Ten male Wistar rats (age, 6–8 weeks; weight 100–120 g) susceptible to developing CIA were purchased from Vital River Laboratories (Beijing, China). All rats were kept under controlled environmental conditions with a mean temperature of 22±3°C, 12 h dark-light cycle, relative humidity of 40% and access to food and water ad libitum. The experimental protocol was approved by the Ethics Committee for the use of animals of Shandong Academy of Medical Science (Jinan, China). All efforts were made to minimize discomfort and reduce the number of experimental animals used. All procedures conformed to the ethical guidelines regarding the care and use of laboratory animals, published by the International Association for the Study of Pain and the National Institutes of Health (12). After acclimatization for one week in their cages, the rats received a sub-cutaneous injection of 100 µg native bovine type II collagen (Chondrex, Inc., Redmond, WA, USA) emulsified in Freund's complete adjuvant (Chondrex, Inc.) into the base of the tail. A second sub-cutaneous boost of 100 µg type II collagen in Freund's complete adjuvant was given 21 days later. The animals were anesthetized followed by a lethal dose of sodium pentobarbital (Euthasol; Virbac USA, Fort Worth, TX, USA) 20 days after the second immunization when the paws were notably swollen.

The synovial tissues of rats with CIA was finely chopped and incubated with type II collagenase (1 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco's modified Eagle's medium (DMEM, HyClone, Thermo Fisher Scientific, Waltham, MA, USA) for 6 h in an incubator at 37°C with 5% CO₂. The tissue was treated with 0.25% trypsin (Solabio, Beijing, China) in phosphate-buffered saline (PBS), in a volume equivalent to that of DMEM. Cells were filtered and cultured overnight in DMEM supplemented with 10% fetal bovine serum (FBS; HyClone) as well as penicillin (100 IU/ml) and streptomycin (100 µg/ml) (Gibco-BRL, Invitrogen Life Technologies, Carlsbad, CA, USA) for three passages. FLS of rats with CIA at passage 4–6 were used for the present study, which were negative for CD14, CD3, CD19 and CD56 expression as identified by flow cytometric analysis using a Coulter Epics XL flow cytometer (Beckman Coulter, Brea, CA, USA). Phycoerythrin (PE)-conjugated CD14 (cat. no. 12-0149), fluorescein isothiocyanate (FITC)-conjugated CD3 (cat. no. 11-0039), FITC-conjugated CD19 (cat. no. 11-0199) and PE-conjugated CD56 (cat. no. 12-0567) antibodies were obtained from eBio-science Inc. (San Diego, CA, USA) and used at a 1:50 dilution.

siRNA targeting PCSK6 (target mRNA sequence: 5′-GCAGAGAAGAAUGUAUUCATT-3′) was designed and synthesized by GenePharma Co. Ltd (Shanghai, China). Cultured FLS were transfected with siRNA at 160 nmol/l using a HiPerFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The cells were harvested for analysis at 24 h following the transfection. A negative siRNA (sequence: 5′-UUCUCCGAACGUGUCACGUTT-3′) was designed and synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). was used as the negative control; treatment with transfection reagent only was used as the Mock group.

Total RNA was extracted from the cultured cells and the human tissue using a Total RNA kit (R6834; Omega Bio-Tek, Inc., Norcross, GA, USA) and reverse-transcribed using a ReverTra Ace qPCR RT kit (FSQ-101; Toyobo, Osaka, Japan) according to the manufacturer's instructions. qPCR was performed using the LightCycler 480 (4887352001; Roche Diagnostics, Basel, Switzerland) using the following amplification protocol: Denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 sec followed by annealing at 60°C for 1 min and extension at 72°C for 1 sec. The comparative threshold cycle (Ct) method was used to analyze the relative expression of mRNA (13). The relative target gene expression was normalized to GAPDH mRNA levels. Primers for qPCR were also designed according to the consensus sequence as determined using AlignX (Vector NTI Advance 11.0; Invitrogen Life Technologies). The primers for the amplification of PCSK6 were as follows: Forward, 5′-ACTCCAGAAGAAGAGGAAGAGTA-3′ and reverse, 5′-ACCATCGCAGCCTTTATCA-3′. The primers for the amplification of GAPDH were as follows: Forward, 5′-TGAACGGGAAGCTCACT-3′ and reverse, 5′-CATGTCAGATCCACAACGGATA-3′. All primers are synthesized by Bio-Asia Diagnostics Co., Ltd. (Shanghai, China). For all PCRs, standard curves, dissociation curves and migration of PCR products on acrylamide gels were performed to confirm the specificity of the products. The specificity of the qPCR assay was evaluated by melting curve analysis, which showed that the PCSK6 amplification product generated a melting peak at 81.20±0.34°C without primer-dimers or non-specific products.

The CIA FLS were seeded onto 96-well culture plates and cultured at 37°C to 80% confluence. The cultures were treated with PCSK6 siRNA (160 nmol/l). After incubation for 24, 48 or 72 h, 20 µl 5 mg/ml MTT in PBS was added to each well, and cultures were incubated for 4 h at 37°C in the incubator. MTT solution was removed and 150 µl dimethyl sulfoxide was added to extract the MTT-formazan products at room temperature for 10 min. The absorbance was measured in triplicate at 490 nm using a spectrophotometer (DNM-9602G; Prolong Group, Beijing, China).

The invasive ability of the cells was tested using Transwell plates (BD Biosciences, Franklin Lakes, NJ, USA). FLS were seeded into the upper chamber of the Transwell plate at a density of 3×10⁴ cells/well and incubated at 37°C with 160 nmol/l siRNA for 8 h. The upper and the lower chambers were then filled with medium without FBS, followed by incubation for 12 h. Subsequently, the lower chamber was filled with 20% FBS in DMEM, followed by a further incubation for 24 h. The non-invaded cells at the upper surface of the membrane were removed with cotton swabs and the invaded cells on the lower side were stained with Giemsa (Solabio). The cell number of cells that had transgressed through the filter was quantified in five random fields at x100 magnification and the average number was calculated. Cells were observed using a XDS-1B microscope (Nikon, Tokyo, Japan)

A wound-healing assay was performed to test the migration ability of FLS. Cells were plated onto 24-well plates and cultured at 37°C until reaching 80% confluence. The cell monolayers were scratched linearly in multiple areas with a cell scraper (Corning Life Sciences, Tewksbury, MA, USA), and cells were subsequently transfected with 160 nmol/l PCSK6 siRNA or control siRNA. After 24 h of incubation, the number of cells migrated into the scratched area was calculated using a method identical to that described above.

FLS were transfected with 160 nmol/l siRNA for 24 h, and the culture medium was collected and centrifuged at 800 × g for 5 min at 4°C. 100 µl medium was added to a 96-well microplate (Corning-Costar, Corning, NY, USA), which was stored overnight at 4°C. After gently washing with PBS containing Tween 20 (Solabio, Beijing, China), 1% bovine serum albumin (Solabio) plus 5% sucrose (Solabio) was used for blocking for 1 h at 37°C. Following three washes with PBS, antibodies against IL-1α, IL-1β, IL-17 and TNF-α (all from Abcam, Cambridge, MA, USA; dilution, 1:1,000) were applied to the plate for overnight incubation. The plate was washed, blocked and incubated with a 1:2,000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (ProteinTech, Chicago, IL, USA) for 3 h at 37°C. Staining was developed using a TMB kit (CW0050; CWBIO, China). The absorbance at 450 nm was measured using a plate reader (Synergy HT; BioTek, Winooski, VT, USA). FLS treated with transfection reagent only were used as a control.

Cells were seeded onto six-well culture plates (Corning-Costar) at 1.0×10⁶ cells/well and the cells were treated with siRNA as described above. After removal of the culture medium, the cells were harvested at 24 h by trypsinization, washed twice with ice-cold PBS and fixed overnight with 70% ethanol at 4°C. Prior to analysis, the fixed cells were rinsed with PBS, re-suspended in PBS and stained for 30 min at 37°C with 1 ml 0.05 mg/ml propidium iodide solution (Digguo Biotech, Beijing, China) containing 10 µg/ml RNase (Sigma-Aldrich) for 30 min at 37°C. Cells were analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter, Brea, CA, USA) and the DNA content was determined using EXPO32 software (Beckman Coulter).

All results were confirmed in at least three independent experiments and were expressed as the mean ± standard deviation. Multiple comparisons were performed using one-way analysis of variance. All statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference between values.

Since the present study hypothesized that PCSK6 may be involved in the progression of RA, RNA interference was used to knockdown PCSK6 expression in order to assess the resulting effects on FLS from rats with CIA. PCSK6 expression was determined by RT-qPCR 24 h after transfection with either control siRNA or PCSK6 siRNA. The mRNA expression of PCSK6 was significantly decreased following transfection with PCSK6 siRNA (P=0.0001) (Fig. 1A), demonstrating effective knockdown. To evaluate the roles of PCSK6 in the proliferation, migration and invasion of FLS in RA, MTT, wound healing and Transwell assays were performed after PCSK6 knockdown. Transfection with siRNA-PCSK6 reduced the proliferation of FLS from rats with CIA compared to that of the control- or Mock-transfected cells with normal PCSK6 expression (P=0.0001) (Fig. 1B). In the cell migration assay, monolayers of FLS were scratched and subsequently incubated with siRNA-PCSK6, control siRNA or transfection reagent only for 24 h. Significantly fewer FLS were present in the wounded area when PCSK6 was knocked down (P=0.0001) (Fig. 1C). Furthermore, the invasion assay showed that significantly fewer FLS transgressed through the Transwell filter when PCSK6 was knocked down (P=0.0001) (Fig. 1D).

As RA is a chronic inflammatory condition, pro-inflammatory cytokines have prominent roles in the disease. In particular, TNF-α, IL-1β and IL-17 are important pro-inflammatory cytokines associated with synovitis and joint destruction (5). TNF-α, IL-1 α, IL-1β and IL-17 levels were compared in the supernatant of FLS following treatment with 160 nmol/l PCSK6 siRNA or control siRNA. The secretion of IL-6, TNF-α and IL-1β by siRNA-PCSK6-transfected FLS was significantly lower as compared with that in the controls (P=0.0001) (Fig. 1E).

To further study the effects of PCSK6 on the proliferation of FLS from rats with CIA, flow cytometric cell cycle analysis was performed. The ratio of G₀/G₁-phase cells was significantly higher in FLS with PCSK6 knockdown as compared with that in the controls after 12, 48 and 72 h (Fig. 2A), which suggested that downregulation of PCSK6 inhibited the cell cycle in FLS of mice with CIA.

To gain further insight into the role of PCSK6 in the pathology of RA, the expression levels of genes associated with proliferation, invasion, migration and inflammation were detected by RT-qPCR. A total of eight genes were found to be down-regulated after PCSK6 knockdown (Fig. 3). Among these genes, IGF-2 was associated with cell proliferation, while CXCL9 was associated with inflammation. Other downregulated genes including, NOSTRIN, MMP2 and MMP9, were associated with angiogenesis, while MPZL2 is involved in cell adhesion. Of note, HIF-1α and PADI4, which are closely associated with hypoxia, the main contributor to RA and CIA (14,15), were also downregulated after PCSK6 knockdown.

The present study also examined the expression of Furin, which has a protective role in immune response-induced arthritis (8), following PCSK6 knockdown. However, silencing of PCSK6 had no significant effect on the expression of Furin.