Patients and samples
Samples were procured from patients with RA and controls at the First Affiliated Hospital of Wenzhou Medical University. RA diagnosis was based on the 2010 American College of Rheumatology (ACR) criteria. The clinical information of patients was presented in Table S1. Synovium selection, rheumatoid arthritis activity, and severity assessment were accomplished prior to any process. The control group obtained from patients with osteoarthritis was matched for age and sex.
Cells
RAW264.7 cells, HUVEC, HEK293, and HEK293T cells were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences in Shanghai, China. The cells were cultured in DMEM (ThermoFisher Scientific, USA) supplemented with 10% Fetal bovine serum (FBS; Sigma, USA), 100 ug/mL penicillin, and 100 ug/mL streptomycin (Mediatech, USA) and incubated at 37 ℃ with 5% CO2. RANKL (100 ng/mL) was used to induce RAW264.7 cell differentiation.
Screening of human ScFv phage antibody—THY-1 Ab
Human ScFv phage antibody library was purchased from Abiocenter (Beijing, China). The THY-1 extracellular domains protein were synthesized and purified by Sino Biological (Beijing, China). The purified THY-1 extracellular domains protein and PBS were successively coated on the microtitration plate overnight at 4 °C, and then a coating buffer was added. The plate was sealed with 5% skim milk (0.4 mL per tube) at room temperature for 1 h. Phage library (5×1011 pfu/tube) was added and incubated for 2 h at 37 °C. Then, 0.1% TBST was used to wash wells three times, and bound phage was eluted with glycine-HCl (0.1 mL/tube) for 8 min and neutralized with Tris-HCl until its pH 7 was achieved. To obtain phages with high affinity, we performed a more stringent panning process. Specific selection conditions are shown in Table 1. Phage THY-1 Ab DNA sequencing was performed by BGI Genomics (Shenzhen, China).
Table 1: The specific selection condition
Rounds
|
Coated antigen (μg/mL)
|
Washing buffer
|
Washing times
|
Elution
|
1
|
THY-1 protein (10)
|
0.1%PBST
|
3
|
Glycine-HCl
|
2
|
THY-1 protein (10)
|
0.1%PBST
|
6
|
Glycine-HCl
|
3
|
THY-1 protein (10)
|
0.1%PBST
|
8
|
Glycine-HCl
|
4
|
THY-1 protein (10)
|
0.1%PBST
|
8
|
Glycine-HCl
|
Protein expression and purification
The THY-1 Ab plasmid was mixed with transfection reagent TF2 and added to HEK293 cells. M293TI-1 serum-free medium was added on days 1, 3, and 5 after transfection. Transfection reagent TF2 and M293TI-1 serum-free medium were purchased from Abiocenter (Beijing, China). The collected serum-free media were added to an affinity chromatography column (GE Healthcare, the USA). Purified THY-1 Ab was obtained and analyzed through mass spectrometry. Thermo Velos Pro quadrupole ion trap mass spectrometer was used.
Surface plasmon resonance analysis
The kinetic parameters of the interaction between THY-1 Ab and THY-1 were determined using OpensprTM (Nicoyalife,Canada). Briefly, THY-1 Ab was immobilized on a CM5 dextran sensor chip in 10 mM sodium acetate buffer (pH 4.5). An amine coupling kit (Sigma, the USA) was used, and the flow rate was 20 µL/min. THY-1 Ab in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20) was then injected for over 2 min at a flow rate of 20 µL/min. Data were evaluated using TraceDrawer (Ridgeview Instruments ab, Sweden), and the association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD, kd/ka) were calculated and used in determining binding affinity.
Design and construction of the plasmid vector
The THY-1 Ab vector was designed according to the sequencing results of phage THY-1 Ab. Gene sequence information is presented in Table S2. The mutated THY-1 Ab (THY-1 Ab MUT) sequences were designed according to the binding sites of THY-1 Ab and THY-1 extracellular domains mimicked by molecular docking (GLY-199, PRO-182, GLN-39, TYR-95, GLN-107, GLU-6, and THR-122). Gene sequence information is presented in Table S2. The plasmid vector used in constructing THY-1 extracellular domains, THY-1 Ab, and mutated THY-1 Ab plasmids was pcDNA3.1 (plasmid map and sequence information are provided in Fig.S1 and Table S2, respectively), which was made by Tsingke (Beijing, China).
Molecular docking
The amino acid sequences of THY-1 Ab and THY-1 extracellular domains were uploaded to the Robetta Server (https://robetta.bakerlab.org/) for homologous modeling. Five models were established for each sequence. The protein models were evaluated using SAVES v6.0 Server. The Model1 of the THY-1 Ab amino acid sequence and Model3 of the THY-1 extracellular domain amino acid sequence satisfied the requirements of the server and were thus selected as the receptor and ligand protein, respectively. The modeled protein was uploaded to the ZDOCK server, and were used them in protein docking without specifying the docking modes of the amino acid residues. The combination mode with the highest score was selected for analysis from the top 10.
Immunofluorescence co-location detection
Sterile cell slide was placed at the bottom of the orifice plate, and RA FLS cells were implanted into 12-well plates at 2×106/well and treated with THY-1 Ab. After membrane breaking treatment and sealing, THY-1 rabbit antibody (D3V8A, CST, and USA) and THY-1 Ab-customized mouse antibody (Sino Biological, Beijing, China) were incubated with RA FLS at 4 °C overnight. The cells incubated with secondary antibody (CoraLite488-conjugated Affinipure Goat Anti-Mouse (H+L), CoraLite594-conjugated Goat Anti-Rabbit (H+L), Proteintech,China) for 1 h. After nonspecific stain was removed, the cells were incubated with DAPI for 10 min. The cell images were obtained using a confocal microscope at 400× magnification.
ELISA
RA FLS cells were plated into 6-well plates with 5×105 cells per well and cultured in incubators overnight. The αvβ3 purchased from Sino biological (Beijing, China), pcDNA3.1 THY-1 shRNA was designed and synthesized by ribobio (Guangzhou, China). Sequence details were shown in Table S2. After three kinds of THY-1 shRNA knocked down THY-1, we chose shTHY-1#1 to use it as a follow-up knock down THY-1. THY-1 overexpression vector is the THY-1 extracellular domain vector described previously. The corresponding substances were added, cultured for 48h, and the supernatant was collected. The serum samples were centrifuged at 3000 rpm for 5 min and diluted 20 times. The specific operation shall be carried out according to the ELISA Kit (biolegend, the United States) instructions. Read at 450nm and measure the absorbance value of each group.
HUVEC tube-formation assay
HUVEC tube formation assay was performed for the evaluation of angiogenic activity in vitro. Matrigel (Bjunique, Beijing, China) was diluted with DMEM in a ratio of 1:1, and then 96-well plates were coated with Matrigel and incubated at 37 °C for about 1 h to promote gelling. As previously mentioned, freeze-dried powder made from the supernate of FLS treated with THY-1 Ab were added after dissolution to each well containing human umbilical vein endothelial cells (HUVEC). After 4, 8, and 16 h of incubation, the results of the tube formation assay were examined using the number of branch points in each field in each micrograph. Finally, the results were sorted out and analysed by ImageJ software,and then the mean ± standard error (SE) was calculated for each experimental condition. Two-tailed t-test was used in evaluating statistical significance.
Chick embryo chorioallantoic membrane assay
A total of 30 fertilized eggs, aged 3–5 days, were incubated for 7 days at 37 °C. Grouping is the same as previously. On the seventh day, eggs were removed from the incubator and opened, treated with moderate saline solution, and the dissolved freeze-dried, which were forced in sterile Whatman GB/B glass fiber filter disks (6 mm in diameter; Reeve-Angel, Clifton, NJ, USA). The eggs were then sealed with tape and incubated for 72 h. The growth condition of the CAM within the Whatman GB/B glass fiber filter disks was observed and recorded twice under a stereomicroscope at 48h and 72h respectively. The vascular and CAM areas were assessed with ImageJ 2.43, and the percentage of angiogenic area was calculated using the following equation: Vascular area / CAM area × 100%.
Osteoclast induction and TRAP staining
RAW264.7 cells were inoculated to 24-well plate at a density of 1.0 × 105 cells/mL. Conditioned medium containing 100 ng/mL soluble RANKL was then added to RAW264.7 cells as positive controls and PBS in equivalent volume was added as a negative control. The treatment of the experimental group is as described previously. On the seventh day of cultivation, after cell fixation, TRAP staining kit (Solarbio Science & Technology) was used in observing and counting osteoclast-like cells. More than three nuclei in each cell were positively stained. Each experiment was performed in duplicate. The counting step was repeated three times.
Bone resorption assay
The bone resorption assay was performed using bone slices (BioVendor). RAW264.7 cells were inoculated into the bone slices in each well at a density of 2.0 × 104 cells/cm2 at 37 °C in a humidified 5% CO2 atmosphere. The treatment is the same as the above osteoclast induction and TRAP staining assay. Fixed with 4% paraformaldehyde for 30 min after trypsin digestion, and dehydrated with gradient alcohol (85%, 95%, and 100%). Ten areas were selected for each bovine bone slice without showing overlaps between areas. The pit area was evaluated by using an Eclipse 80i microscope, and images were analyzed using Image-pro Plus6.0.
CIA model
DBA/1 mice (male, 6–8 weeks old, 18–20g) were acquired from SLAC Laboratory Animal Co. (Shanghai, China). All mice were maintained in specific pathogen–free conditions on a 12-hour reversed light/dark cycle. Male DBA/1 mice were immunized with collagen and then treated with THY-1 Ab (300 µg/kg) or PBS by intraperitoneal injection every 2 days for 48 days. From day 21 to day 48, the mean clinical score of each mouse was assessed according to a standardized method. Mean clinical scores (0-4) were assigned as in Table 2. Joint scores and the number of diseased limbs were calculated three times a week. Mice were sacrificed on day 49. Serum and joint tissues were harvested for further studies.All experimental procedures were approved by the institutional animal care and use committee of Wenzhou Medical University.
Table 2: Clinical score of arthritis degree
Score
|
Incidence
|
0
|
No symptoms
|
1
|
Erythema and slight swelling limited to the ankle joint and toes
|
2
|
Erythema and slight swelling spreading from the ankle to the midfoot
|
3
|
Erythema and severe swelling spreading from the ankle to the metatarsal joints
|
4
|
Ankylosing deformity with joint swelling
|
Histopathology evaluation
Paraffin section of knee joints were deparaffinized, rehydrated, and stained with H&E, Safranin O/Fast Green, and TRAP. The semiquantitative scores of inflammatory cell infiltration, synovial hyperplasia, and bone destruction were performed after H&E staining. Histological score was evaluated on a scale of 0 (normal) to 3 (severe).
ChIP assay
After RA FLS cell samples were collected, the nuclei were prepared using a ChIP kit (MERK, USA). The specific procedure was carried out according to the instructions. Ultrasound was performed on the prepared sample for 20 min (Power 250W, 1S on and 1S off). ChIP antibody was added to the IP group, and IgG antibody was added to the IgG group. ProteinA/G-beads were used to combine with antibodies. After heating in a water bath at 65 °C for 15 min, the beads were harvested with a magnetic grate. DNA was de-cross-linked and precipitated as instructed. The harvested DNA was verified through qPCR.
Dual-luciferase reporter assay
PmirGLO, dual-luciferase plasmids of hsa_circ_0094342, mutated hsa_circ_0094342, SPI, and mutated SPI were constructed by Tsingke Biotechnology Co., Ltd. Specific sequences were provided in supplemental materials. Targeting at hsa_circ_0094342, miRNA-155-5P, and SPI1, transfection assay was carried out. After the transfection of 293T cells for 48 h, protein lysis was conducted, and the supernatant was collected. A dual-luciferase reporter gene detection kit was purchased from Beyotime Biotechnology. After the addition of the firefly luciferase detection reagent as instructed, the microplate reader was used to detect luminescence wavelength of 560 nm. Luminescence wavelength of 465 nm was detected when renilla luciferase was used as the internal reference. The RLU value determined by firefly luciferase was divided by the RLU value determined using sea kidney luciferase. Therefore, the activation levels of reporter genes were compared. Experiments were conducted independently in triplicate.
Statistical analysis
The significant differences between mean values obtained from at least 3 independent experiments. Each value is represented as a dot, with a minimum of 3 replicates. Student’s t-test was used for comparisons, with P < 0.05 considered significant.