Human FLS culture
Synovial tissue samples were taken from the knees of three active RA and OA patients. RA patients were diagnosed according to 2010 the Rheumatoid arthritis classification criteria . All OA patients were diagnosed based on American College of Rheumatology Subcommittee Guidelines for Osteoarthritis . Biopsy samples were obtained during the knee joint arthroscopy. Synovial tissues were sectioned into 1–2 mm3 pieces and cultured with DMEM containing 10% fetal calf serum, 100 U/ml of penicillin and 100 μg/mL of streptomycin, and incubated in a humidified incubator containing 5% CO2. The culture medium was changed every 3–4 days. FLS was maintained as monolayers and serial passages between 3 to 6 were used for experiments described herein.
Total RNA was isolated from RA-FLS and OA-FLS using Trizol reagent (Invitrogen, USA) according to manufacturer’s protocols. Reverse transcription was carried out using the first-strand cDNA synthesis kit (TaKaRa, China). To assess the RHAMM mRNA, IL-6 and IL-8 expression, real-time PCR was performed using a SYBR Premix ExTaq kit (TaKaRa, China). The primers for RHAMM are listed as follows: Forward-CAGGTCACCCAAAGGAGTCTCG, Reverse-CAAGCTCATCCAGTGTTTGC, IL-6: Forward-CCGGGAACGAAAGAGAAGCT, Reverse-GCGCTTGTGGAGAAGGAGTT, IL-8: Forward-ATGACTCAGATGTGCTCTCAAAGG and Reverse-GCTTGCATCATGTCAGAGGAAATTC, β-actin: Forward-AACTACCTTCAACTCCATCA, Reverse-GCCAGACTCGTCATACTC.
Proteins were extracted from RA-FLS and OA-FLS with an extraction buffer. The samples were loaded on 8% polyacrylamide Tris/glycine gels and separated at 90 V for 1 h, then transferred to a nitrocellulose membrane at a setting of 90 V for 1 h. After blocking, the membrane was incubated over-night with primary Rabbit antibodies specific to RHAMM (1:10000, Santa Cruz, USA) or Mouse anti-β-actin (1:5000, Santa Cruz, USA) at 4 °C for overnight, and then incubated with Goat-anti-Rabbit HRP (1:5000, Santa Cruz, USA) or Equine-anti-Mouse HRP (1:10000, Santa Cruz, USA) respectively. After chemiluminescent staining and fixing, gel images were scanned and analyzed using image processing software (Image J).
Collagen antibody-induced arthritis
Ten weeks-old C57BL/6 mice were obtained from the animal laboratory of Southern Medical University, Guangzhou,China. The mice were housed in a standard environment (25 °C and 12 h light/dark cycle), and the mice were fed with standard food and water ad libitum. All the animal treatments were conducted in accordance with the ethical guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals . Experiments were approved by the Ethics Committee of Southern Medical University (license no.SCXK 2011–0015). Mice were randomly divided into two groups (n = 5/group), CAIA and control groups. In the CAIA group mice were injected intravenously with 150 μL (1.5 mg) of five anti-type II collagen monoclonal antibodies at day 0, and 70 μL (35 μg) LPS after 3 days. Every mouse in the control group was injected intravenously with 150 μL of 0.9% saline and 70 μL of 0.9% saline after 3 days.
Mice were scored for arthritis from days 0 to 12 by using 5-point scale scoring system: 0 = no arthritis; 1 = mild joint deformity and swelling; 2 = moderate joint deformity and swelling; 3 = severe swelling in the toe, foot and ankle and 4 = severe inflammation.
On day 12, all the mice were sacrificed by decapitation, and their ankle, lung and kidney tissues were collected and fixed in 4% paraformaldehyde at room temperature for 6 h, dehydrated, embedded in paraffin and sectioned. Paraffin sections were HE stained for the evaluating the morphology of the joint. After heat-mediated antigen retrieval, sections were incubated with primary antibody for RHAMM (1:50) at 4 °C for 12 h, and then with secondary antibody (1:50) at room temperature for 30 min. For antigen visualization, the sections were immersed in 3-amino-9-ethylcarbazole+substrate-chromogen for 30 min, and counter-stained with Gill’s haematoxylin for 30 min.
The RNA interference assay
In this assay, the small interfering RNA (siRNA) for against RHAMM (AM16708, Invitrogen) and the no-target control siRNA (AM4611, Invitrogen) were used. FLS were transiently transfected with the indicated combinations of the siRNAs using Lipofectamine™ 2000 transfection reagent (Invitrogen, USA) according to the manufacturer’s recommendations. The efficiency of siRNA-mediated gene silencing was assessed using real-time quantitative PCR and Western blot.
Enzyme linked immunosorbent assay
After treatement with RHAMM siRNA, 2 × 104 RA-FLS or OA-FLS were stimulated in 24 wells using human IL-1 alpha (10 ng/ml, Invitrogen, USA) for 24 h, supernatant was collected and used for the detection of IL-6 and IL-8 levels by ELISA (Thermo Fisher Scientific, USA). The optical density (OD) of each sample was measured at 450 nm. Recombinant IL-6 and IL-8 cytokines were used as standards.
Migration and invasion assay of FLS
Wound scratch assay
To demonstrate the effects of siRNA on the migratory capacity of FLS, wound scratch assay was performed. After treatment with RHAMM siRNA, 2 × 104 RA-FLS and OA-FLS were seeded in 24-well plates. After serum starvation for 24 h, 200 μL pipette tip was used to make a perforation. FLS were viewed for 0–24 h. The rate of migration was calculated by measuring the distance of the wound after scratching as follows: rate of migration in % = [distance moved (migrating cell front)/total distance (wound margin)] × 100.
Transwell migration and invasion assay
The directed chemotaxis assay was performed using transwell chambers with 8 μm pores (Corning, BD Biosciences) coated with bovine serum albumin (BSA). Briefly, 10% fetal bovine serum (FBS)/DMEM as a chemoattractant was placed in the lower chambers. A total of 2 × 104 FLS were suspended in serum-free DMEM in the upper chambers for 48 h. After non-migrated cells were removed with a cotton swab, the membranes were fixed with 4% paraformaldehyde for 30 min and then stained with 0.1% crystal violet. Migration was quantified by using an optical microscope and counting the stained cells that migrated to the lower side of the filter. The number of migrated cells was presented as the migration index, and this value was calculated by normalizing relative to the media control,. For the invasion assay, similar experiments were performed using inserts coated with a Matrigel membrane matrix. Briefly,the FLS were seeded at a density of 2 × 104 and grown in DMEM for 48 h. Cells that invaded through the matrix to the basolateral side of the membrane were fixed and stained. For further calculations, means values of the number of invading cells from six field-of-view images normalized relative to the control were used.
Data are presented as the mean ± standard deviation. The Mann–Whitney U and the Kruskal-Wallis statistic tests using GraphPad Prism 5 were used to analyze the differences between groups. Differences were considered to be statistically significant when p < 0.05 at 95% confidence interval.