Patients’ and Samples
We enrolled 12 age and gender matched medical staff as healthy controls. Their blood samples as well as 61 RA and 20 OA patients’ samples were used for measuring HAPLN1 levels by ELISA. Synovium samples were collected by arthroscopic surgery done with 20 RA and 17 OA patients and used for IHC staining of HAPLN1. The inclusion and exclusion criteria and general information such as age, gender, disease activity and disease course were reported earlier21 and summarized in supplementary table 1 and 2.
Enzyme linked immunosorbent assay (ELISA)
Blood samples of HC, OA and RA patients were centrifuged after standing at room temperature for 2 h at 1,500 g for 10 min to collect the plasma and HAPLN1 levels were detected by ELISA (RayBiotech, USA). Plasma AMPK levels in RA patients were also evaluated by ELISA according to the manufacturer’s protocol (Jianglaibio, China). The SuPerMax 3000FA absorbance microplate reader (Flash Co. Ltd., China) was used to read the optical density (OD) value at 450 nm and concentrations of specific proteins were calculated based on the standard curve.
Immunohistochemical (IHC) staining for HAPLN1
Synovium samples collected from 20 RA and 17 OA patients for IHC staining of HAPLN1 were prepared as reported earlier17. Rabbit monoclonal anti-HAPLN1 antibody (Abcam, USA) was added as primary antibody (1:50) and incubated for 2 h at 37℃. Biotin-conjugated goat anti-rabbit antibodies (ZSGB-Bio, China), streptavidin-peroxidase conjugate, and diaminobenzidine were used as the detecting system. IHC stained sections were semi-quantified under a microscope. The staining intensity was counted as none (0 points), weakly positive (1+), moderately positive (2+) and strong positive (3+), and the percentage of positive cells was obtained to calculate the H-Score, using the formula H-Score = (% at 0) × 0 + (% at 1+) × 1+ (% at 2+) × 2+ (% at 3+) × 3. The range of H score for each slice was between 0-30022.
Isolation and culture of RA-FLSs
Primary RA-FLSs were acquired from 3 untreated RA patients having appropriate diagnostic criteria. Isolation and culture of RA-FLSs were reported as before17. Briefly, FLSs were isolated by enzyme digestion and subsequently cultured in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS, Invitrogen) and antibiotics (penicillin and streptomycin) at 37°C with 5% CO2. Cells cultured between passages 4 and 9 were used in this study.
Small Interfering RNA (siRNA) HAPLN1 preparation and transfection
RA-FLSs at 60 - 70% confluency were transfected with siRNAs (Ribobio Company, China) at 50 nM with Lipofectamine™ 3000 reagent (Invitrogen, USA). The following siRNA sequences were used: control siRNA (confidential sequence information) and 3 siRNAs of HAPLN1, si-1 (5’-AGGGTAGAGTGTTTCTGAA-3’), si-2 (5’-CCTGGAAAATTCTCGGATA) and si-3 (5’-ACCTCACTCTGGAAGATTA-3’). All the three siRNAs effectively silenced HAPLN1 expression (supplementary figure 2A), and si-1 was selected randomly and used in subsequent studies.
HAPLN1 over-expression vector preparation and transfection
For HAPLN1OE RA-FLSs experiments, HAPLN1 over-expression plasmid and its control were constructed and packaged by Ubigene Biosciences (Guangzhou, China). The stbl3 strain plasmid cytomegalovirus vector infected cells were cultured in LB medium (QDRS biotec, China) with 100 µg/ml of ampicillin under 37℃, 225 rpm for 24 h. The HAPLN1OE plasmid vector and its control were then isolated with Genopure Plsmid Maxi Kit (Roche, USA). RA-FLS at 60 - 70% confluency were transfected with HAPLN1OE vector or its negative control with Lipofectamine™ 3000 reagent (Invitrogen). The effects of HAPLN1OE plasmid vector were shown in the supplementary figure 2B.
MTT assay
MTT assay was used to ascertain FLSs viability transfected with si-HAPLN1, HAPLN1OE or their corespondent controls, or treated with rHAPLN1 (Recombinant human HAPLN1 protein, Abcam, USA) at different concentrations (0, 25 and 50 ng/ml). FLSs samples (si-HAPLN1 vs its negative control, HAPLN1OE vs its negative control, or treated with different concentrations of rHAPLN1) digested using 0.25% pancreatin were transferred to 96-well plates with 3-5 × 103 cells/well. At different time points (24, 48 and 72 h), proliferation viability was measured using MTT assay kit (Abcam).
CCK-8 assay
Cell viability after transfection with si-HAPLN1, HAPLN1OEor their respective controls, or treated with different concentrations of rHAPLN1 was also determined using Cell Counting Kit-8 (CCK-8, Molecular Technology, Japan) assay.
TUNEL assay
FLSs transfected with si-HAPLN1, HAPLN1OE or their corespondent controls, or treated with rHAPLN1 (0 or 50 ng/ml) were digested and transferred to 6-well plates with 2-3 × 105 cells/well, cultured for 48 h, and stained by One Step TUNEL Apoptosis Assay Kit (Beyotime, China). Apoptosis rate was calculated under fluorescence microscope (Leica, Germany) with the excitation wavelength at 550 nm (Cy3), and the emission wavelength at 570 nm (red fluorescence).
Flow cytometry for FLSs apoptosis
FLSs apoptosis treated with rHAPLN1 was measured using Annexin V-FITC/PI Cell Apoptosis Detection Kit (Vazyme, China) by flow cytometry. After FLSs were treated with rHAPLN1 (0 or 50 ng/ml) for 48 h in 6-well plates, the cells were collected (3×105/well), washed twice with PBS, re-suspended in 500 µl 1 X binding buffer, mixed with Annexin-V-fluorescein isothiocyanate (FITC, 5 µl) and propidium iodide (PI, 5 µl), and analyzed using a flow cytometer (BD FACSCantoTM Ⅱ, USA). The scatter diagram was distributed as follows: Q3, healthy cells (FITC-/PI-); Q2, apoptotic cells at an advanced stage (FITC+/PI+); Q4, apoptotic cells at an early stage (FITC+/PI-). Apoptosis rate was calculated as ratio of apoptotic cells in P2 (Q4 + Q2).
Wound healing assay
Wound healing assay was done to evaluate the migration viability of FLSs transfected either with si-HAPLN1, HAPLN1OE or their corespondent controls, or treated with different concentrations (0, 25 and 50 ng/ml) of rHAPLN1. FLSs samples (si-HAPLN1 vs its negative control, HAPLN1OE vs its negative control, or treated with different concentrations of rHAPLN1) were transferred to 6-well plates with 3 × 105 cells/well and cultured with serum free - RPMI 1640 medium. At different time points, migration viability was measured by wound healing assay as previously reported17.
Transwell assay
Transwell assay was also performed to evaluate the migration capacity of FLSs transfected with si-HAPLN1, HAPLN1OE or their respective controls, or treated with rHAPLN1. FLSs in each set of experiment were re-suspended after culturing for 24 h. Transwell assay procedure was described earlier17.
Quantitative real-time polymerase chain reaction (qPCR)
Total RNA from FLSs transfected with si-HAPLN1, HAPLN1OE or their respective controls, or treated with rHAPLN1 was prepared using TRIzol® Reagent (Thermo Scientific, USA) and quantified using Qubit (Thermofisher, USA). RNA was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Takara, Japan). The reaction mixture contained 5 µl of 2 x TB Green Premix Ex Taq II (Takara, Japan), 3 µl of nuclease-free water, 1 µl of cDNA, 0.4 µl of each gene-specific primer and 0.2 µl of ROX reference dye. The qRT-PCR analysis was performed using Applied Biosystems ViiA™ 7 Real-Time PCR System (Thermofisher, USA). Each value represents an average from three independent biological replicates. GAPDH gene expression was used for data standardization. The fold change was calculated using 2−ΔΔCt method. Primers of GAPDH, AMPK-ɑ, TNF-ɑ, IL-6, TGF-β, ACAN, fibronectin, collagen II, MMP1, MMP3, MMP9, cyclin-D1 and Ki-67 are given in the supplementary Table 3.
Automated western blot analysis
Total proteins from FLSs transfected with si-HAPLN1, HAPLN1OE or their respective controls for 48 h were extracted with Cell Lysis Buffer (Cell Signaling, USA) and their concentration was measured using a BCA Protein Assay Kit (Merck, USA). Relative changes in HAPLN1, pAMPK-α, IL-6, TNF-α, MMP1, MMP3 and MMP9 protein levels were determined. Expression of β-tublin was selected as internal reference. Capillary electrophoresis and western blot analysis were carried out using reagents provided in the kit and following instructions in the user manual (ProteinSimple WES, USA) as previously reported17. Rabbit anti-HAPLN1 antibody (Abcam, USA), Rabbit anti-TNF-α, pAMPK-α, MMP-1, MMP-3, IL-6, and β-tublin specific mAbs (Cell Signaling, USA) were used (1:100). Goat anti-rabbit secondary antibodies were provided in the ProteinSimple WES kit and applied as instructed. Data were analyzed using an in-built Compass software SW 4.0. The truncated and full-length target protein intensities (area under the curve) were normalized to that of tubulin peak (control). In most of the figures, electropherograms are represented as pseudo-blots, generated using Compass software.
Proteomics study
Label-free proteomics study was applied to FLSs transfected by si-HAPLN1, or treated by rHAPLN1 (50 ng/ml) and their controls for 48 hours (for management of each group is seen in table 4) by PTMBiolabs, Inc. (Hangzhou, China). Each concentration with 3 biological replicates. Cell samples processed as reported23. LC−MS/MS proteomics analysis was performed on an EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system, followed by MS/MS using Q Exactive Plus (ThermoFisher Scientific, USA) coupled online to the UPLC system. The MS/MS data were retrieved by the Maxquant search engine (v1.6.6.0). A human database was searched (Swiss-Prot). The decoy database antilibrary was used to the reduce false positive rate (FDR). The FDR was adjusted to < 1%, and the minimum score for modified peptides was set > 40. Proteins with a fold-change ≥1.50 or ≤0.67 between si-HAPLN1, rHAPLN1 and their controls were considered as expression significant. Based on the protein sequence alignment method, the protein domain functions were defined by InterProScan (http://www.ebi.ac.uk/interpro/). Functional annotation enrichment of DEGs were performed by Gene Ontology (GO) annotation analysis and KEGG analysis. The enrichment significant was identified as p < 0.05 in Fisher’s exact test and q < 0.05 in Benjamini-Hochberg’s procedure.
High-throughput mRNA sequencing
High-throughput RNA sequencing was performed using FLSs after treatment with rHAPLN1 (0 and 50 ng/ml) for 48 h. Each concentration was tested thrice. RNA-seq and high throughput sequencing were conducted by Seqhealth Technology Co., Ltd (Wuhan, China). Total RNA (2 µg) was used for stranded RNA sequencing library preparation using KCTM Stranded mRNA Library Prep Kit for Illumina® (Seqhealth Co., Ltd. China). PCR products corresponding to 200-500 bps were enriched, quantified and sequenced with Novaseq 6000 sequencer (Illumina), PE150 model. Raw sequencing data was first filtered by Trimmomatic (v. 0.36), and low-quality reads were discarded. The reads contaminated with adaptor sequences were trimmed. Clean data were mapped to the human reference genome from UCSC (https://genome.ucsc.edu/) using STRA software (v. 2.5.3a) with default parameters. Reads mapped to the exon regions of each gene were counted by feature Counts (Subread-1.5.1; Bioconductor) and then RPKMs were calculated. DEGs between groups were identified using the edgeR package (v. 3.12.1) in R studio software (version 3.6). A p-value cutoff of 0.05 and fold-change cutoff of 2.0 were used to judge the statistical significance of gene expression differences. The volcano plot was drawn with the ggplot2 package in R studio. Heatmaps of pathway enrichment analysis of DEGs were generated using Metascape (http://metascape.org)24 and p value less than 0.05 was considered to be statistically significant. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis for DEGs were done uisng KOBAS software (v. 2.1.1) with a p value cutoff of 0.05. To compare transcriptome characteristics of rHAPLN1 and PBS groups, GSEA software (version 4.0.0) was used. Annotated pathway files (c5.go.bp.v7.4.symbols.gmt) were downloaded in the MSigDB database (http://www.gsea-msigdb.org/gsea/msigdb/collections.jsp). Pathways with P value less than 0.05 and false discovery rate (FDR) less than 0.2 were considered to be significantly enriched.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.0 software. All the data were given as mean ± SD. Differences between two groups were evaluated for statistical significance using Student’s t-test. One-way ANOVA with Tukey’s multiple comparisons test was used to evaluate the differences between three or more groups. Pearson correlation coefficient was calculated using “cor” function in R studio. Correlations were evaluated using liner regression and correlation tests. p < 0.05 was considered as statistically significant.