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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

MicroRNA, a class of noncoding RNA, play a role in human diseases. MicroRNA-146a (miR-146a) is a negative regulator of immune and inflammatory responses, and is strongly expressed in rheumatoid arthritis (RA) synovium and peripheral blood mononuclear cells (PBMCs). This study was undertaken to examine whether miR-146a expression inhibits osteoclastogenesis, and whether administration of miR-146a prevents joint destruction in mice with collagen-induced arthritis (CIA).

Methods

PBMCs from healthy volunteers were isolated and seeded in culture plates. The following day, double-stranded miR-146a was transfected and cultured in the presence of macrophage colony-stimulating factor and either tumor necrosis factor α or RANKL. After 3 weeks, tartrate-resistant acid phosphatase (TRAP)–positive multinucleated cells were counted. Three days after miR-146a culture, the expression of c-Jun, nuclear factor of activated T cells c1 (NF-ATc1), PU.1, and TRAP was evaluated by quantitative reverse transcriptase–polymerase chain reaction. After the onset of distinct arthritis in mice with CIA, double-stranded miR-146a or nonspecific double-stranded RNA was administered twice by intravenous injection. Radiographic and histologic examinations were performed at 4 weeks.

Results

The number of TRAP-positive multinucleated cells in human PBMCs was significantly reduced by miR-146a in a dose-dependent manner. The expression of c-Jun, NF-ATc1, PU.1, and TRAP in PBMCs was significantly down-regulated by miR-146a. Administration of miR-146a prevented joint destruction in mice with CIA, although it did not completely ameliorate inflammation.

Conclusion

Our findings indicate that expression of miR-146a inhibits osteoclastogenesis and that administration of double-stranded miR-146a prevents joint destruction in arthritic mice. Administration of miR-146a has potential as a novel therapeutic target for bone destruction in RA.

Systemic joint destruction by chronic synovial inflammation is a major problem in rheumatoid arthritis (RA). Osteoclasts, the major bone resorptive cells, play a crucial role in joint destruction and are formed as multinucleated giant cells by the fusion of hematopoietic cells of the monocyte and macrophage lineage at or near the bone surface (1, 2). Macrophages in the RA synovium are capable of differentiating into osteoclasts by initiating a cascade of cellular signals, such as proinflammatory cytokines (3, 4). Prevention of osteoclast differentiation in inflamed joints is one of the best therapeutic strategies for RA patients (5).

MicroRNA (miRNA) are a class of noncoding RNA that regulate gene expression by binding the 3′-untranslated region (3′-UTR) of their target messenger RNA (mRNA), leading to translational repression or mRNA degradation (6–9). Several miRNA exhibit a tissue-specific or developmental stage–specific expression pattern and have been reported to be associated with human diseases, including RA (10–15). MicroRNA-146 (miR-146) has been shown to be strongly expressed in RA synovium and peripheral blood mononuclear cells (PBMCs) and to inhibit the expression of interleukin-1 receptor–associated kinase 1 (IRAK1) and tumor necrosis factor receptor–associated factor 6 (TRAF6) by binding to the 3′-UTR of their mRNA (13–16).

It has been reported that expression of miR-146a/b plays a role as a negative regulator of constitutive activity of NF-κB (16). NF-κB, which activates osteoclast precursor cells, is one of the most important molecules in osteoclastogenesis (17–19). TRAF6, which is one of the target genes of miR-146a, is one of the most important mediators of osteoclastogenesis, and therefore, there is a possibility that regulation of miR-146a could lead to suppression of osteoclastogenesis and subsequently to the prevention of joint destruction.

The aim of this study was to determine whether overexpression of miR-146a inhibits osteoclastogenesis. First, we confirmed that transfection of miR-146a into human PBMCs suppresses osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and either tumor necrosis factor α (TNFα) or RANKL in vitro. Second, we investigated the degree of efficacy of intravenous administration of double-stranded miR-146a for the prevention of joint destruction in vivo in mice with collagen-induced arthritis (CIA).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Isolation of human PBMCs.

Peripheral blood was collected from 4 healthy volunteer donors with written permission, and then added to Dulbecco's phosphate buffered saline (DPBS) (5 mM:0.5M EDTA) drop by drop and mixed well. This mixture was loaded slowly onto Histopaque (Sigma) in another tube and centrifuged at 1,000g at room temperature for 10 minutes. PBMCs accumulated as the middle white monolayer. After the supernatant was discarded, only the white monolayer cells were aspirated and added to DPBS-E, then centrifuged at 400g at 4°C for 10 minutes. The supernatant was discarded, DPBS-E and ammonium chloride (The Cell Experts) were added at a rate of 1:3, and the mixture was allowed to stand at room temperature for 10 minutes. After centrifuging at 400g at 4°C for 10 minutes, the supernatant was discarded, then DPBS-E was added and mixed well. This final process was repeated several times. The remaining white cells were PBMCs.

Culture for osteoclastogenesis and transfection of double-stranded RNA.

Isolated PBMCs were seeded at 5 × 105 cells in each well of 96-well culture plates. The following day, the adherent cells were used for in vitro studies. The adherent cells were confirmed to have abundant CD14-positive cell fractions by immunofluorescence (results not shown). Double-stranded miR-146a (sequences 5′-UGA-GAA-CUG-AAU-UCC-AUG-GGU-U-3′ and 3′-CCU-GUG-AAA-UUC-AGU-UCU-UCA-G-5′) (B-Bridge International) or nonspecific double-stranded RNA (small interfering RNA [siRNA] negative control; B-Bridge International) were transfected into the cells using Lipofectamine 2000 according to the recommendations of the manufacturer (Invitrogen). Cultures were maintained in 200 μl of α-minimum essential medium containing 10% fetal bovine serum, 50 IU/ml penicillin, and 50 μg/ml streptomycin for 21 days with 50 ng/ml M-CSF (Sigma) and either 50 ng/ml TNFα (R&D Systems) or 50 ng/ml RANKL (PeproTech) at 37°C in a humidified atmosphere of 95% air and 5% CO2. Half of the culture medium was replaced with fresh medium containing M-CSF and either TNFα or RANKL every 3 days.

Osteoclast differentiation assay.

Three weeks after transfection, cells were cytochemically stained for tartrate-resistant acid phosphatase (TRAP) using a commercially available kit according to the recommendations of the manufacturer (Sigma). TRAP-positive multinucleated cells containing >3 nuclei were identified as osteoclasts. All experiments were carried out in triplicate at least 3 times. To examine the suppression of osteoclastogenesis by transfection of double-stranded miR-146a, the number of TRAP-positive multinucleated cells was counted under a microscope. The cell number in 30 microscopic fields was counted in each well at 200× magnification.

Pit-formation assay.

A bone resorption assay was performed to assess osteoclast activity. After PBMCs were cultured for 3 weeks on dentin slices, the slices were rinsed with PBS and left overnight in 1M ammonium hydroxide to remove all cells and their debris. The slices were then washed with PBS and stained with 0.5% toluidine blue. Pit formation was analyzed by light microscopy.

Real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR).

To examine the expression of the representative osteoclast marker genes c-Jun, nuclear factor of activated T cells c1 (NF-ATc1), PU.1, and TRAP, real-time qRT-PCR assays were performed using SYBR Green (Invitrogen). Three days after transfection of double-stranded RNAs into PBMCs, RNA was extracted using TRIzol according to the recommendations of the manufacturer (Invitrogen). One microgram of total RNA was reverse-transcribed using a QuantiTect Reverse Transcription kit according to the recommendations of the manufacturer (Qiagen). Reverse transcriptase reactions of mature miRNA contained a sample of total RNA, 50 nM stem–loop RT primer, 10× RT buffer, 100 mM of each dNTP, 50 units/μl MultiScribe reverse transcriptase, and 20 units/μl RNase inhibitor. Fifteen- microliter reactions were incubated in a thermocycler (Bio-Rad) for 30 minutes at 16°C, 30 minutes at 42°C, 5 minutes at 85°C, and held at 4°C. Real-time qRT-PCR was performed using a Mini Opticon Real-time PCR System (Bio-Rad) in a 10-μl PCR mixture containing 1.33 μl RT product, 2× TaqMan Universal PCR Master Mix, 0.2 μM TaqMan probe, 15 μM forward primer, and 0.7 μM reverse primer. Each SYBR Green reaction was performed with a 1.0 μl template complementary DNA, 10 μl SYBR Green Mix, 1.5 μM primer, and water to adjust the final volume to 20 μl.

Primer sequences were as follows: for PU.1, 5′-AAG-ACC-TGG-TGC-CCT-ATG-ACA-3′ (forward) and 5′-GGT-GGA-AGT-CCC-AGT–AAT-GGT-C-3′ (reverse); for NF-ATc1, 5′-GCA-TCA-CAG-GGA-AGA-CCG-TGT-C-3′ (forward) and 5′-GAA-GTT-CAA–TGT-CGG-AGT-TTC-TGA-G-3′ (reverse); for TRAP, 5′-CGC-ACA-GGT-AGG-CAG-TGA-C-3′ (forward) and 5′-CTA-CCC-CGT-GTG-GTC-CAT-AG-3′ (reverse); for c-Jun, 5′-GCG-TTA-GCA-TGA-GTT-GGC-AC-3′ (forward) and 5′-CGC-ATG-AGG-AAC-CGC-ATC-GC-3′ (reverse); and for GAPDH, 5′-CTG-TTC-GAC-AGT-CAG-CCG-C-3′ (forward) and 5′-AAT-CCG-TTG-ACT-CCG-ACC-TTC-3′ (reverse).

All reactions were incubated in a 48-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute, performed in triplicate. The U18 or GAPDH gene was used as a control to normalize differences in total RNA levels in each sample. Ct was observed in the exponential phase of amplification, and quantification of relative expression was performed using standard curves for target genes and the endogenous control. Geometric means were used to calculate the ΔΔCt values and expressed as 2math image. The value of each control sample was set at 1 and used to calculate the fold change of target genes.

Immunocytochemical analysis.

Three days after transfection of double-stranded RNAs into PBMCs, immunofluorescence analysis was performed. Cells were fixed in cold methanol for 2 minutes and washed in PBS for 10 minutes at room temperature. Afterward, the cells were washed 3 times in PBS and incubated in blocking buffer for 30 minutes at room temperature. Cells were washed in PBS and then incubated overnight at 4°C with TRAF6 primary antibody (Alexis Biochemicals). Cells were washed in PBS again and then incubated with Alexa Fluor 568–conjugated goat anti-rabbit IgG (Molecular Probes/Invitrogen) as a secondary antibody for 1 hour at room temperature. DAPI solution (Dojindo) was applied for 5 minutes for nuclear staining.

Induction of CIA.

All animals were housed individually and fed a commercial diet. This study was reviewed and approved by the Ethics Committee for Experimental Animals of Hiroshima University, and all animals were treated according to the guidelines of the Institutional Animal Care and Use Committee. Eight-week-old male DBA/1 mice were used. Lyophilized chicken type II collagen (Chondrex) was dissolved overnight at 4°C in 0.05M acetic acid under constant stirring. The collagen was emulsified on ice with Freund's complete adjuvant (Chondrex) to a final concentration of 2 mg/ml, using electric homogenization. Mice were injected in the tail with 0.1 ml of emulsion. Lipopolysaccharide (LPS; 50 μg) was injected intraperitoneally on day 28. Arthritis developed no later than 2 days after the LPS injection. Development of arthritis was monitored using the scoring system described previously (20), where 0 = no inflammation; 1 = mild, but definite redness and swelling of the ankle or wrist, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits; 2 = moderate redness and swelling of ankle or wrist; 3 = severe redness and swelling of the entire paw including digits; 4 = maximally inflamed limb with involvement of multiple joints (maximum possible score 16).

Intravenous injection of double-stranded miR-146a or scrambled siRNA.

An equal volume of atelocollagen (AteloGene systemic use; Koken) (in PBS at pH 7.4) and miRNA solution (20 μg/25 μl) was combined and mixed by rotation at 4°C for 20 minutes. The nonspecific double-stranded RNA–atelocollagen complex was prepared in the same manner. Beginning on day 30, mice were injected in the tail vein with atelocollagen mediated with double-stranded miR-146a (n = 5) or nonspecific double-stranded RNA (n = 5) at 1-week intervals. Four weeks after the first injection, mice were killed by an overdose of anesthesia, and a radiograph was obtained. Next, the limbs were dissected and fixed with 4% paraformaldehyde for 24 hours. The samples were decalcified in 0.5M EDTA (pH 7.5) for 14 days and embedded in paraffin. The sections were prepared at a thickness of 6 μm and stained with hematoxylin and eosin or TRAP.

Histologic examinations were performed independently by 2 evaluators (YN and TN). Sections were graded according to the system described previously, where 0 = no inflammation, 1 = slight thickening of the synovial cell layer and/or some inflammatory cells in the sublining, 2 = thickening of the synovial lining, infiltration of the sublining, and localized cartilage erosions, and 3 = infiltration into the synovial space, pannus formation, cartilage destruction, and bone erosion (21).

Immunofluorescence analysis.

For immunofluorescence analysis of proinflammatory cytokines in sections from mice, each section was washed in PBS and treated with retrieval solutions (Dako) for 20 minutes at 90°C. After blocking with blocking reagent for 30 minutes, sections were incubated with the following primary antibodies: TNFα (R&D Systems), interleukin-1β (IL-1β; Santa Cruz Biotechnology), and IL-6 (Santa Cruz Biotechnology). The secondary antibodies for each immunostaining were as follows: Alexa Fluor 568–conjugated rabbit anti-goat IgG (Molecular Probes/Invitrogen) for TNFα and IL-6 and Alexa Fluor 488–conjugated goat anti-rabbit IgG (Molecular Probes/Invitrogen) for IL-1β. DAPI solution was applied for 5 minutes for nuclear staining. The negative control was prepared in the same manner without the primary antibody.

Statistical analysis.

Comparisons among the 4 groups were performed using the Tukey-Kramer post hoc test, and the Mann-Whitney U test was used for the detection of differences between the control siRNA group and the miRNA group. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Suppression of osteoclastogenesis in human PBMCs by transfection of miR-146a.

To examine whether overexpression of miR-146a suppresses osteoclastogenesis, double-stranded miR-146a was transfected into human PBMCs at various concentrations, and osteoclastogenesis was induced by 2 different methods: M-CSF and TNFα or M-CSF and RANKL. At 3 weeks, the number of TRAP-positive large multinucleated cells was significantly reduced by transfection of miR-146a in a dose-dependent manner, in both osteoclastogenesis culture systems (Figures 1A, 1C, 2A, and 2C). In both osteoclastogenesis culture systems, little bone resorption was observed on the dentin slice after transfection of double-stranded miR-146a at a concentration of 50 nM (Figures 1B and 2B).

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Figure 1. Osteoclastogenesis mediated by macrophage colony-stimulating factor and tumor necrosis factor α in human peripheral blood mononuclear cells (PBMCs). A, Tartrate-resistant acid phosphatase (TRAP) staining 3 weeks after induction of osteoclastogenesis. The number of TRAP-positive large multinucleated cells was decreased by overexpression of microRNA-146a (miR-146a), in a dose-dependent manner. Bars = 200 μM. B, Pit-formation assay. A resorption pit on the dentin slice was barely visible in PBMCs after transfection of double-stranded miRNA-146a (ds miR-146a) at a concentration of 50 nM. Bars = 200 μM. C, Number of osteoclasts. The number of TRAP-positive large multinucleated cells was significantly decreased by transfection of miR-146a, in a dose-dependent manner. D, Quantitative reverse transcriptase–polymerase chain reaction for the evaluation of osteoclast marker genes. Transfection of miR-146a significantly down-regulated the expression of c-Jun, PU.1, TRAP, and nuclear factor of activated T cells c1 (NF-ATc1). Bars show the mean ± SD. ∗ = P < 0.05 versus control nonspecific double-stranded small interfering RNA (ds Nega). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Figure 2. Osteoclastogenesis mediated by macrophage colony-stimulating factor and RANKL in human PBMCs. A, TRAP staining 3 weeks after induction of osteoclastogenesis. The number of TRAP-positive large multinucleated cells was decreased by overexpression of miR-146a, in a dose-dependent manner. Bars = 200 μM. B, Pit-formation assay. A resorption pit on the dentin slice was barely visible in PBMCs after transfection of double-stranded miR-146a at a concentration of 50 nM. Bars = 200 μM. C, Number of osteoclasts. The number of TRAP-positive large multinucleated cells was significantly decreased by transfection of miR-146a, in a dose-dependent manner. D, Quantitative reverse transcriptase–polymerase chain reaction for the evaluation of osteoclast marker genes. Transfection of miR-146a significantly down-regulated the expression of c-Jun, PU.1, TRAP, and NF-ATc1. Bars show the mean ± SD. ∗ = P < 0.05 versus control nonspecific double-stranded small interfering RNA. See Figure 1 for definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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The expression of c-Jun, NF-ATc1, PU.1, and TRAP was significantly down-regulated 3 days after the transfection of double-stranded miR-146a in both osteoclastogenesis culture systems (Figures 1D and 2D). Osteoclastogenesis was suppressed via the down-regulation of c-Jun, NF-ATc1, PU.1, and TRAP by transfection of double-stranded miR-146a.

TRAF6 is one of the validated targets of miR-146a, and a major factor in osteoclastogenesis. Therefore, immunocytochemical analysis was performed to examine the expression of TRAF6 protein in M-CSF and RANKL–induced osteoclastogenesis in PBMCs after the transfection of miR-146a. TRAF6 was well detected in the nonspecific double-stranded RNA group, but hardly observed in the miR-146a group (Figure 3). These results suggested that one of the mechanisms of suppression of osteoclastogenesis was down-regulation of TRAF6 by miR-146a.

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Figure 3. Immunofluorescence analysis of tumor necrosis factor receptor–associated factor 6 (TRAF6) expression in human PBMCs in which osteoclastogenesis was induced by macrophage colony-stimulating factor and RANKL. TRAF6 was well detected after transfection of nonspecific double-stranded RNA, while it was hardly visible after transfection of double-stranded miR-146a. Original magnification × 200; bars = 100 μm. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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Prevention of cartilage and bone destruction in mice by administration of double-stranded miR-146a.

To examine the degree of efficacy of administering double-stranded miR-146a for the prevention of joint destruction, an intravenous injection of double-stranded miR-146a was administered twice to arthritic mice. Intravenous injections were performed after the onset of full-blown arthritis to examine the potential role of miR-146a in preventing cartilage and bone destruction in mice through the suppression of osteoclastogenesis.

The symptoms of arthritis, such as swelling and redness, were virtually identical in mice treated with nonspecific double-stranded RNA and mice treated with double-stranded miR-146a. However, 4 weeks after the first injection, radiographic examination of the hind paw revealed that the extent of bone destruction in mice that received the double-stranded miR-146a injection seemed to be suppressed compared with that in mice that received the nonspecific double-stranded RNA injection (Figures 4A and B). A section from the hind paw of a representative mouse treated with nonspecific double-stranded RNA revealed extensive pannus formation with synovial hyperplasia, infiltration of a large number of inflammatory cells at the joint space, and cartilage and bone destruction (Figure 4C). In contrast, neither cartilage nor bone structure was destroyed in a section from the hind paw of a representative mouse treated with double-stranded miR-146a (Figure 4D). However, synovial hyperplasia, such as infiltration of inflammatory cells, was not completely inhibited.

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Figure 4. Effect of systemic administration of double-stranded miR-146a in mice with collagen-induced arthritis. A and B, Representative radiographs of the hind paws of mice treated with nonspecific double-stranded RNA (A) or double-stranded miR-146a (B). Arrows indicate joint destruction. C and D, Hematoxylin and eosin staining of sections from the hind paws of mice treated with nonspecific double-stranded RNA (C) or double-stranded miR-146a (D). Extensive pannus formation with synovial hyperplasia, infiltration of a large number of inflammatory cells into the joint space, and cartilage and bone destruction were observed in mice treated with nonspecific double-stranded RNA. In contrast, neither cartilage nor bone structure was destroyed in the section from the hind paw of a mouse treated with double-stranded miR-146a. Bars = 200 μm. E and F, TRAP staining of sections from the hind paws of mice treated with nonspecific double-stranded RNA (E) or double-stranded miR-146a (F). A number of TRAP-positive cells were observed at the border of the pannus and bone in the mice injected with nonspecific double-stranded RNA, while few TRAP-positive cells were observed in the mice injected with double-stranded miR-146a. Arrows indicate TRAP-positive cells. Bars = 200 μm. G, Histologic scores of sections from mice injected with double-stranded RNA and mice injected with double-stranded miR-146a. Scores in the group treated with double-stranded miR-146a were significantly lower than those in the group treated with nonspecific double-stranded RNA. Bars show the mean ± SD. ∗ = P < 0.05. See Figure 1 for definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131.

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A number of TRAP-positive cells were observed at the border of the pannus and bone in the group treated with nonspecific double-stranded RNA, while few TRAP-positive cells were observed in the group treated with double-stranded miR-146a (Figures 4E and F). There was no significant difference in the arthritis score between the 2 groups, although the histologic score in the double-stranded miR-146a injection group was significantly lower than that in the nonspecific double-stranded RNA injection group (Figure 4G).

To examine whether miR-146a has as an antiinflammatory effect in mice with arthritis, immunofluorescence analysis of TNFα, IL-1β, and IL-6 expression was performed. Although TNFα, IL-1β, and IL-6 levels in the synovium of mice treated with double-stranded miR-146a were low compared to those in the synovium of mice treated with nonspecific double-stranded RNA, administration of double-stranded miR-146a did not completely suppress the expression of proinflammatory cytokines (Figure 5).

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Figure 5. Immunofluorescence analysis of tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), and IL-6 expression in sections from mice treated with nonspecific double-stranded RNA and mice treated with double-stranded miR-146a. Low levels of TNFα, IL-1β, and IL-6 were observed in the synovium of mice treated with double-stranded miR-146a, while abundant levels of TNFα, IL-1β, and IL-6 were observed in synovial hyperplasia in mice treated with nonspecific double-stranded RNA. Asterisks indicate bone. Bars = 100 μm. See Figure 1 for other definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Recently, miRNA has been attracting attention due to its crucial role in human disease and is shaping up to be a new therapeutic target. It has gradually become clear that miRNA participate in the pathogenesis of RA. Stanczyk et al reported that miR-146 and miR-155 are strongly expressed in RA synovial fibroblasts compared with osteoarthritis (OA) fibroblasts (13). We previously demonstrated that miR-146 is strongly expressed in RA synovial tissue compared with OA and normal synovial tissue. We also demonstrated that miR-146 is predominantly expressed in CD68+ macrophages but is also expressed in some CD3+ T cell subsets and CD79a+ B cells in RA synovial tissue (14). Pauley et al reported that PBMCs from RA patients exhibit high expression of miR-146a, miR-155, miR-132, and miR-16 compared with healthy individuals and control patients (15). This evidence, which proves that miRNA play a role in RA pathogenesis, may lead to a novel treatment strategy.

Several therapeutic trials to regulate miRNA in vivo have been undertaken. Tazawa et al demonstrated that local injection of double-stranded miR-34a into a tumor suppresses tumor growth in mice (22). We previously showed that local injection of miR-1, miR-133, and miR-206 accelerates muscle regeneration in the rat model of skeletal muscle injury (23). We have also shown that intraarticular injection of double-stranded miR-15a induces apoptosis in the synovium of arthritic mice (24). A novel therapy targeting miRNA might be developed for RA.

Taganov et al reported that the induction of miR-146a/b is regulated by NF-κB, and that miR-146a/b plays a role in fine-tuning innate immune responses by providing negative feedback with down-regulation of TRAF6 and IRAK1 genes (16). In RA synovium, the expression of miR-146a might be induced by NF-κB to regulate the expression of proinflammatory cytokines by negative feedback. Proinflammatory cytokines, especially TNFα, are one of the most important factors in osteoclastogenesis (25, 26).

Osteoclastogenesis induced by TNFα occurs independently of RANK signaling. Biologic agents such as TNFα blockers have been shown to successfully decrease joint destruction. In the present study, overexpression of miR-146a inhibited osteoclastogenesis induced by both TNFα and RANKL, although TNF induced osteoclastogenesis independently of TRAF6, which is recognized as the target gene of miR-146a. However, the mechanism by which miR-146 inhibits osteoclastogenesis remains unclear. Bhaumik et al demonstrated that expression of miR-146a/b in the metastatic human breast cancer cell line functions to negatively regulate NF-κB activity (27). When miR-146a is overexpressed in PBMCs in the osteoclastogenesis culture system induced by TNFα, miR-146a has the potential to inhibit osteoclastogenesis via negative regulation of NF-κB activity independently of the TRAF6 pathway. It is possible that miR-146a also inhibits the expression of TRAF6, although TRAF6 is recognized to be the critical signaling molecule in RANKL-mediated osteoclastogenesis (28–30).

The number of target genes of miRNA is estimated to range between one and several hundred, based on target predictions using the bioinformatics approach (31). Several factors other than TRAF6 and IRAK1 might contribute to the inhibition of osteoclastogenesis by miR-146a. Moreover, the mechanism of action of osteoclastogenesis has not been elucidated.

In this study, since osteoclastogenesis was successfully inhibited by transfection of double-stranded miR-146a in vitro, double-stranded miR-146a was administered to mice in vivo via intravenous injection. Administration of double-stranded miRNA-146a prevented joint destruction in mice. However, it did not completely ameliorate joint inflammation. Although bone and cartilage destruction were prevented in mice by administration of miRNA-146a, histologic analysis showed that synovitis was not completely abrogated. However, there was no significant difference in the arthritis score between the mice that received nonspecific double-stranded RNA and those that received miR-146a.

Immunofluorescence analysis showed that administration of miR-146a to mice did not completely suppress the expression of TNFα, IL-1β, or IL-6. First administration of double-stranded miR-146a was conducted after the onset of distinct arthritis. MicroRNA suppressed the production of protein such as TNFα by targeting mRNA, but miRNA did not suppress the protein that had already been secreted.

Administering double-stranded miR-146a by intravenous injection might not have a great enough antiinflammatory effect; this may explain the lack of a significant difference in arthritis score between mice that received nonspecific double-stranded RNA and those that received miR-146a. The combination of a biologic agent and disease-modifying antirheumatic drugs might be more effective. Khoury et al reported that an intravenous injection of a cocktail of 3 siRNA for IL-1, IL-6, and IL-18 ameliorated joint inflammation and degradation (32). The combination of several miRNA, which play a crucial role in RA pathogenesis, has greater potential to effectively reduce inflammation.

Moreover, the methods of administration of synthetic miRNA, including dosage and delivery system, should be examined to determine which methods ameliorate arthritis most efficiently. We examined the distribution of double-stranded miR-146a 24 hours after injection using real-time qRT-PCR, as previously described (24). The results revealed that miR-146a was up-regulated in the liver, spleen, and kidney. However, there was no notable difference in the hind paws between mice that received an injection of miR-146a and mice that received no injection (data not shown). There is a possibility that miR-146a affects circulating cells that play a role in arthritis, including bone destruction; however, the functional mechanism of double-stranded miR-146a could not be elucidated. The tissue distribution and cell type that takes up the administered double-stranded miRNA should be examined in the future.

This is the first study to evaluate the systemic administration of double-stranded miRNA for arthritis. Although this preliminary study might provide a useful optional tool for RA treatment, further studies are needed to determine more comprehensive therapeutic strategies.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Nakasa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Nakasa, Ochi.

Acquisition of data. Nakasa, Shibuya, Nagata.

Analysis and interpretation of data. Nakasa, Shibuya, Nagata, Niimoto.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES