Patients and clinical samples

A total of 45 patients with SLE and 44 age- and sex-matched healthy controls (HCs) were recruited from Southwest Hospital, the First Affiliated Hospital of Third Military Medical University (Chongqing, China), between October 2019 and September 2020. The healthy donors had no history of autoimmune diseases or immunosuppressive therapy. Patients with concurrent infections were excluded from the study. All the participants fulfilled the American College of Rheumatology (ACR) classification criteria for SLE. Detailed information on the characteristics of all participants is presented in Table 1. The study was approved by the Research Ethics Committee of Southwest Hospital, First Affiliated Hospital of the Third Military Medical University (approval no. KY2019119). Informed consent was obtained from all the participants.

Table 1 Main clinical and laboratory parameters of objectionsIsolation of PBMCs

PBMCs were isolated from the SLE patients and healthy controls according to the human lymphocyte isolation solution protocol (TBD, Tianjin, China) by gradient centrifugation at 1800 × g for 30 min at room temperature. Cells in the interphase were collected and washed twice with Hank’s balanced salt solution (HBSS; HyClone, Logan, UT, USA). PBMCs in 1 mL TRIzol (Invitrogen, Waltham, MA, USA) and serum samples were stored at − 80 °C for subsequent experiments.

Cell culture and transfection

Jurkat T cells were cultured in RPMI 1640 (Gibco, Waltham, MA, USA), and HEK-293 T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco) and maintained at 37 °C and 5% CO2. The miRNA mimics, inhibitors, and controls were purchased from Ruibo Biosciences (Guangzhou, China). pcDNA3.1 lentivirus products LV-circRNA, sh-circRNA, and the corresponding negative controls were constructed in Jurkat T cells. Stably transfected cells were verified by quantitative real-time PCR (qRT‒PCR).

Cell counting kit-8

Cell proliferation was measured using a standard cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, 6 × 103 cells were plated in 96-well plates and cultured for 0, 24, 48, and 72 h in an incubator. A total of 10 μL CCK-8 solution was added to each well and incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using a standard microplate reader.

Apoptosis assays

Cell apoptosis was determined using the Annexin V Apoptosis Detection Kit PE (Thermo Scientific, Waltham, USA) according to the manufacturer’s instructions. Briefly, 6 × 105 cells were harvested and resuspended in 500 μL of 1 × binding buffer. After adding 5 μL Annexin V-PE and 10 μL 7-aminoactinomycin D (7-AAD) to each tube, the cells were incubated in the dark at room temperature for 10 min. Then, the samples were analyzed using flow cytometry (BD Biosciences, San Jose, CA, USA).

Cell cycle analysis

Cell cycle progression was determined using a cell cycle detection kit (MULTI SCIENCES, Hangzhou, China). Briefly, 5 × 105 cells were harvested, washed twice with phosphate-buffered saline (PBS), collected by centrifugation at 300 × g for 5 min at room temperature, and treated with 10 μL permeabilization solution and 1 mL DNA staining solution. Finally, the cells were incubated for 30 min in the dark at room temperature and analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA).

CeRNA analysis and target prediction

We constructed a circPTPN22-miRNA‒target gene network using Cytoscape software (https://cytoscape.org) to visualize interactions based on high-throughput RNA sequencing data. In the network, the circRNA-miRNA interaction was analyzed using the databases of RNAhybrid (bibiserv.techfak.uni‑bielefeld.de/rnahybrid) and miRanda (http://www.microrna.org/microrna/home.do). MiRNA target genes were predicted using the TargetScan (http://www.targetscan.org/vert_71/), miRDIP (http://ophid.utoronto.ca/mirDIP/), miRDB (http://mirdb.org) and miRWalk (http://mirwalk.umm.uni-heidelberg.de) databases. A protein‒protein interaction (PPI) network was constructed using the STRING database (https://string-db.org/).

RNA extraction and quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. Subsequently, RNA concentration and purity were measured using a NanoDrop spectrophotometer (Invitrogen, Waltham, MA, USA) and 1% agarose gel. Total RNA (1000 ng) was reverse-transcribed into complementary DNA (cDNA) using a PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Beijing, China). Quantitative real-time PCR was performed using cDNA and SYBR Premix Ex Taq II (Takara Bio, Beijing, China) in a CFX96 Thermal Cycler (Bio-Rad, Shanghai, China) following the manufacturer’s instructions. The relative expression levels were calculated using the 2−△△Ct method. The primers used are listed in Table 2, and the miRNA reverse primers used were universal primers from the Mir-X miRNA qRT‒PCR TB Green Kit (Takara Bio, Beijing, China).

Table 2 Primer sequences for RT‒PCREnzyme-linked immunosorbent assay (ELISA)

Jurkat T cells were stimulated with IL-2 (50 ng/mL) for 30 min, and then human TNF-α and IL-6 in the Jurkat T-cell supernatant and serum in patients and healthy controls were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (Elabscience, Wuhan, China).

RNA-binding protein immunoprecipitation (RIP) assay

The RIP assay was performed using an RNA Immunoprecipitation Kit (BersinBio, Guangzhou, China). The cell lysate was incubated with RIP buffer containing magnetic beads conjugated with Argonaute-2 (AGO2) or IgG control antibody (Abcam, Waltham, MA, USA). The expression of circPTPN22 in immunoprecipitates was determined by real-time PCR.

Dual-luciferase activity assay

The wild-type or mutated binding sites of miR-4689 in the 3'-untranslated region (3'-UTR) of S1PR1 mRNA and full-length circPTPN22 were cloned into a luciferase reporter vector (psiCHECK2). HEK-293 T cells were seeded in 96-well plates at a density of 5 × 103 cells/well and incubated overnight. Then, the recombinant constructs were transfected into HEK-293 T cells together with miR-4689 or NC mimic using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA). Firefly and Renilla luciferase activities were measured 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer’s instructions using Promega GloMax 96 (Promega, Madison, WI, USA).

Fluorescence in situ hybridization (FISH)

The expression of circPTPN22 and miR-4689 was detected using a FISH kit (RuiBoBio, Guangzhou, China) according to the manufacturer's instructions using a specific Cy3-labelled mix and Fam-labelled DNA oligo probes, respectively. Briefly, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.25% Triton X-100 for 15 min at 4 °C. In situ hybridization was performed overnight at 37 °C using 20 μM circPTPN22 or miR-4689 probes in hybridization buffer. Then, the cells were washed continuously with 4 × , 2 × , and 1 × saline-sodium citrate buffer. Finally, nuclei were stained with DAPI, and images of the cells were captured using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany).

Western blot analysis

Cells were treated with cell lysis buffer containing 1 µL of protease inhibitor and 2 µL of phosphate inhibitor. After denaturation, 30 µg of protein in the loading buffer was subjected to SDS‒PAGE for isolation and then transferred to PVDF membranes. Membranes were blocked with 5% bovine serum albumin for 1 h at room temperature. Primary antibodies against S1PR1 (rabbit, 1:1000) and GAPDH (rabbit, 1:1000) from Thermo Fisher Scientific (Invitrogen, Waltham, MA, USA) were added and incubated with the PVDF membranes at 4 °C overnight and specific secondary antibodies for 1 h at room temperature. The protein band intensities were detected using an enhanced chemiluminescence kit (Thermo Scientific, Waltham, USA).

Statistical analysis

One-way ANOVA or Student’s t test was used to analyze the data. SPSS 26.0 and GraphPad software (version 8.0) were used for the statistical analysis. Statistical significance was set at P < 0.05.