, S. A., 2007. Rheumatoid Arthritis: Diagnosis and Management. Clinical Symposia. 120, 936-939. Müllerladner, U., Kriegsmann, J., Franklin, B. N., et al., 1996. Synovial fibroblasts of patients with rheumatoid arthritis attach to and invade normal human cartilage

16

Journal Pre-proof

when engrafted into SCID mice. American Journal of Pathology. 149, 1607-1615. Niwa, Y., Kanda, H., Shikauchi, Y., et al., 2005. Methylation silencing of SOCS3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene. 24, 6406-6417.

oo

f

O'Shea, J. J., Murray, P. J., 2008. Cytokine signaling modules in inflammatory responses. Immunity. 28, 477-487.

pr

Scott, D. L., 2010. Rheumatoid arthritis. Lancet. 376, 1094-1108.

e-

Shouda, T., Yoshida, T., Hanada, T., et al., 2001. Induction of the cytokine signal

Pr

regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis. Journal of Clinical Investigation. 108, 1781-1788.

al

Stanczyk, J., Pedrioli, D. M. L., Brentano, F., et al., 2008. Altered expression of

rn

MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis.

Jo u

ARTHRITIS & RHEUMATISM. 58, 1001-1009. Tamiya, T., Kashiwagi, I., Takahashi, R., Yasukawa, H., Yoshimura, A., 2011. Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: regulation of T-cell inflammation by SOCS1 and SOCS3. Arterioscler Thromb Vasc Biol. 31, 980-985. Walker, J. G., Smith, M. D., 2005. The Jak-STAT pathway in rheumatoid arthritis. Journal of Rheumatology. 32, 1650-1653. Warren S, A., 2002. Suppressors of cytokine signalling (SOCS) in the immune system.

17

Journal Pre-proof

Nature Reviews Immunology. 2, 410-416. Weber, A., Hengge, U. R., Bardenheuer, W., et al., 2005. SOCS3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions

Jo u

rn

al

Pr

e-

pr

oo

f

and causes growth inhibition. Oncogene. 24, 6699-6708.

18

Journal Pre-proof

Figure legends Figure 1 The morbidity state of rats after RA modeling. A: The appearance of the knee joint in the control group. Knee joints are smooth and bright. B: The internal synovium for the control group. The synovial membrane in the joint is smooth and transparent with no sputum. C: The appearance of the knee joint in

oo

f

the RA model group. Knee joints are swollen and dim. D: The internal synovium in the RA model group. The synovial membrane in the joint is not smooth and opaque. The

pr

synovial membrane proliferates and infiltrates the cartilage. HE staining for WT (E) and

e-

RA model (F).

Pr

Figure 2 Immunofluorescence analysis of the synovial fibroblasts. A: Merge 400×, stained with Vimentin antibody and DAPI. B: Vimentin 400×, stained with the Vimentin

al

antibody only. C: DAPI 400×, stained with the DAPI only. Red represents the synovial

rn

fibroblasts stained positively for the Vimentin. Blue represents the DAPI.

Jo u

Figure 3 SOCS3 mRNA level using qPCR. WT: wild type rats; RA: rheumatic arthritis rats. ** P < 0.01, *** P < 0.001. Figure 4 SOCS3 protein expression detected using western blotting (WB). WB experiments were repeated in triplicate. **P<0.01, compared with WT group. Figure 5 The results of the methylation-specific analysis (MSP). A: PCR electrophoresis, B: relative expression value by U primer, C: relative expression value by M primer. The experiments were repeated for 3 times.

19

Journal Pre-proof

Highlights: 4. The RA model was successfully demonstrated. 5. SOCS3 level was significantly increased in the RA rat.

rn

al

Pr

e-

pr

oo

f

There was no difference of SOCS3 methylation between two groups.

Jo u

6.

20