In silico study (bioinformatic tools)
dbSUPER (https://asntech.org/dbsuper/) is a database that describes 82,234 SEs in 102 human and 25 mouse tissue/cell types. RA-susceptibility genes were detected by GWAS, as reported previously . BioGPS (http://biogps.org/#goto=welcome) is a database that summarizes the patterns of mRNA expression specific to human and mouse tissues and cells that have been identified by oligonucleotide array analysis.
Preparation of CD4+ T cells
CD4+ T cells were prepared as described previously . First, CD4+ T cells were collected from peripheral blood mononuclear cells (PBMC) of 11 healthy donors [male to female=3:8; mean age, 42.5±7.3 (±SD) years] and of 24 RA patients (8:16; 49.3±5.1 years, respectively), with clinical disease activity index (CDAI) of 136±49, simplified disease activity index for rheumatoid arthritis (SDAI) 140±51, and disease activity score-28 (DAS28) of 6.1±1.2. The T cells were prepared using CD4+ microbeads of the CD4+ T Cell Isolation kit (#130-096-533; Miltenyi Biotec, Auburn, CA) (Supplementary Table 1). CD4+ T cells were sorted into subsets by fluorescence-activated cell sorting (FACS) gating. Cells were then seeded onto six-well plates at a density of 1 × 106 cells/well and incubated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum and 1× antibiotics at 37°C under 5% CO2 atmosphere. The positive control Jurkat cells were treated with anti-CD28 (PV1) or anti-CD3 (2C11) for 6 h to activate TCR signaling .
CD4+ T cells were prepared from PBMC of 3 healthy donors (male to female 1:2) and 3 patients with RA (male to female 1:2). We also used CD4+ T cell line Jurkat as a positive control. Immunoblotting was performed as described previously . Briefly, whole-cell extracts were prepared from lysis of PBMC in TNE buffer (50 mM Tris-hydrochloride [pH 7.4], 0.5% [v/v] Nonidet P-40, 150 mM sodium chloride, 5 mM ethylenediaminetetraacetic acid, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). Proteins (7 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Protran BA83; GE Healthcare, Chalfont St. Giles, UK) for blocking. The membranes were incubated with anti-phospho-NF-κB antibody (Ser 536, #3033; Cell Signaling Technologies, Danvers, MA), anti-NF-κB antibody (#8242; Cell Signaling Technologies), anti-UBASH3A antibody (GTX116432; GeneTex; Irvine, CA), and anti-β actin antibody (A1978; Sigma-Aldrich, St. Louis, MO). The bound antibodies were visualized with secondary antibodies against mouse and rabbit immunoglobulin G (IgG, GE Healthcare) conjugated with horseradish peroxidase and chemiluminescence reagent (ECL™ Prime western blotting Detection Reagent; GE Healthcare).
Cytometric bead array (CBA)
CBA was performed as described previously . Briefly, the supernatant from cultured CD4+ T cells of 3 RA patients (male to female 1:2) and 3 healthy donors (male to female 1:2) was collected 24 h after transfection of the cells with plasmid cloning DNA (pcDNA) 3.1-UBASH3A (courtesy of Professor Patrick Concannon) and empty vector. Next, 20 μL of the supernatant was reacted with 0.2 μL of capture beads of the Human Flex Set (#558279 for IL-1 beta, #558276 for IL-6, #560383 for IL-17A, and #558273 for TNF; BD Biosciences, San Diego, CA) and 19.8 μL of RPMI medium for 1 h at room temperature. Then, 0.2 μL of phycoerythrin detection reagent and 19.8 μL of RPMI medium were added, and the mixture was allowed to react for 1 h. The supernatant was rinsed twice with flow cytometry staining (FACS) buffer, centrifuged, and suspended in 150 μL of FACS buffer. Finally, each cytokine was detected by FACS, and its concentration was analyzed by FCAP Array software.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed as described previously . Briefly, the total RNA was extracted from cultured CD4+ T cells of 3 RA patients (male to female 1:2) and 3 healthy donors (male to female 1:2) using the RNeasy Mini kit (Qiagen, Hilden, Germany) and reverse-transcribed using SuperScript®VILO™ Master Mix (Life Technologies). On a Step One Plus system (Applied Biosystems), qPCR was performed with TaqMan®Fast Universal PCR Master Mix (Applied Biosystems) and the TaqMan® Gene Expression Assay (Applied Biosystems) primer/probe pairs to measure the expression of the following genes (Supplementary Table 2): UBASH3A, interleukin-1 beta (IL-1β), IL-6, IL-17A, tumor necrosis factor-alpha (TNF-α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
A single patient with RA and a single patient with dermatomyositis, who met the diagnostic criteria of the two conditions, consented to provide lymph node samples. This study was conducted with the approval of the ethics committee of the University of Occupational and Environmental Health. Lymph node tissue samples were collected, fixed in 10% formaldehyde, and embedded in paraffin. Immunofluorescence staining was performed as described previously . Briefly, the biopsy samples were immersed in phosphate-buffered saline with Tween (PBST; 0.05% [v/v], pH 6.0) containing sodium citrate (5 mM) to inactivate antigens. The samples were blocked with a serum-free protein block (Dako, #2016-08) and reacted with polyclonal antibodies that recognize human CD4 (dilution, 1:200; #ab67001; Abcam, Cambridge, MA) and UBASH3A (dilution, 1:200; #ab197168, Abcam). The slides were rinsed with PBST and then incubated for 1 h with an anti-rabbit IgG secondary antibody labeled with fluorescein isothiocyanate and an anti-mouse IgG secondary antibody labeled with rhodamine (dilution, 1:500; DakoCytomation, Glostrup, Denmark). Nuclei were stained with 4’,6-diamidino-2-phenylindole (dilution, 1:200; Merck). The stained samples were examined under an all-in-one fluorescence microscope. The results of the staining were quantified using ImageJ software as described previously .
Chromatin immunoprecipitation-PCR (ChIP-PCR)
ChIP-PCR was performed as described previously . CD4+ T cells collected from 5 healthy donors (male to female 1:4) and 5 RA patients (male to female 1:4) were cultured in RPMI 1640 medium. Chromatin was cross-linked with 1% formaldehyde for 10 min and fragmented to 200–500 bp by sonication. DNA was extracted using the EZ ChIP Kit (Merck Millipore, Taufkirchen, Germany) using the protocol provided by the manufacturer. DNA was immunoprecipitated using a series of antibodies. PCR was performed using specific primers (Supplementary Table 2).
Following the instructions provided by the manufacturer, we transfected pcDNA3.1-UBASH3A and empty vector into CD4+ T cells (1 × 105 cells) collected from 3 healthy donors (male to female 1:2) and 3 RA patients (male to female 1:2) using Lipofectamine 3000 and P3000 reagents (Thermo Fisher Scientific, Waltham, MA). In contrast, locked nucleic acid (LNA) was designed to knock down the expression of eRNA (Supplementary Table 2). LNA targeting eRNA and control LNA were transfected into CD4+ T cells (1 × 105 cells) with RNAi MAX (Thermo Fisher Scientific) reagents for 72 h, using the protocol provided by the manufacturer.
SNP-PCR was performed according to the instructions supplied by the manufacturer to determine the genotypes of rs1893592-A/C, as described previously . On a Step One Plus system (Applied Biosystems), PCR was performed in 10 μL of reaction volume containing 1 μL of genomic DNA (20 ng/μL) from 14 RA patients (male to female 4:10), 5 μL of TaqMan Universal PCR Master Mix, 0.25 μL of VIC/FAM-labeled probe (200 nM), and 3.75 μL of double-distilled water. Supplementary Table 2 lists the probes used in this process. The series of reactions was analyzed using the Allelic Discrimination Sequence Detection Software (Applied Biosystems).
All quantified data are expressed as mean ± standard deviation. Differences between the two groups were tested for statistical significance by the Student’s unpaired two-tailed t test or Dunnett’s multiple comparison test. Spearman’s test was used for the correlation analysis between two variables of interest. The statistical significance was set at P<0.05. All statistical analyses were performed using SPSS statistical software (v. 21.0; IBM Corp., Armonk, NY).