MicroRNA-9 ameliorates destructive arthritis through down-regulation of NF-κB1-RANKL pathway in fibroblast-like synoviocytes

Clinical Immunology 212 (2020) 108348

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MicroRNA-9 ameliorates destructive arthritis through down-regulation of NF-κB1-RANKL pathway in fibroblast-like synoviocytes

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Wen Shi Leea, Shinsuke Yasudaa,b, , Michihiro Konoa, Yuki Kudoa, Sanae Shimamuraa, Michihito Konoa, Yuichiro Fujiedaa, Masaru Katoa, Kenji Okua, Tomohiro Shimizuc, Tomohiro Onoderac, Norimasa Iwasakic, Tatsuya Atsumia a

Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan Department of Rheumatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan c Department of Orthopedic Surgery, Faculty of Medicine, Hokkaido University, Sapporo, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Rheumatoid arthritis microRNA-9 RANK ligand Joint destruction NF-kB1 Collagen-induced arthritis

We investigated the effect of miR-9 on fibroblast-like synoviocytes (FLS) from RA patients and animal arthritis model. The binding of miR-9 to NF-κB1 3’UTR was analyzed by luciferase reporter assay and immunoprecipitation. ChIP assay and luciferase promoter assay were performed to identify the binding of NF-κB1 to RANKL promoter and its activity. FLS were treated with miR-9/anti-miR-9 to evaluate cell proliferation and the expression of RANKL. Therapeutic effect of intra-articular miR-9 was evaluated in type-II collagen-induced arthritis in rats. miR-9 bound to the 3’-UTR of NF-κB1 and downregulated NF-κB1. NF-κB1 bound to RANKL promoter and increased the promoter activity of RANKL. RANKL was downregulated by miR-9. Proliferation of FLS was increased by miR-9 inhibitor. miR-9 dampened experimental arthritis by lowering inflammatory state, reducing RANKL and osteoclasts formation. Our findings revealed miR-9-NF-κB1-RANKL pathway in RA-FLS, further, miR-9 ameliorated inflammatory arthritis in vivo which propose therapeutic implications of miR- 9 in RA.

1. Introduction Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that is characterized by synovial inflammation and hyperplasia, autoantibody production, and cartilage/bone destruction [1]. Fibroblast-like synoviocytes from RA patients (RA-FLS) have been known to play an essential role in the pathophysiology of RA [2]. RA-FLS actively attach to and invade articular cartilage and bone tissue, expressing increased amounts of adhesion molecules and proinflammatory cytokines, chemokines, matrix-degrading proteases, as well as receptor activator of nuclear factor κB ligand (RANKL) [3]. RANKL expressed on RA-FLS, rather than T cells, has been known as predominantly responsible for the formation of osteoclasts and erosions during disease progression of active RA [4]. The pathogenic roles of RANKL have been keenly investigated and clarified; RANKL binds RANK, then activates NF-κB inflammatory pathway [5]. NF-κB pathway is also activated by TNF-α and recognized as one of the most important inflammatory

pathways in RA [6]. NF-κB1/p105 is processed by proteasome and produces NF-κB1/p50 which enters into the nucleus, acting as a transcription factor with p65 [7,8]. However, the regulation mechanisms of RANKL production have not been established. MicroRNAs (miRNAs) are small non-coding RNAs with approximately 22-nucleotide length with critical functions in a wide range of biological and pathologic processes. Functional studies have revealed that dysregulation of miRNAs is causal in autoimmune diseases, including RA [9]. In addition, several miRNA-based therapeutics have reached clinical development in treating cancer, including miRNA mimics and anti-miRNAs [10]. Several miRNAs show different expression in the plasma of RA patients compared with healthy controls (HCs) [11,12]. Among these miRNAs, miR-9-5p has lower expression in RA patients. Also in Tregdepleted mice, miR-9 was markedly decreased in the sera, suggesting its potential role in autoimmunity [11]. Moreover, miR-9 has been reported to target NF-κB1 gene in bone marrow-derived mesenchymal

Abbreviations: FLS, fibroblast-like synoviocytes; microRNA, miR; NF-κB, nuclear factor κB; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor κB ligand ⁎ Corresponding author at: Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine, Hokkaido University, N15W7, Kita-ku, Sapporo 060-8638, Japan. E-mail address: [email protected] (S. Yasuda). https://doi.org/10.1016/j.clim.2020.108348 Received 6 December 2019; Received in revised form 20 January 2020; Accepted 20 January 2020 Available online 21 January 2020 1521-6616/ © 2020 Published by Elsevier Inc.

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and miR-9a-5p mimics (mirVana™, ThermoFisher) or negative control miR by lipofectamine 2000 (ThermoFisher) for 48 h, and luciferase assays were performed with the dual-glo luciferase reporter assay system (Promega). Luminescent signals were quantified by a luminometer (Glomax, Promega).

stem cells (BMSCs) and hence inhibit the NF-κB signaling pathway [13]. Further, upregulation of miR-9 could promote proliferation of chondrocyte from osteoarthritis (OA) patients in vitro, meanwhile downregulation of miR-9 increased the expression of matrix metalloproteinase-13 (MMP-13). These findings suggest the important role of miR-9 in joint or bone/cartilage homeostasis [14,15]. Therefore, we speculated that miR-9 suppresses NF-κB pathway in RA-FLS and preserve cartilage in inflammatory arthritis. In addition, based on JASPAR database, the binding of NF-κB onto RANKL promoter was predicted, which may result in up-regulated RANKL expression. In this study, we identified the existence of NF-κB-RANKL pathway in RAFLS, which was dampened by miR-9. We then examined if intraarticular administration of miR-9 could protect joint destructions in a collageninduced arthritis (CIA) animal model.

2.3. Western blot analysis To evaluate the effect of miR-9 on NF-κB1 at protein level, following immunoblotting was done. After transfection of miR-9a-5p mimics or control miR with lipofectamine RNAiMAX (ThermoFisher) for 24 h, the cells were lysed immediately for 5 min at 95 °C in buffer containing 62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, and 5 mM β-mercaptoethanol. Cells lysates were subjected to SDS-PAGE and transferred to PVDF (Merck) membranes. Blots were probed with anti-NF-κB1 p105/p50 antibody (Cell Signaling Technology), followed by peroxidase-conjugated goat anti-rabbit antibody as the secondary antibody (Sigma Aldrich). Monoclonal anti-β-actin antibody conjugated with peroxidase was used as internal control (Sigma Aldrich).

2. Methods 2.1. Human sample collection and FLS isolation Synovial tissue specimens were collected in phosphate buffered saline (PBS) during knee joint replacement surgery from 10 RA patients and 10 osteoarthritis (OA) patients with written informed consent. Demographic characteristics of patients are summarized in Table 1. This study was approved by the Human Ethics Committee of Hokkaido University Hospital (approval number: 008–0103). All participants fulfilled the American College of Rheumatology (ACR)/European League Against Rheumatism 2010 classification diagnosis criteria [16]. FLS isolation was done as described in previous report [17,18]. After isolation, the cells were cultured in Iscove's modified Dulbecco's medium (IMDM; Sigma), supplemented with 10% fetal bovine serum (FBS, Cosmo Bio), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma) at 37 °C, in 5% CO2 humidified atmosphere. FLS at passages 3–8 were used in this study, served to the following experiments.

2.4. Prediction of potential transcription factor which binds to RANKL promoter The prediction of transcription factors binding onto RANKL promoter lesion was performed by the algorithm of JASPAR datasets (http://jaspar.genereg.net/). We screened all Homo sapiens transcription factors which match the RANKL promoter sequence from Genbank™ (accession no. AF 333234.1) by JASPAR and the results showed that NF-κB1/p50 is one of the potential transcription factors. Predicted binding sequences of NF-κB1/p50 of whole RANKL promoter sequence were listed in Supplementary File, Table S1.

2.2. NF-κB1 3′-UTR and miR-9 binding analysis by dual luciferase reporter assays

2.5. Chromatin immunoprecipitation assay Predicted binding of NF-κB1 onto the RANKL promoter was confirmed by Chromatin immunoprecipitation (ChIP) assay. RA-FLS were transfected by miR-9 mimic or negative control and stimulated by 10 ng/ml of TNF-α for 24 h before crosslinking with 1% formaldehyde at room temperature for 10 min. Chromatin immunoprecipitation (ChIP) assay was performed using SimpleChIP enzymatic ChIP kit Cell Signaling) according to the manufacturer's instructions. The DNA-protein complexes were immunoprecipitated using anti-NF-κB1 p105/p50 antibody with a concentration of 1:10 or normal rabbit IgG (Cell Signaling). The bound DNA fragments were subjected to real-time PCR with SYBR Green using primers spanning RANKL gene in the region of promoter (Forward: 5′-CATATATGAAGCTCCTGGGT-3′, Reverse: 5′-CCTCAGGTTACAAGAAAGTCCC-3′).

Previous reported effect of miR-9 on NF-κB1 was confirmed in RAFLS. The segments of the wild-type/mutant 3′UTR of genes of interest, NF-κB1/p50 containing predicted target sites of miR-9 from TargetScan (http://www.targetscan.org/vert_72/) were cloned into the pmirGLO dual-luciferase reporter (Promega). HEK293 and RA-FLS from patients were used for analysis. The cells were co-transfected with cloned vector Table 1 Characteristics of the RA and OA patients in this study.

No. of women/men Age at operation, years Serum CRP mg/dL MMP-3, mg/dL RF, units/mL ACPAs, units/mL Swollen joint count Tender joint count DAS28-ESR HAQ DI score Methotrexate use, no (%) Biologic agent use, no (%)

RA patients

OA patients

8/2 65 (56–78) 0.60 (0.02–5.69) 97.1 (31.3–449.9) 46.0 (1.1–156.8) 187.1 (4.0–300) 3 (1–16) 3 (0–16) 4.40 (2.99–6.86) 1.25 (0–2.875) 6 (60) 5 (50)

10/0 73 (64–80) 0.07 (0.02–0.61) NA NA NA NA NA NA NA 0 (0) 0 (0)

2.6. RANKL gene promoter construction and promoter activity The RANKL gene promoter region was selected from the promoter sequence position of −1 to -2 kb from Genebank™ (accession no. AF 333234.1) according to a previous report [19]. Mutant construct sites were selected based on the predicted NF-κB1 binding sites. Each of the construct was sub-cloned into a pGL4.10 basic luciferase vector. The RANKL-luciferase reporter plasmid constructs (2 μg), pGL4.73 rluc SV40 and NF-κB1 vector (OHu22499D, GenScript) were transiently transfected into HEK293 or RA-FLS using lipofectamine 2000. Control siRNA or NF-κB1 siRNA (ThermoFisher) was co-transfected with RANKL-luciferase reporter plasmid and pGL4.73 rluc SV40 by lipofectamine 2000 to RA-FLS. Cells were harvested 48 h after transfection for analyzing promoter activity by using before-mentioned Dual-glo® luciferase assay system.

Except where indicated otherwise, values are the median (range). ACPAs, anti–citrullinated protein antibodies; CRP, C-reactive protein; DAS28-ESR, Disease Activity Score in 28 joints using the erythrocyte sedimentation rate; HAQ DI, Health Assessment Questionnaire disability index; MMP-3, matrix metalloproteinase 3; NA, not available; OA, osteoarthritis; RA, rheumatoid arthritis; RF, rheumatoid factor, † Two patients (20%) were taking tocilizumab, 2 patients (20%) were taking infliximab, and 1 patient (10%) was taking etanercept. 2

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Fig. 1. NF-κB1 is a direct target of miR-9. (A) The predicted miR-9 binding site on NF-κB1 mRNA 3’UTR and the NF-κB1 3’UTR mutation containing a mutated miR-9 ‘seed-region’ binding site are shown (mark in grey). (B) Dual luciferase reporter assay revealed that miR-9 significantly decreased the luciferase activity of NF-κB1 wildtype 3’UTR in both HEK293 (n = 5) and RA-FLS (n = 7). *P < .05, **P < .01, by one-way ANOVA. (C) RA-FLS (n = 5) were transfected with miR-9a-5p mimic or negative control miR, and the protein expression of NF-κB1 was evaluated by Western blot. WT, wildtype; MUT, mutant. *P < .05, ***P < .001, by Student's t-test. Values are the mean ± S.D. in RA-FLS or the mean ± S.E.M. in HEK293.

2.7. Quantitative real-time PCR and flow cytometry analysis

with PE (Biolegend) or corresponding isotype control.

We evaluated the effect of miR-9 and its inhibitor on RANKL at the transcript/protein levels. The cells were transfected with miR-9 mimic, miR-9 inhibitor and their corresponding negative controls for 24 h with or without 10 ng/mL TNF-α stimulation for 6 h. mRNA levels of RANKL were analyzed by quantitative real-time PCR. Primers were purchased from ThermoFisher (TNFSF11 Taqman™, 4,331,182; GADPH Taqman™, 4,351,370). Similar transfection was done for 24 h, with or without 10 ng/mL TNF-α and/or 100 ng/ml IFN-γ stimulation for 72 h and the cell surface RANKL expression was analyzed by flow cytometry (FACS Calibur, BD Biosciences) with anti-human RANKL antibody conjugated

2.8. Collagen-induced arthritis and treatment of miR-9 in rats To evaluate the effects of miR-9 on NF-κB1 - RANKL system in vivo, Collagen-induced arthritis rat model was introduced, as previously reported [17]. This study was approved by the Animal Ethics Committee of the Hokkaido University Hospital (approval number: 17–0031). Briefly, twelve 8-week-old female Lewis rats were injected intradermally with 1 mg/ml solution of porcine type II collagen (Chondrex) dissolved in 0.05 M acetic acid and emulsified 1:1 in Freund's incomplete adjuvant (Chondrex), followed by a booster injection on day 3

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Fig. 2. NF-κB1 directly bound to the promoter region of RANKL (TNFSF11). (A) NF-κB1 binding sequence (Matrix profile MA0105.1) predicted by JASPAR using RANKL promoter template. (B) The RANKL-NF-κB1 binding sites were confirmed by ConSite. (C) RANKL promoter sequence and NF-κB1 binding sites which were predicted by the programs JASPAR and ConSite are marked with red. Red-underlined sequences are the primers selected for ChIP assay. Coordinates are given relative to the translational start site (shown as +1). (D) ChIP assay using RA-FLS: Real time-PCR for RANKL promoter using immunoprecipitated samples. For real time-PCR, each ChIP DNA fraction Ct value was normalized to the input DNA fraction Ct value, the results were then normalized by control IgG. Values are the mean ± SD from five independent experiments. ***P0.001 as compared with negative control IgG, by Student's t-test. (E) Dual luciferase reporter assay of RANKL promoter activity in both HEK293 and RA-FLS. Values are mean of four independent experiments in HEK293 and three independent experiments in RA-FLS. WT, wildtype; MUT, mutant; si, siRNA. Values are presented as mean ± S.E.M. in HEK293 and mean ± S.D. in RAFLS. *P < .05, **P < .01, ****P < .0001, by one-way ANOVA analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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blotting using RA-FLS, confirming that miR-9 mimic successfully downregulated the expression of NF-κB1/p105 and NF-κB1/p50 at protein levels, the former more evidently (Fig. 1C).

7. On day 14, the rats were randomized into control group or miR-9 group to receive an intraarticular injection with 60 μL of solution containing 5 μM miRNA-atelocollagen complex (Atelocollagen), into both hind ankle joints. Atelocollagen-RNA complexes are resistant to nucleases and efficiently transduced into cells, and are prevented from diffusing out of the injected sites. Theses interventions were done near the onset of arthritis, mimicking treating early RA patients. One group of rats received mirVana™ miRNA mimic negative control while the other group received mirVana™ miRNA mimic miR-9a-5p.

3.2. NF-κB1 directly binds to the promoter region of RANKL in RA-FLS NF-κB1 binding sequence (Matrix profile MA0105.1) was predicted by JASPAR using RANKL promoter template (−2 kb to +1) and the predicted binding sites were evaluated using ConSite (Fig. 2A, B). The full sequence of RANKL promoter has 7 predicted binding sites in total (Supplementary Table S1). The promoter sequences used for further experiments were -2 kb to +1, with the predicted NF-κB1 binding sequences marked in red (Fig. 2C). We performed ChIP assay to confirm the direct binding of NF-κB1 to the promoter region of RANKL. The primers for real time-PCR were selected within -2 kb to +1 of the promoter sequences of RANKL. Using RA-FLS, direct binding of NF-κB1 to the RANKL promoter sequences was demonstrated, showing significant difference compared to the control IgG (P < .001) (Fig. 2D). NF-κB1-overexpressed HEK293/RA-FLS transfected with RANKL wildtype promoter had the highest promoter activity. Mutant RANKL promoter showed decreased RANKL-luciferase activity in both HEK293 cells and RA-FLS, in the latter cells with intrinsic activity, presumably by their inflammatory nature. Reverse experiments using co-transfection of NF-κB1 siRNA and RANKL-luciferase reporter plasmid showed significant suppression of RANKL promoter activity (Fig. 2E).

2.9. Clinical assessment and radiological examinations of arthritis Rats were monitored for development of arthritis by digital microcaliper measurements of diameter of hind ankle in a blinded manner by three independent researchers. In addition, the arthritis score was used as described in previous report [20]. The accuracy of the scoring system was verified by micro-computed tomography (micro-CT) coupled with histological analysis of representative arthritic limbs. Radiographic severity in rats with CIA was assessed in a blinded manner on day 31. Micro-CTs of the ankles were performed on all rats. The treated rats were given a joint destruction scores based on the extent of soft tissue swollenness, joint space narrowness, bone destruction, and periosteal new bone formation in the 2D image [21]. 3D micro-CT image bone erosion score was used as described in previous report [22]. 2.10. Conventional histology, immunohistochemistry and TRAP staining

3.3. miR-9 down-regulates RANKL expression at mRNA levels and at protein levels

Rats were euthanized on day 31 for histopathological and immunohistochemical analysis. The ankles were decalcified and embedded in paraffin, and serial sections were stained with anti-RANKL antibody (Abcam), anti-cadherin antibody (ThermoFisher), TRAP, H&E, or safranin O for microscopic examination. Inflammatory exudates and infiltrates were scored in H&E-stained sections as described [17]. For TRAP staining, the number of osteoclasts (three or more nuclei TRAP+ cells) was quantified per ×100 field. The RANKL+ lining area was adjusted to the linear horizontal length (μm2/μm). The evaluation score of safranin O-fast green staining was used according to previous report (Supplementary File, Table S2) [23]. All scores were assessed in a blinded manner.

We investigated the expression of RANKL after miR-9a-5p mimic transfection in RA-FLS. RANKL mRNA expression was significantly down-regulated by miR-9 transfected RA-FLS compared to control RAFLS (Fig. 3A). Slight increment without statistical significance was found in the expression levels of RANKL by 6 h' stimulation with 10 ng/ mL of TNF-α. RANKL mRNA levels in RA-FLS transfected with miR-9a5p inhibitor was significantly upregulated compared to the control group when stimulated with TNF-α (Fig. 3B). This tendency did not reach statistical significance without TNF-α. The cell surface expression of RANKL was also analyzed by flow cytometry. RA-FLS express some amount of RANKL on their surface, even without cytokine stimulation, which was enhanced by dual-stimulation with TNF-α or IFN-γ (Fig. 3C). miR-9 significantly down-regulated the cell-surface RANKL, regardless of the presence or absence of TNF-α and/or IFN-γ stimulation (Fig. 3C and D). This suppressive effect was incomplete in RA-FLS with dualstimulation.

2.11. Statistical analysis The expression levels of the miRNA and mRNA were calculated using the 2-ΔΔCt method, in which the value represents the n-fold change in the test samples relative to their corresponding control. Statistical analysis was performed using Graphpad Prism® software. Student's ttest was applied when there were only two groups of samples. In the case of more than two groups of samples, one-way analysis of variance (ANOVA) was used for analyzation. The data are expressed as the mean ± SD in vivo FLS experiments and mean ± S.E.M in vitro study and in vivo HEK293 experiments. For FLS, we compared and normalized the value of each patient while HEK293 was compared and normalized by one of the repetitive data.

3.4. miR-9 reduced the inflammation and joint destruction in CIA rats Intra-articular injection of miR-9 significantly improved the clinical arthritis score and ankle diameter in CIA rats compared to the control miRNA group (Fig. 4A, B). Micro-CT revealed that CIA rats injected with miR-9a-5p mimic had significantly lower erosion scores compared to CIA rats injected with negative control miRNA, using two different scoring system from previous reports (Fig. 4C, D). Histopathology revealed that the inflammatory exudates and infiltrates within the ankles of miR-9 treated rats were significantly reduced compared to control CIA rats. Further, there were obvious pannus formation in control group but little or no pannus formation in miR-9a-5p mimics treatment group. Safranin O-fast green staining specimen revealed that miR-9a-5p mimics protected the cartilage from degradation in CIA rats. TRAP stain as well as the lining RANKL+ area was both significantly decreased in relevant tissues of rats that received miR-9a-5p mimics compared with rats that received control miRNA, indicating suppressed osteoclastogenesis in miR-9a-5p mimics-treated rats. We have selected the pannus-bone interface (control) or subchondral bone area (in miR-9 treated group which has no pannus

3. Results 3.1. NF-κB1 is a direct target of miR-9 in RA-FLS The predicted miR-9 binding site NF-κB1 mRNA 3’UTR from TargetScan has a perfect seed sequence (Fig. 1A). Dual luciferase reporter assay revealed that miR-9 significantly decreased the luciferase activity of NF-κB1 wildtype 3’UTR in both HEK293 (P < .01) and RAFLS (P < .05) (Fig. 1B). miR-9 did not suppress the luciferase activity of mutated NF-κB1 mRNA 3’-UTR, showing comparable activity with those in cells treated with miR-9 plus pmir-GLO empty vector. To further elucidate the effect of miR-9 to NF-κB1, we performed western 5

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Fig. 3. miR-9 regulates RANKL expression. (A) RANKL mRNA levels evaluation in miR-9a-5p mimic transfected RA-FLS in the presence or absence of TNF-α. (B) RANKL mRNA levels evaluation in anti-miR-9a-5p transfected RA-FLS in the presence or absence of TNF-α. Values are the mean of six independent experiments. (C) Cell surface RANKL expressions on RA-FLS analyzed by flow cytometry. Histograms are representative of five independent experiments using RA-FLS from different patients. (D) Cell surface expression levels of RANKL were quantified and presented as mean fluorescent intensity (MFI) ratio. All values are presented as mean ± S.D. *P < .05, **P < .01, ****P < .0001, by one-way ANOVA analysis.

formation) to score TRAP+ cells and RANKL+ area as RANKL protein has been reported to express at these sites in rheumatoid arthritis [24]. Cadherin 11 positive cells were considered as RA-FLS in synovial tissues, had a relatively similar position with RANKL positive area (Fig. 5A, B).

enhancing the degradation of IκBα, the inhibitor protein of NF-κB [31,32]. In our flow cytometry data, as expected, the expression of RANKL was upregulated by synergistic effect of TNF-α and IFN-γ. We identified that miR-9 has the ability to reduce RANKL expression which we considered as an indirect effect via the reduction of NF-κB1. Finally, the protective effect of miR-9 against destructive arthritis was confirmed using CIA rat model. TRAP+ osteoclasts and RANKL expression were significantly dampened in miR-9 treated joints compared to those treated with control miRNA. Although we did not set active positive treatment control, previously we performed similar experiment using RasGRP4-specific siRNA [17]. Comparing to RasGRP4 knockdown, the extent of the therapeutic effects by miR-9 mimic were comparable. The intact cartilage in miR-9 treated CIA rats suggests the protective effect of miR-9 via NF-κB1 and protegenin on chondrocytes [14,33]. Beside chondrocytes, previous study has reported that synovial fluid cells in mouse tibial plateau fracture model has lower miR-9 expression after joint injury and the reduced miR-9 in osteoclast precursors has a potential in promoting osteoclasts survival which is compatible to our TRAP stain results [34]. The inflammatory parameters in clinical, micro-CT images and histology specimens further emphasized the importance of miR-9 regulation in RA. The limitation of this study is that as miR-9 has multiple targets, total effects might be different according to the cell types and/or conditions. Different from knocking down a specific molecule using more than one siRNA, we could not deny the possibility that mir-9 mimic acting on multiple molecules but NF-κB1 in our arthritis model, because miR itself targets multiple molecules in general. For example, Makki et al reported that increased miR-9 in OA chondrocytes binds to monocyte chemoattractant protein-induced protein 1 (MCPIP1) and upregulates the production of IL-6 which increases the disease activity [35]. The stability and delivery of miR-9 should be further explored for its clinical use. However, treatment strategy using miR has been increasingly improved in the field of malignancy, indicating the feasibility of miR-9 for the treatment of RA.

4. Discussion The expression levels of miR-9 have been reported to be lower in plasma from RA patients compared to those in healthy individuals [12]. Previous study also reported that miR-9 has lower expression in OA cartilage tissues compared to the normal tissues which were collected from trauma patients [14]. Further, miR-9 has proliferative effects on chondrocytes, exerting its ability to protect cartilage in OA joints [14]. We compared the miR-9 levels in FLS between RA and OA patients, finding scarce miR-9 expression in both groups (Supplementary File, Fig. S1). Although we could not obtain FLS from healthy individuals, our data on FLS has no inconsistency with the previously reported serum data and also partly support the concept of miR-9 administration as a therapeutic strategy. MiR-9 at the same time has the ability to proliferate or induce apoptosis according to different cell types [25]. Hence, we analyzed the proliferation rate of RA-FLS treated with miR-9 mimics or inhibitors, finding that inhibition of miR-9 significantly increased the proliferation rate of RA-FLS (Supplementary File, Fig. S2). As our results have shown that miR-9 suppresses NF-κB1, we hypothesized that RA-FLS would decrease proliferation rate by miR-9, as inhibition of NF-κB signaling results in suppressed proliferation of RA-FLS [26]. These results indicate that miR-9 might work protectively both in RA and OA, by suppressing the proliferation of FLS and by inhibition of cartilage degradation. We confirmed the binding of miR-9 to the 3’-UTR sites of NFκB1 and hence the reduction of NF-κB1 expression in RA-FLS. Thus, miR-9 is supposed to dampen inflammatory nature of RA-FLS via degradation of NF-κB1 transcripts. The transcription factor NF-κB1 has been well recognized as an important regulator of inflammation, development of T cells, functions of RA-FLS and differentiation and activation of bone resorbing activity of osteoclasts [27]. Among these functions, we focused on the expression of RANKL in RA-FLS that induces bone resorption via differentiation/activation of osteoclasts. Previous study reported that TNF-α induces IL-6 expression and the combination of IL-6/sIL-6R directly increases the expression of RANKL in FLS [28]. We revealed that NFκB1 binds onto the promoter region of RANKL and acts as a transcription factor that enhances RANKL promoter activity. In our luciferase assay, RANKL promoter had the highest level of activity when cotransfected with NF-κB1, which was suppressed by NF-κB1-specific siRNA. The mutated RANKL promoter showed reduced activity in HEK293 but only slight decrease in RA-FLS, compared to wildtype RANKL. These results might due to the endogenous NF-κB1 activity in RA-FLS or to only little mutations in RANKL promoter we designed, as NF-κB1 has more than one predicted binding site to RANKL promoter. Nonetheless, this novel NF-κB1–RANKL axis in RA-FLS may function as an osteoclastogenesis/inflammatory accelerator in RA synovium. We further analyzed the effects of miR-9 on the expressions of RANKL in RA-FLS in the absence or presence of cytokines. Stimulation with TNF-α and IFN-γ was selected as both cytokines have been reported to activate NF-κB signaling pathway [29,30]. Previous reports have shown that TNF-α and IFN-γ synergistically activates NF-κB signaling pathway by inducing NF-κB -like factor binding elements or

5. Conclusion In conclusion, the present study revealed that the treatment with miR-9 in RA-FLS reduced the levels of NF-κB1 as well as RANKL. The protective effects of miR-9 in preventing joint destruction and reducing inflammation were confirmed in in vivo arthritis model. At least in RAFLS, novel activation mechanism of RANKL induced by NF-κB1 as a transcription factor was discovered. Taken together, evidences from both in vitro and in vivo assessment strongly suggest the potential of miR-9 in preventing joint destruction in RA patients. Therefore, miR-9 as an inhibitor of NF-κB1/RANKL pathway would be a possible therapeutic strategy in some of the treatment resistant patients suffered from RA.

Acknowledgements The authors acknowledge Dr. Masaaki Murakami and members (Department of Molecular Neuroimmunology) for providing and assisting the use of luminometer. The authors would like to thank all the members and laboratory assistants of Department of Rheumatology, Endocrinology and Nephrology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University for their technical support and valuable discussion. 7

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Fig. 4. Intra-articular injection of miR-9a-5p mimic to ankle of rats with collagen-induced arthritis (CIA) reduced the inflammation and severity of joint destruction. (A, B) Arthritis scores and ankle diameters of ankle joints were monitored for 31 days in the CIA rats. The rats received intra-articular injection of negative control miRNA (circles, n = 12 ankles) and miR-9a-5p mimic (squares, n = 12 ankles) respectively. Arthritis scores: 0 = normal; 1 = mild but definite redness and swelling of the ankle/digits; 2 = moderate redness and swelling of ankle; 3 = severe redness and swelling of the entire paw including digits; and 4 = maximally inflamed limb with involvement of multiple joints. (C) Representative 3D and 2D micro-CT images of the rats' ankles as well as their corresponding erosion scores (D). 2D micro-CT images joint destruction score: 0 = normal; 1 = soft tissue swelling only; 2 = soft tissue swelling and early erosions; 3 = severe cartilage and bone erosions for each ankle. 3D micro-CT images bone erosion score: 0 = normal, no signs of erosion; 1: roughness of bone surface; 2: pitting/indentations; 3: full thickness holes. Ctrl, control; miR, miRNA; Ta, talus; Ti, tibia. Values are the mean ± S.E.M. *P < .05; **P < .01; ***P < .001; ****P < .0001 versus negative control miR, by by two-way ANOVA analysis in Fig. 4A, B while the others are analyzed by Student's t-test.

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Fig. 5. Histological results of CIA rats after miR-9a-5p treatment. (A) Representative ankle joints specimens stained with H&E, safranin O-fast green, TRAP, or immunohistochemistry using anti-RANKL antibody and anti-cadherin 11 antibodies in the CIA rats. (B) Quantification of parameters to determine inflammation or joint destruction in CIA rats in both control group and miR-9a-5p mimic group. Exudate scores and infiltrate scores were used to determine inflammation state with a scale of 0 = no inflammation to 3 = severe inflamed joint, depending on the number of inflammatory cells. Safranin O-fast green was used to determine the destruction of cartilage. Cells with 3 or more nuclei were considered as TRAP stain positive osteoclasts and the quantification was analyzed by number of osteoclasts per x100 field. The RANKL+ area in the synovial lining was determined in both groups. Ctrl, control; CDH11, cadherin 11; miR, miRNA; MNCs, multinucleated cells. All values are means of 12 rats' ankles of each group and are presented as mean ± S.E.M. *P < .05, **P < .01, ***P < .001, by Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Funding [13]

This work was supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology [grant number: 18K08380].

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Disclosure statement [15]

TA received grants and personal fees from Astellas, grants and personal fees from Takeda, grants and personal fees from Mitsubishi Tanabe, grants and personal fees from Chugai, grants and personal fees from Pfizer, grants and personal fees from Daiichi Sankyo, grants from Otsuka, personal fees from Eisai, personal fees from AbbVie, outside the submitted work. SY received grants and personal fees from Chugai Pharmaceutical, grants and personal fees from Bristol Myers Squibb, personal fees from Tanabe-Mitsubishi Pharmaceutical, grants from Novartis Pharmaceutical, personal fees from Pfizer, outside the submitted work.

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Authors contributions [20]

Study design: W.S.L., S.Y., Michihiro K., Michihito K. and M.Kato. Study conduct: W.S.L., S.Y., Michihiro K, Michihito K, Y.K. Data collection: W.S.L., S.Y., Michihiro K., Michihito K., Y.K., T.S., T.O. Data analysis: W.S.L., S.Y., Michihiro K., Michihito K., S.S., T.S., Data interpretation: W.S.L., S.Y., Michihiro K., Michihito K., M.Kato, Y.F., K.O., S.T., N.I. and T.A. Drafting manuscript: W.S.L. and S.Y. Revising manuscript content: Michihito K, N.I. and T.A.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.clim.2020.108348.

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