Study population

All blood samples in this study were obtained from participants in an observational study of circulating non-coding RNAs in acute ischemic stroke (AISRNA) (www.clinicaltrials.gov, NCT04175691), which was a multiset, hospital-based, case–control study for the detection of noncoding RNA expression in AIS. LncRNA microarray profiling data were obtained from our previous study [17]. One hundred and seventy-three AIS patients were enrolled within 48 h after onset of stroke, which was confirmed by a high-density lesion on diffusion weighted imaging (DWI) of magnetic resonance (MR) or a new low-density lesion on a brain computed tomography scan. We excluded individual participants with a history of infectious disease within the 2 previous weeks, immune diseases, hemorrhagic infarction, and progressive malignancy. A total of 116 participants who were matched with the AIS patients for age, sex and medical history were enrolled as healthy controls (HCs) in the physical examination center. All participants offered informed consent. The study protocol was approved by the Ethics Committee of Nanjing Medical University (No. (2019)695) and Southeast University (No. 2018ZDKYSB193), and followed the tenets of the Declaration of Helsinki.

Blood sampling and processing

Human blood samples (4–5 ml) were collected in a K2-EDTA containing tube (Vacutainer, BD, USA) within 48 h after onset of stroke. PBMCs were subsequently isolated within 2 h as previously reported [17]. Plasma was extracted from individual participants into a procoagulant tube (Vacutainer, BD, USA) for measurement of cytokine protein levels. Mouse blood samples were obtained from the retro-orbital plexus by enucleation to rupture the ophthalmic artery. Peripheral blood samples (1–1.5 ml) were collected in a K2-EDTA containing tube (Vacutainer, BD, USA) and processed within 2 h. After centrifugation at 1500 rpm for 10 min to remove plasma, blood samples were subjected to Ficoll density centrifugation (TBD Science, Tianjin, China) to isolate PBMCs according the manufacturer’s instructions.

RNA isolation and quantitative real-time PCR (qRT-PCR)

RNA isolation and qRT-PCR were performed according our previous study [17]. Briefly, total RNA was extracted from cells (human and mouse) and brain tissues of mice with TRIzol reagent (Invitrogen, CA, U.S.A.) following the manufacturer’s protocol. The purity and concentration of RNAs were determined by a NanoDrop spectrophotometer (Thermo Scientific, MA, USA). Subsequently, RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara, Dalian, China) and quantified using SYBR Premix Ex TaqTM II Kit (Takara, Dalian, China). Analysis was performed using GAPDH as the internal control. Expression levels of candidate RNAs were calculated with ΔCT or 2−ΔΔCt values if necessary. Primers for qRT-PCR were synthesized by GENEray (Generay Biotech Co, Shanghai, China) and are listed in Additional file 1: Table S1.

Cytokine protein analysis

Plasma samples from a total of 173 AIS patients and 116 corresponding HCs were used for measurement of cytokine protein levels. The concentrations of IL-4, IL-10, IL-6 and tumor necrosis factor α (TNF-α) in plasma were analyzed with a Navios flow cytometer (Beckman Coulter, California, USA). Mouse peripheral blood (1–1.5 ml) acquired from the retro-orbital plexus was collected in a K2-EDTA tube (Vacutainer, BD, USA) and centrifuged at 1500 rpm for 10 min. The plasma was separated and stored at a temperature of -80 °C when necessary, avoiding freeze–thaw cycles. The concentrations of the cytokines IL-4, IL-10, IL-6 and TNF-α in mouse plasma were measured with enzyme-linked immunosorbent assay (ELISA) Kits (Joyee Biotechnics Co. Ltd., Shanghai, China) according to the manufacturer’s protocol.

Separation and purity of monocytes/macrophages

Human PBMCs were re-suspended for cell sorting. We selected CD14+ monocytes/macrophages by using CD14 MicroBeads and Isolation Kit as previously reported [18]. Unmarked lymphocytes were also collected after magnetic cell sorting. Subsequently, monocytes/macrophages were treated with a FITC-conjugated anti-human CD14 antibody (Biolegend, CA, USA). The purity of monocytes/macrophages was confirmed by flow cytometry (BD FACSCanton-II, BD Bioscience, Franklin Lakes, NJ, USA) following the manufacturer’s protocol. Data were analyzed with FlowJo 7.6 software.

Cell culture

Human monocytes/macrophages were isolated after magnetic cell sorting and re-suspended in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). Human THP-1 monocytes, mouse RAW264.7 cells and HEK293T cells were obtained from the Cell Bank of the National Academy of Sciences (Shanghai, China) and incubated in RPMI 1640 medium supplemented with 10% fetal bovine serum for further study. THP-1 cells were treated with PMA (100 ng/ml, Sigma, Germany) in serum-free medium for 24 h to induce differentiation into macrophages.

RNA FISH and protein immunofluorescence staining

The fluorescence in situ hybridization (FISH) array assay was performed using a Fluorescence In Situ Hybridization Kit (C10910; RiboBio, China) according to its protocol. The 5’ FAM-labeled SNHG15 probe was designed and synthesized by GenePharma (Suzhou, China). The probe sequences are shown in Additional file 1: Table S2. After incubation with the SNHG15 probe, cells were treated with a mouse monoclonal anti-CD14 antibody (Abcam, Cambridge, UK) or anti-TRAF2 antibody (Cell Signaling Technology, MA, USA) for 2 h at 37 °C and were then washed three times with PBS. These cells were incubated with a fluorescently labeled secondary antibody (Proteintech, Chicago, USA) for 1 h at 37 °C and washed three times prior to nuclear staining with DAPI for 10 min. Finally, the microscopy sides were mounted with anti-fade mounting medium (Abcam, Cambridge, UK).

Nuclear and cytoplasmic isolation

Nuclear and cytoplasmic RNA was isolated from sorted monocytes/macrophages, THP-1 cells and RAW264.7 cells using a PARIS Kit (Invitrogen, USA) according to the manufacturer’s protocol.

RNA pull-down and mass spectrometry

The biotin-labeled SNHG15 (sense and anti-sense) probe was purchased from RiboBio (Guangzhou, China). The SNHG15 probe was incubated with THP-1 cell lysates overnight at 4 °C, and complexes were then isolated with streptavidin magnetic beads (Invitrogen) for 1 h at 4 °C. The isolated complexes were processed by silver staining (Sangon Biotech, Shanghai, China) for mass spectrometry, and a specific band in the experimental lane was subsequently selected for further analysis. Additionally, the isolated complexes were analyzed by immunoblotting using an antibody specific for TRAF2 (Cell Signaling Technology, MA, USA).

RNA immunoprecipitation and co-immunoprecipitation

RNA immunoprecipitation (RIP) and co-immunoprecipitation (Co-IP) assays were performed using an EZ Magna RIP Kit (Millipore, USA) according to the protocol. Briefly, cell lysates were incubated with RIP buffer containing magnetic beads that were preincubated with an anti-TRAF2 or anti-STAT6 (Cell Signaling Technology, MA, USA) antibody or IgG for 30 min at room temperature. The cell lysates were incubated with beads overnight at 4 °C. RNA/protein complexes were precipitated with protein G Dynabeads. Finally, the RNA or protein was purified and analyzed by qRT-PCR or western blotting.

Ubiquitination assay

The expression plasmids HA-K48-UB (with all lysines except lysine 48 mutated to arginine) and HA-K63-UB (with all lysines except lysine 63 mutated to arginine) were purchased from Hanbio Technology (Shanghai, China). Ubiquitination assays were performed by immunoprecipitation and immunoblotting. Briefly, cell lysates were incubated with an anti-TRAF2 antibody (Cell Signaling Technology, MA, USA) overnight at 4 °C. Bound proteins were analyzed by immunoblotting with an antibody specific for ubiquitin (Invitrogen, MA, USA) after washing three times.

Luciferase reporter assay

To confirm the association of signal transducer and activator of transcription 6 (STAT6) and the SNHG15 promoter, a pmirGLO reporter vector containing a constitutive or partially mutated promoter of SNHG15 was constructed. Monocytes were plated in 6-well plates and transiently cotransfected with the SNHG15 promoter or negative control using Lipofectamine 2000 (Invitrogen, MA, USA). Finally, luciferase activity was measured with a Dual-Luciferase Reporter Assay System (Promega, Wisconsin, USA).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software Inc., CA, USA). Band intensities from the immunoblots were analyzed with ImageJ Software. Data are presented as the mean ± standard deviation (S.D.) values. Student’s t test (n = 2 groups) or analysis of variance (ANOVA) (n > 2 groups) was used to assess differences between groups. A p value less than 0.05 was considered statistically significant.

A description of methods, including construction of lentiviral and plasmid, western blot, the transient middle cerebral artery occlusion (tMCAO) model of mice, adenoviral vector construction and injection, neurological deficits evaluation, triphenyltetrazolium chloride (TTC) staining and infarct volume assessment, and PBMCs isolated from mice, are available in Additional file 1.

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