Patients and controls
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 . 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.
Demographic and clinical features of the study subjects*
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 (). 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.
Synthesis of complementary DNA (cDNA)
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 (real-time) PCR
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.
Histologic analysis and in situ hybridization
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).
Double staining combining in situ hybridization and immunohistochemistry
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.
Isolation and culture of human RA synovial fibroblasts (RASFs)
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.
Induction of miR-146a expression in RASFs by TNFα and IL-1β
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.