Patients and synovial tissue samples
All the synovial tissue specimens for Tandem Mass Tag (TMT) relative quantitative proteomics and further experiments were obtained from the patients with RA or OA undergoing surgical joint replacement at the clinical of joint surgery (Xi'an Hong Hui Hospital, Xi'an Jiaotong University, China). The diagnosis of the patients were accorded to the criteria of the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) in 2010. This study was approved by the Ethics Committee in Hong Hui Hospital, and the Medical Ethics Committee of Xi’an Jiaotong University. The detailed patient clinical tests were presented in Table 1.
Protein extraction, digestion and TMT labeling
The synovial tissues were extracted with SDT buffer (4 %(w/v) SDS, 100 mM Tris-HCl, pH 7.6, 0.1 M DTT). And then the proteins were digested with FASP after BCA quantification. Peptides were quantified with Nanodrop 2000 via OD280. A total of 100 μg of peptides from each sample was labeled with a TMT kit following the kit instructions (ThermoFisher,USA). Briefly, acetonitrile and 0.1% TFA were used to balance the reversed-phase chromatography column, and then the equally mixed peptide fractionation was loaded onto the column with ddH2O. After adding ddH2O, the salt was desalted by low-speed centrifugation. Finally, the column-bound peptides were eluted with a high-pH acetonitrile solution with sequentially increasing concentrations. Each eluted peptide was completely dried and resuspended in 12 μL of 0.1% TFA.
LC-MS/MS analysis
Peptides were dissolved in 0.1% TFA and were directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific) at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). The peptides were subjected to HCD followed by tandem mass spectrometry (MS/MS) in Q Exactive (ThermoFisher,USA). Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using 28% NCE, ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 1.5E4 in the MS survey scan with 60.0 ms dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were used to generate the MS/MS spectra. For MS scans, the m/z scan range was 300 to 1800 Da.
Peptides data processing
The MS data were processed using Mascot and Proteome Discovery (version 1.4) search engines. Tandem mass spectra were searched against the UniProt_human database (161584 sequences) concatenated with the reverse Decoy Database. Trypsin/P was specified as a cleavage enzyme allowing up to 2 missed cleavages. Mass error was set to 20 ppm for precursor ions and 0.1 Da for fragment ions. Carbamidomethylation on cysteine was specified as a fixed modification, and oxidation and TMT were specified as variable modifications. A 1.0% false discovery rate (FDR) was used to filter the identified peptides. All the other parameters in Mascot were set to their default values.
GO analysis and KEGG pathway analysis
The online analysis database, Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david.ncifcrf.gov/, Version 6.8), to analyze the Gene Ontology (GO) annotation and the enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for these data [18,19]. The p value < 0.05 and gene counts >2 were criteria for the both.
Western blotting assay
The synovial tissue from OA and RA patients were described in the synovial tissue segment. Such tissue samples were extracted in RIPA buffer with protease inhibitor cocktail. After 20 min incubation in ice, all the samples were centrifuged at 12000 rpm for 30 min at 4 ℃ and then quantified with BCA kit. 40 ug protein of each samples were loaded on 8-10% SDS-PAGE gels. The antibodies used for western blotting were OLFM4 (NOVOS, NBP2-24535), MZB1 (Abcam-ab181205) and DERL3 (Abcam-ab78233), S100P (Abcam-ab225543), and LAP3(ABclonal-A7101), rabbit anti-ACTB. All the first antibodies were diluted at 1:1000, and the HRP conjugated goat anti-rabbit IgG antibody was diluted at 1:10000. Signal intensity was determined by Supersignal® West Pico Kit (Thermo Scientific). And the images for western blotting were taken by Syngene G. All the density of the bands was measured and normalized to β-actin. Final data are expressed by showing one representative image and quantitative result of samples from various patients.
Immunohistochemistry
The paraffin-embedded RA and OA synovial tissues were sectioned and mounted on positively charged slides. All the slides were incubated at 60°C for overnight, followed by de-paraffinization and rehydration. These slides were transferred to the Leica Bond-IIITM and were treated for antigen retrieval at 100°C for 20 minutes in a retrieval solution. Endogenous peroxidase was blocked using peroxidase-blocking reagent, followed by primary antibody incubation for 60 minutes. The blocker buffer were used for blocking the unspecific reaction for 1 hour at room temperature. Then the first antibody of OLFM4, MZB1 (Marginal Zone B and B1 Cell Specific Protein) and DERL3 (Derlin 3), and S100P (S100 Calcium Binding Protein P) were diluted at 1:1000 and then applied to the tissue at 4 ℃ overnight. The next morning all the slides were taken out and balanced at room temperature for 30 min, then washed with PBS for times. Then the secondary antibody were used for 30min at room temperature, followed by the horseradish peroxidase reaction with DAB reaction.
Fluorescence immunohistochemistry was performed using the primary antibodies, anti-OLFM4, and anti-Vimentin. DAPI (1:100000, Sigma-Aldrich, D9542) were used for staining the nucleic.
RT-qPCR
The total RNA were isolated from RA and OA patients’ synovial tissues, and then quantified. The cDNA were reversed by using First Strand cDNA Synthesis Kit (Roche, USA). The synovial fibroblast were isolated from RA synovial tissues. The FLS were treated with different pro-inflammatory cytokines, 10 ng/mL TNFα, 2 ng/mL IL-1β or 1 ng/mL LPS for 24 hours. All the RNA were extracted with Trizol, quantified and then the mRNA were reversed to cDNA by using First Strand cDNA Synthesis Kit (Roche, USA). The real time PCR was performed with Fast Start Universal SYBR Green Master (ROX) (Roche, USA) by Agilent Mx3000. PCR efficiency and specificity of each primer pair was examined by a standard curve of serially diluted cDNA and melting curve functionality respectively. Fold change was calculated based on 2−ΔΔ Ct method after normalization to the transcript level of housekeeping gene ACTB. Primer sequences used in the RT-PCR could be found in Table 3.
RNAi
The siRNA kit for the target protein OLFM4 were purchased from Santa Cruz. The 20uM siRNA (siNC or siOLFM4, Santa Cruz) were transfected Lipofectamine™ 2000 Transfection Reagent (Thermo Scientific, 11668019) to the FLS for 72 hours. Then the cell were collected either for RT-qPCR or for cell proliferation assay.
Cell proliferation assay
The transfected NC and siOLFM FLS cell were incubated with PE-labeled cell trace dye (diluted at 1:1000) for 72hours. After 72 hours cultured, all the cell were collected and detected by flow cytometer. The unstained NC cell were used as a negative control. The MFI were measured to determine the cell proliferation rate after knocking down the OLFM4 gene.
Statistics
Statistics were carried out using SPSS and Graphpad Prism software. The student’s t-test of the Mann-Whitney U test and unpaired student’ s t-tests were used to determine statistical differences between two groups. The Fisher’s tests were used to test for enrichment or one-way ANOVA depletion of specific annotation terms among members of the resulting protein cluster. P<0.05 was considered to indicated a statistically significant difference. Data were presented as mean ± SEM.