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Several microRNA, which are ~22-nucleotide noncoding RNAs, exhibit tissue-specific or developmental stage–specific expression patterns and are associated with human diseases. The objective of this study was to identify the expression pattern of microRNA-146 (miR-146) in synovial tissue from patients with rheumatoid arthritis (RA).
The expression of miR-146 in synovial tissue from 5 patients with RA, 5 patients with osteoarthritis (OA), and 1 normal subject was analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR) and by in situ hybridization and immunohistochemistry of tissue sections. Induction of miR-146 following stimulation with tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) of cultures of human rheumatoid arthritis synovial fibroblasts (RASFs) was examined by quantitative PCR and RT-PCR.
Mature miR-146a and primary miR-146a/b were highly expressed in RA synovial tissue, which also expressed TNFα, but the 2 microRNA were less highly expressed in OA and normal synovial tissue. In situ hybridization showed primary miR-146a expression in cells of the superficial and sublining layers in synovial tissue from RA patients. Cells positive for miR-146a were primarily CD68+ macrophages, but included several CD3+ T cell subsets and CD79a+ B cells. Expression of miR-146a/b was markedly up-regulated in RASFs after stimulation with TNFα and IL-1β.
This study shows that miR-146 is expressed in RA synovial tissue and that its expression is induced by stimulation with TNFα and IL-1β. Further studies are required to elucidate the function of miR-146 in these tissues.
Rheumatoid arthritis (RA) is characterized by chronic inflammation of synovial tissue, causing destruction of cartilage and bone (1). Synovial tissue from RA patients shows infiltration by macrophages, T cells, and B cells, proliferation of the lining cells, and production of inflammatory cytokines, such as tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β). Inhibiting these cytokines ameliorates clinical symptoms, which strongly supports the important roles played by cytokines in RA (2,3).
The transcription factor NF-κB is a key regulator of inflammation (4,5). Several studies have revealed that activated NF-κB is detected in RA synovial tissue, and its expression contributes to the initiation and maintenance of chronic inflammation (6–8). Not only does NF-κB regulate the expression of the inflammatory cytokines TNFα and IL-1β, but it also promotes the secretion of IL-2, IL-12, and interferon-γ (IFNγ) from Th1 cells, which subsequently activates macrophages. In addition, NF-κB activation promotes synovial hyperplasia by stimulating cell proliferation and inhibiting c-myc–induced apoptosis (9,10).
MicroRNA are a family of ~22-nucleotide non-coding RNAs identified in organisms ranging from nematodes to humans (11–13). Many microRNA are evolutionarily conserved across phyla, regulating gene expression by posttranscriptional gene repression. Long primary transcripts (primary microRNA) are transcribed by RNA polymerase II, processed by the nuclear enzyme Drosha, and released as an ~60-bp hairpin precursor micro. Precursor microRNA are processed by the RNase III enzyme Dicer to ~22 nucleotides (mature microRNA) and then incorporated into RNA-induced silencing complex (RISC). The microRNA–RISC complex binds the 3′-untranslated region of target messenger RNA (mRNA) and either promotes translational repression or mRNA degradation (14–17). Several microRNA exhibit a tissue-specific or developmental stage–specific expression pattern and have been reported to be associated with conditions such as cancer and viral infection (18,19).
Taganov et al (20) reported that microRNA-146a/b (miR-146a/b) is induced in response to lipopolysaccharide (LPS) and proinflammatory mediators and that miR-146a induction is regulated by NF-κB. They also found that miR-146a/b targets were TNF receptor–associated factor 6 (TRAF6) and IL-1 receptor–associated kinase 1 (IRAK1) genes and concluded that miR-146 plays a role in fine-tuning innate immune responses by negative feedback, including down-regulation of TRAF6 and IRAK1 genes.
Until now, there has been no report of miR-146 expression in human disease. RA is a representative inflammatory disease involving proinflammatory cytokines, such as TNFα and IL-1β. We therefore sought to determine whether miR-146 is expressed in RA synovial tissue.
Five patients who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) classification criteria for RA (21) were included. Their clinical characteristics are shown in Table 1. All RA patients were treated with low-dose corticosteroids; 2 of the patients (RA3 and RA5) were also treated with the disease-modifying antirheumatic drugs (DMARDs) methotrexate and sulfasalazine, respectively. Patient RA3 had mutilating disease, with severe joint destruction. Patient RA5 showed more erosive disease, with severe destruction in the large joints. Patients RA1 and RA4 had the least erosive disease. The disease in patient RA1 was well controlled, and severe joint destruction was localized to the small joints of the wrists and feet. Patient RA4 had end-stage joint destruction, accompanied by vasculitis; the vasculitis was controlled with 10 mg of corticosteroids per day. Patient RA2 had more erosive disease, but was treated with steroids only because she was trying to become pregnant; thus, in this patient, disease control was poor and joint destruction severe.
In addition, 5 patients with knee osteoarthritis (OA) diagnosed according to typical clinical features and 1 patient undergoing leg amputation, but whose knee joint was normal, were included. All OA synovial tissue samples were obtained by total knee arthroplasty.
Clinical research was conducted in compliance with the Declaration of Helsinki. Written permission was obtained from all subjects who participated in the study.
Synovial tissue was obtained from 5 patients with RA and 5 patients with OA who were undergoing open synovectomy or total joint replacement, as well as from a patient with a normal joint who was undergoing above-the-knee amputation because of angiosarcoma (Table 1). Three synovial tissue specimens were obtained from random sites during surgery. Each sample was inspected visually to ensure that only inflamed tissue was included. Tissue samples were stored at −70°C until analyzed.
For polymerase chain reaction (PCR) analysis, total RNA was isolated from tissue samples that had been homogenized on ice with Isogen reagent (Nippon Gene, Toyama, Japan). For histopathologic analysis, the tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin.
One microgram of total RNA was reverse-transcribed using 0.5 μg/μl of oligo(dT) primer and First-Strand Reaction Mix Beads (GE Healthcare, Little Chalfont, UK). The reaction mixture was incubated for 60 minutes at 37°C.
Quantitative reverse transcription–PCR (RT-PCR) assays were performed using a TaqMan microRNA assay kit (Applied Biosystems, Foster City, CA) for the mature microRNA and using SYBR Green (Applied Biosystems) for the primary miR-146a/b and TNFα. RT reactions of mature microRNA contained a sample of total RNA, 50 nM stem-loop RT primer, 10× RT buffer, 100 mM each dNTPs, 50 units/μl of MultiScribe reverse transcriptase, and 20 units/μl of RNase inhibitor. Reaction mixtures (15 μl) were incubated in a thermocycler (Applied Biosystems) for 30 minutes at 16°C, 30 minutes at 42°C, and 5 minutes at 85°C and then maintained at 4°C.
Real-time PCR was performed using an Applied Biosystems 7900HT Sequence Detection System in a 10-μl PCR mixture containing 1.33 μl of 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 1.0 μl of template cDNA, 10 μl of SYBR Green mixture, 1.5 μM primer, and water to adjust the final volume to 20 μl.
Primer sequences were as follows: for primary miR-146a, 5′-CAG-CTG-CAT-TGG-ATT-TAC-CA-3′ (forward) and 5′-GCC-TGA-GAC-TCT-GCC-TTC-TG-3′ (reverse); for primary miR-146b, 5′-AGA-CCC-TCC-CTG-GAA-TAG-GA-3′ (forward) and 5′-CAC-CTG-GCT-GGG-AAG-TTG-3′ (reverse); for TNFα, 5′-GAG-TGA-CAA-GCC-TGT-AGC-CCA-3′ (forward) and 5′-AGC-TCC-ACG-CCA-TTG-GC-3′ (reverse); and for GAPDH, 5′-CAT-TGG-CAA-TGA-GCG-GTT-C-3′ (forward) and 5′-GGT-AGT-TTC-GTG-GAT-GCC-ACA-3′ (reverse). All reactions were incubated in a 96-well plate at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute; all were performed in triplicate. The let-7a or GAPDH gene was used as a control to normalize differences in total RNA levels in each sample. A threshold cycle (Ct) was observed in the exponential phase of amplification, and quantification of relative expression levels was performed using standard curves for target genes and the endogenous control. Geometric means were used to calculate the ΔΔCt values and were expressed as 2−ΔΔCt. The value of each control sample was set at 1 and was used to calculate the fold change in target genes.
Paraffin-embedded tissue was sectioned at 5 μm and stained with hematoxylin and eosin. For in situ hybridization, primary miR-146a fragments were derived from PCR products, cloned using the Qiagen PCR cloning kit into the pDrive vector (Qiagen, Chatsworth, CA), and sequenced. Primer sequences for primary miR-146a were 5′-TAT-TGG-GCA-AAC-AAT-CAG-CA-3′(forward) and 5′-GCC-TGA-GAC-TCT-GCC-TTC-TG-3′(reverse).
Digoxigenin (DIG)–labeled riboprobes were transcribed with a DIG RNA labeling kit and T7 polymerase (Roche, Mannheim, Germany). After deparaffinization, each section was fixed in 4% paraformaldehyde for 10 minutes at room temperature, washed 3 times in phosphate buffered saline (PBS) for 3 minutes, and subsequently treated with 600 μg of proteinase K for 10 minutes at room temperature. After treatment in 0.2% glycine-PBS for 10 minutes, sections were refixed in 4% paraformaldehyde for 10 minutes, washed 3 times in PBS for 3 minutes each, and acetylated with 0.25% acetic anhydride in 0.1M triethanolamine hydrochloride for 10 minutes. After washing in PBS for 30 minutes, sections were prehybridized for 1 hour at 65°C with prehybridization buffer (50% formamide and 5× saline–sodium citrate [SSC]). Hybridization with DIG-labeled riboprobes was performed overnight at 65°C in hybridization buffer (50% formamide, 5× SSC, 5× Denhardt’s solution, and 250 μg/ml of Baker’s yeast transfer RNA). After hybridization, sections were washed in 5× SSC for 30 minutes at 65°C, 0.2× SSC for 2 hours at 65°C, and 0.2× SSC for 5 minutes at room temperature. Blocking was performed overnight at 4°C with 4% horse serum and alkaline phosphatase–conjugated Fab anti-DIG antibody (Roche) in 1% sheep serum. Staining was performed using BCIP and nitroblue tetrazolium (NBT; Roche).
Sections stained with BCIP and NBT and washed in PBS were treated for 20 minutes at 90°C with retrieval solutions (Nakalaitesque, Tokyo, Japan). After blocking for 30 minutes with blocking reagent (Nakalaitesque), sections were incubated with primary antibody at appropriate dilutions for 1 hour at room temperature. For primary antibodies, monoclonal mouse anti-human antibody against CD68 (Dako, Carpentaria, CA) and CD3ε (BD PharMingen, San Diego, CA), and monoclonal rabbit anti-human antibody against CD79a (Spring Bioscience, Fremont, CA) were used. After washing, sections were incubated with Alexa Fluor 594 conjugate for CD68 and CD3, and with Alexa Fluor 569 conjugate for CD79a (Invitrogen, Carlsbad, CA) for 30 minutes at room temperature, washed, and then incubated with 4′,6-diamidino-2-phenylindole (Dojindo Laboratories, Kumamoto, Japan). The negative control was prepared in the same manner, but without the primary antibody.
Fresh synovial tissue was obtained from a separate group of 4 RA patients. Synovial cells were isolated from the synovial tissue and cultured as described elsewhere (22). After the third passage, cells appeared to be morphologically homogeneous fibroblast-like cells. RASFs at passages 4–6 were used for the experiments.
Cells were seeded at 1.0 × 105/well into a 6-well plate containing 2 ml of Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum and 1% penicillin/streptomycin. After cells became adherent, they were treated with both recombinant human TNFα (1 ng/ml) and IL-1β (10 ng/ml) (R&D Systems, Minneapolis, MN) and then incubated for 24 hours under an atmosphere of 5% CO2. Cells were washed twice with cold PBS, and then total RNA was isolated with Isogen reagent. Real-time PCR was performed in triplicate with the TaqMan microRNA assay kit to analyze the expression of mature miR-146a or with SYBR Green to analyze the expression of primary miR-146a/b. RT-PCR was conducted to analyze primary miR-146a/b and TNFα.
Data were analyzed statistically using the Mann-Whitney U test. P values less than 0.05 were considered statistically significant.
In the pathogenesis of RA, TNFα is an essential mediator of inflammation. To examine a potential link between miR-146a/b and RA inflammatory activity, mRNA for primary miR-146a/b and TNFα were analyzed by quantitative RT-PCR in normal synovial tissue and in synovial tissue from RA and OA patients (Figures 1A–C). Both primary miR-146a and miR-146b, and the mature form of miR-146a (Figure 1D) were strongly expressed in patients RA1, RA2, RA3, and RA5. TNFα expression (Figure 1C) was also up-regulated in synovial tissue from these patients. In synovial tissue from patient RA4, who had lower levels of RA activity compared with that in the other RA patients, neither the primary miR-146a/b nor TNFα mRNA was highly expressed.
In contrast, in OA synovium, expression of primary miR-146a/b and TNFα mRNA was low. Expression of primary miR-146a/b or TNFα was hardly detected in normal synovial tissue. These observations suggest that primary miR-146a/b expression may accompany synovial inflammation caused by TNFα.
We next examined the expression of mature miR-146a processed by Dicer using real-time PCR of synovial tissue specimens. Mature miR-146a was intensely expressed in patients RA1, RA2, RA3, and RA5 (Figure 1D). In these patients, the expression pattern of mature miR-146a was similar to that of primary miR-146b, suggesting that miR-146a/b up-regulation occurs at a transcription, rather than a maturation, step.
To examine the expression of primary miR-146a in synovial tissue from RA and OA patients, we performed in situ hybridization. Primary miR-146a expression was seen in synovial tissue cells in the superficial and sublining layers of samples from all RA patients examined (Figure 2), except for patient RA4, in which the expression of miR-146 and proinflammatory cytokines as determined by RT-PCR was low (Figure 1). Hematoxylin and eosin staining of synovial tissue from patient RA4 revealed fibrosis and little infiltration of inflammatory cells in synovial tissue. Synovial tissue from the other RA patients showed vigorous proliferation of synovial cells and infiltration of inflammatory cells typical of the histopathologic changes of RA.
In synovial tissue from OA patients, hematoxylin and eosin staining revealed little hyperplasia and infiltration of inflammatory cells in the superficial and sublining layers. Superficial and sublining layers of the tissue from these patients showed little expression of primary miR-146a.
To identify the cells that expressed miR-146 in RA synovial tissue, we performed immunohistochemical analyses using the markers CD68 for macrophages, CD3ε for T cells, and CD79a for B cells, in combination with in situ hybridization (Figure 3). Expression of miR-146a mRNA was observed in cells distributed along the superficial and sublining layers. Double staining revealed that miR-146a+ cells were primarily CD68+, indicating that they were macrophages, but several CD3+ T cells and CD79a+ B cells were also seen.
We next evaluated the up-regulation of miR-146 expression in RASFs following stimulation with TNFα and IL-1β, as was previously described in THP-1 cells (20). Expression of mature miR-146a and primary miR-146a/b was significantly up-regulated in RASFs after TNFα and IL-1β stimulation (Figures 4A, C, and D). RT-PCR analysis showed that the expression of mRNA for primary miR-146a/b and TNFα was also induced after stimulation with these factors (Figure 4B).
Recently, a potential link between microRNA and several human diseases has been examined. For example, the expression of let-7 has been shown to be lower in lung cancer tissue than in normal lung tissue, and such down-regulation may promote high levels of expression of the Ras gene (23). It has also been shown that the expression of miR-143 and miR-145 is reduced in colon cancer tissue. Evidence of microRNA function in conditions such as leukemia, viral infection, and DiGeorge syndrome has been reported (24–29), and therapeutic trials aimed at silencing microRNA in vivo have been conducted (29,30).
The present study, which reveals that miR-146a/b is highly expressed in RA synovial tissue, is the first to focus on microRNA expression in the tissue from RA patients. Human miR-146a is located in the second exon of the LOC285628 gene on human chromosome 5, and human miR-146b resides on chromosome 10. Taganov et al (20) reported that miR-146a/b, miR-132, and miR-155 were identified among 200 microRNA after exposure of the human monocytic THP-1 cell line to LPS. Those authors focused particularly on miR-146a/b after validating levels of miR-146a/b, miR-132, and miR-155 by quantitative RT-PCR. In our analysis of RASFs, we observed strong induction of miR-146a following TNFα stimulation and did not observe up-regulation of miR-132 or miR-155 (data not shown).
The results of our in situ hybridization and immunohistochemical analyses indicated that miR-146a is expressed in various cell types in the superficial and sublining layers of synovial tissue, including synovial fibroblasts, macrophages, T cells, and B cells. In RA, activated CD4+ T cells stimulate macrophages and synovial fibroblasts. These cells secrete inflammatory cytokines, such as TNFα and IL-1β, which also contribute to the formation of hyperplastic synovium, called pannus. It is possible that miR-146a/b might play a role in these pathologic conditions. Moreover, our results also show that miR-146a/b expression could be induced by stimulation with TNFα and IL-1β, which implies that miR-146 mRNA are expressed in synovial fibroblasts in response to TNFα and IL-1β. In our small series of patients, all of the RA patients were being treated with corticosteroids, and 2 patients were also receiving a DMARD. Thus, the influence of drug therapy on miR-146 expression could not be evaluated in our study. Whether or how drug therapy influences miR-146 expression should be clarified in future studies.
Taganov et al (20) reported that miR-146a/b targets are TRAF6 and IRAK1, which are key molecules downstream of TNFα and IL-1β signaling. Those authors concluded that miR-146a/b might play a pivotal role in the fine regulation of a Toll-like receptor and cytokine signaling through negative feedback involving the down-regulation of TRAF6 and IRAK1. If similar processes occur in the pathogenesis of RA, miR-146a/b may function in the termination of inflammation triggered by TNFα and IL-1β. On the other hand, Monti-celli et al (31), using microarray and Northern blot analysis in a murine hematopoietic system, demonstrated that miR-146 expression is higher in Th1 cells than in Th2 or naive T cells. Several other studies have shown that Th1 cells dominate in the balance of Th1/Th2 cells in RA (32,33). Gerli et al (34) noted that Th1 cells drive the condition in RA and that Th2 cells respond early in the disease process. A subset of Th1 cells that produces IL-2, IL-12, and IFNγ may activate macrophages in RA (35). Relevant to this, our data indicate that accumulated CD3+ cells express miR-146, which suggests that miR-146 might play a role in persistent inflammation in RA via a T cell network. Further functional analyses to determine the precise role of miR-146a/b in the pathogenesis of RA could provide novel diagnostic and/or therapeutic tools.
Supported by the NIH (grants AR-50631 and AR-47360), the Arthritis Foundation, the Japan Science and Technology Agency SORST Project, the Japanese National Institute of Biomedical Innovation, Genome Network Project (MEXT), and DECODE.
AUTHOR CONTRIBUTIONSDr. Asahara 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 design. Nakasa, Miyaki, Asahara.
Acquisition of data. Nakasa, Miyaki, Okubo, Nishida, Ochi,
Analysis and interpretation of data. Miyaki, Okubo, Hashimoto, Nishida, Asahara.
Manuscript preparation. Nakasa, Asahara.
Statistical analysis. Nakasa, Hashimoto.