Sample collection and cell culture
Synovial tissue and fluid samples were collected during knee joint replacement surgery from patients with RA (n = 12, 7 female, age 29 to 72 years old, mean 51 years) and patients with OA (n = 12, 6 female, age 39 to 77 years old, mean 62 years). All of the patients fulfilled the American College of Rheumatology (ACR) diagnosis criteria for RA and OA . The RA patients had disease duration of 3–9 years and were classified as having erosive RA (Larsen class IV–V). These patients presented high levels of C-reactive protein (CRP) (32–298 mg/L, mean 73 mg/L), anti-cyclic citrullinated peptide (anti-CCP) (28–458 U/ml, mean 279.6 U/ml) and rheumatoid factor (RF) (38–316 U/ml, mean 183.2 U/ml). Before arthroplasty, the disease activity of each RA patient was evaluated using the 28-joint disease activity score based on CRP (DAS28-CRP) . The degree of OA was graded by experienced orthopedic surgeons prior to surgery by x-ray classification, and the patients’ Kellgren and Lawrence grades ranged from 2–4. All of the patients provided written informed consent to participate in this study. The Ethics Committee of Shandong Provincial Qianfoshan Hospital approved this study. RASFs were isolated from synovial biopsy specimens of patients with RA as previously described  and cells between passages 4 and 7 were used for further study.
RASFs were plated in 12- and 24-well plates (5 × 104 and 3–5 × 105 cells/well) in Dulbecco’s modified Eagle’s medium, and stimulated for the indicated time points with the following agents: lipopolysaccharide (LPS from Escherichia coli J5; SIGMA, St Louis, MO, USA), recombinant purified Sema4A (H00064218-P01, human, Abnova, Taiwan). The dilution buffer, PBS, was applied as the control to LPS or recombinant human semaphorin 4A (rhSema4A). Activation of signaling pathways was blocked using BAY 11–7082, an NF-κB inhibitor, WP1066, a Stat3 inhibitor, SU11274, a Met inhibitor or PD98059, a mitogen-activated protein kinase (MAPK) inhibitor (all from Calbiochem/EMD Millipore, Billerica, MA, USA).
Small interfering RNA transfection in RASFs
RASFs (2 × 105 cells in 100-mm-diameter dishes or 8.5 × 104 cells in 6-well plates) were transiently transfected with Sema4A small interfering RNA (siRNA, 50 nM, SI00133560, SI04174107, QIAGEN, Hilden, Germany) or negative control (QIAGEN, SI03650318) by Hiperfect transfection reagent (QIAGEN) following the manufacturer’s instructions, and all experiments were performed 24–48 h after transfection. The specific siRNAs targeting p50/p65, Plexin B1, Plexin D1 and Tim-2 were designed and synthesized by GenePharma (Shanghai, China), and the most effective single siRNA was used for further experiments as follows: p65, sense strand: 5’-CCUCCUUUCAGGAGAUGAAUU-3’ and anti-sense strand: 5’-UUCAU CUCCUGAAAGGAGGUU-3’; p50, sense strand: 5’-GGCCUGAACAAAUGUUU CAUU-3’ and anti-sense strand: 5’-UGAAACAUUUGUUCAGGCCUU-3’; Plexin B1, sense strand: 5’-GGUUCUGGAUCAAUAUAAU-3’ and anti-sense strand: 5’-AUUAUAUUGAUCCAGAACC-3’; Plexin D1, sense strand: 5’- GGCCUCAAC UUGAUCUUCU-3’ and anti-sense strand: 5’- AGAAGAUCAAGUUGAGGCC-3’; Tim-2, sense strand: 5’- GCUGGGUUCAAGACUGUUA-3’ and anti-sense strand: 5’- UAACAGUCUUGAACCCAGC-3’. Non-specific negative control siRNAs were also designed and synthesized (sense strand: 5’-UUCUCCGAACGUGUCACG-3’ and anti-sense strand: 5’-ACGUGACACGUUCGGAGAATT-3’). The mock group was defined as that supplemented with the transfection reagent only.
Cell invasion assay
The cell invasion assay was performed in transwell apparatus (Corning, Tewksbury, MA, USA). RASFs at a density of 3 × 104 cells/well were grown to confluence and incubated with 100 nM of siRNA or rhSema4A in the upper compartment of the transwell apparatus for 48 h. After starvation, the lower compartment was filled with DMEM with 10 % FBS and the plates were continually incubated at 37 °C for 24 h. The upper surface of the insert was wiped with cotton swabs to remove non-invading cells, and the bottom surface of the insert was stained with Giemsa (Sigma, St Louis, MO, USA). The number of cells that invaded through the membrane was quantified in five random fields at × 100 magnification.
After various treatments, serum-free conditioned media samples were collected and centrifuged at 10,000 × g for 5 minutes to remove particulates. The supernatant was harvested and assayed for IL-1β, TNF-α, and IL-6 (R&D Systems, Minneapolis, MN, USA) by ELISA according to the manufacturer’s instructions. Sema4A within synovial fluid and culture media were determined as follows: sandwich ELISA was developed utilizing goat anti-Sema4A (C-46258, Santa Cruz, CA, USA) as capture antibody, rabbit anti-Sema4A (ab70178, Abcam, Cambridge, UK) as detection antibody and horseradish peroxidase-labeled goat anti-rabbit IgG antibody (Jackson, West Grove, PA, USA) together with 3,3′,5,5′-tetramethylbenzidin for color development. rhSema4A was tested at concentrations ranging from 1 to 1,000 ng/ml to generate a standard curve.
Western blot analysis
Whole cell lysates were fractionated by 10 % SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Little Chalfont, UK) and probed with antibody to phospho-Stat3, Stat3; phospho-extracellular signal-regulated kinase (phosphor-ERK), ERK; phospho-MET, MET; phospho-NF-κB, NF-κB (Cell Signaling, Danvers, MA, USA), Sema4A (Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz). Bound antibodies were revealed with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) and the blot developed using an ECL Plus detection system (Thermo Scientific, Pittsburgh, PA, USA).
RNA extraction and quantitative (qRT-PCR)
RNA extraction and qRT-PCR was performed as previously described . The details of the primers are as followings: Sema4A: 5’-TAAAGTGAATGAAACCATTT GT-3’ (forward), 5’-GTCTGTGAAATGTTTTACAGTGT-3’ (reverse); GAPDH: 5’-C ACCATCTTCCAGGAGC-3’ (forward); 5’-AGTGGACTCCACGACGTA-3’ (reverse). GAPDH was used as internal loading control. Relative mRNA levels were measured using the 2-Δ cycle threshold (2-ΔCT) method. Three independent experiments were completed and each reaction was performed in triplicate.
Chromatin immunoprecipitation assay (ChIP)
RASFs were untreated or treated with LPS for 24 h before the cells were fixed with 1 % formaldehyde in medium for 10 minutes at room temperature. The sonication conditions were optimized to determine generation of DNA fragments between 300 and 600 bp in length. Briefly, nuclear lysates were sheared with five to eight pulses using an Ultrasonic Processor (80 W) with tubes immersed in iced water. Each pulse cycle comprised 5 s sonication followed by 25 s cooling down. Chromatin was immunoprecipitated with IgG (Sigma) and anti-p65 (ab7970, Abcam). The association of p50 and p65 was measured by RT-PCR on immunoprecipitated chromatin by use of the following primers: 5’-TATACTGGCTGTGACA-3’ (forward) and 5’-TGACAGATCCTGGCTT-3’ (reverse).
Statistical analysis of the data was performed using SPSS V.16 software (SPSS, Armonk, NY, USA). The t test was used to assess statistical differences between two groups. The correlation between Sema4A level and TNF-α, and IL-6 in serum from RA patients was calculated by Spearman’s correlation. P values <0.05 were considered significant. The data are presented as standard deviation.