Patients samples
There are 62 SLE patients and healthy volunteers were recruited from the First Affiliated Hospital of Army Medical University between 2017 and 2020. The SLE patients included fulfilled at least four of the American College of Rheumatology (ACR) 1997 revised criteria of SLE (Hochberg, 1997). Demographic, clinical, and laboratory characteristics of each subject were recorded, and disease activity was evaluated with SLEDAI (Bombardier et al. 1992). The information of SLE patients can be found in (Table 1, Additional file 1: Table 1). All participants were Han Chinese.
Table 1 Clinical features of SLE patients and demographic data of HCs
characteristics
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SLE patents(n=62)
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HCs(n=62)
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Age, years, median (IQR)
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37.0(20.8-54)
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36.0(21.5-53.4)
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Female, n (%)
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55(89)
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55(89)
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SLEDAI score, mean (IQR)
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9.05(2.73-15)
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N/A
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Complement C3, median (IQR)
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0.58(0.25-0.92)
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N/A
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Complement C4, median (IQR)
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0.14(0.04-0.27)
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N/A
|
IQR: interquartile range.
RNA extraction and RNA quantitative real-time polymerase chain reaction
Total RNA was harvested and separated from PBMCs in samples via TRIzol reagent (Invitrogen, United States) and sequentially complementary DNA (cDNA) was synthesized. Two micrograms of total RNAs was used to synthesize cDNA, a portion of which (1 µl, equal to 0.2 µg cDNA) were used in a PCR. The reverse transcription polymerase chain reaction (PCR) was operated with the SYBR Green Super Mix (Roche, Switzerland). Analysis of experimental results was analyzed through the 2−ΔΔCt method. β-actin was an internal control. The sequences of the primers used for qRT-PCR in this study are shown in (Additional file 2: Table 2).
Ribonuclease R treatment
Total RNA (2 μg) of PBMCs were mixed with 3 U/μg of ribonuclease R (EpicentreTechnologies, United States) at 37°C for 20 minutes. The stability of circLOC101928570 and linear LOC101928570 were determined. Relative expression levels were evaluated by qRT-PCR.
Fluorescence in situ hybridization
Hybridization was performed overnight with circLOC101928570 probes. Specimens were analyzed using a Nikon inverted fluorescence microscope. The circLOC101928570 probe for fluorescence in situ hybridization (FISH) is 5′-TGGCTTGAATAGATTGGGACTAATA-3′. The cell suspension was pipetted onto autoclaved glass slides, followed by dehydration with 70%, 80% and 100% ethanol. Then hybridization was performed at 37 °C overnight in a dark moist chamber. After being washed twice in 50% formamide/2×SSC for 5 min, the slices were incubated with the regents in Alexa FluorTM 488 Tyramide SuperBoost™ Kits by Riobio (Guangzhou, China) for 30 min and sealed with parafilm containing DAPI. This assay was repeated three times.
Cell isolation and culture
Whole blood (10 ml) was collected in EDTA collection tubes from each subject, and human PBMCs were isolated by density-gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Biosciences, United States) and cultured in RPMI Media 1640 (Gibco, United States) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL Penicillin, 100 U/mL streptomycin (Gibco, United States) at 37 °C with 5% CO2 for 24 h before transfection.
Cell transfection
The circLOC101928570 overexpression plasmids and empty vector were constructed and designed by Genechem (Shanghai, China). The miR-150-3p mimics, miR-150-5p mimics, miR-150-3p inhibitor, miR-150-5p inhibitor, miRNA mimics NC, miRNA inhibitor NC were chemically synthesized by Riobio (Guangzhou, China). pCDH-CMV-MCSEF1-GFP+Puro (CD513B-1) vector as a negative control plasmid, pCDH-MYB plasmid was fragment inserted into pCDH-CMV-MCSEF1-GFP+Puro (CD513B-1) vector with BamHI and NotI restriction sites. The result of vector construction was verified by direct sequencing. Sequence used was (5‘-CCGGAATTCCGGGAAAGCGTCACTTGGGGAAAA-3’). PLKO.1-puro (Addgene plasmid # 8453) was used to design short hairpin RNA , they were transfected by Lipofectamine 3000 (Invitrogen, United States) into cells. The transfection process lasted 48 hours.
Luciferase reporter assay
293T cells in 24-well plates were cotransfected with miR-150-3p/5p mimic, inhibitor, and negative control, luciferase reporter vectors (SV40-Luc-MCS) with wild type or mutant circLOC101928570 were designed and constructed by Genechem (Shanghai, China). The IL2RA 3’ UTR sequences containing two wild-type c-myb predicted binding sites were inserted into the region directly downstream of a T7 promoter-driven firefly luciferase cassette in a psiCHECKTM-2 vector (Promega, United States). All constructs were verified by sequencing. 293T cells were seeded into 24-well plates and were co-transfected with a mixture of 1ug of luciferase reporter plasmid and PCDH, plasmid pCDH-MYB, shNC, shMYB. After 48h, luciferase activity was measured using Dual-Luciferase® Reporter Assay System (Promega, United States). The relative firefly luciferase activity was normalized to Renilla luciferase activity. All experiments were performed in triplicate.
Short hairpin RNA
To stably knock down circLOC101928570 expression, Jurkat cells were cultured and infected with lentivirus carrying shRNA targeting circLOC101928570 and a negative control vector, PLKO.1-puro was used to design short hairpin RNA, the restriction site were AgeI (R3552S), EcoRI (R3101T), after 1300bp has a single restriction site KpnI (R0142M). For the lentivirus package, HEK-293T cells were transfected with the core plasmid PLKO.1-shRNA, with the psAX2 packaging plasmid and pMD2G envelope plasmid for 48 h to obtain the lentivirus supernatant. The shRNA sequences used was showed in (Additional file 3: Table 3). All constructs were verified by sequence analysis. with no homology to any other human genes.
Apoptosis
Double staining of Annexin V and 7-aminoactinomycin-D (7-AAD) was carried out using a PE Annexin V Apoptosis Detection Kit (BD Pharmingen™, United States) and APC Annexin V Kit (BD Pharmingen™, United States) according to the manufacturer’s recommendations. Briefly, cells were washed with cold phosphate-buffered saline and then resuspended in Binding Buffer at a concentration of 1 x106 cell/ml. Then, 100 μl solution (1 x 105 cells) was transferred to a tube, and 5 μl PE Annexin V and 5 μl 7-AAD were added. After incubation for 15 min at room temperature in the dark and addition of 400 μl of binding buffer, flow cytometric analysis was done by flow cytometer (FACScan , BD Biosciences, San Jose, CA, United States) within 1 hour, and the data were analysed with FlowJo software (Treestar, Ashland, OR). PE Annexin V (+) or APC Annexin V (+), 7-AAD (-) cells represent early stage of apoptosis, whereas PE Annexin V (+) or APC Annexin V (+), 7-AAD (+) cells were in-end stage apoptosis or already dead.
Enzyme-linked immunosorbent assay (ELISA)
Concentrations of IL-4 and IFN-γ in Peripheral blood serum supernatants were analyzed by Human IL-4 instant ELISA Kit (eBioScience, United States) and Human IFN gamma Platinum ELISA Kit (eBioScience, United States) following the manufacturer’s instructions, respectively. The concentrations were calculated according to their corresponding stand curves.
Prediction of ceRNAs for circLOC101928570
Mutually targeted method was applied to predict putative ceRNAs for circRNA. To predict ceRNAs for circLOC101928570, we use software circMir1.0 identified circLOC101928570 targeting miRNAs.
Pull-down assay
Biotinylated circLOC101928570 probe was specifically designed and synthesized for binding to the junction site of circLOC101928570. The biotin-coupled RNA complex was pulled down by incubating the cell lysates with Pierce™Streptavidin Magnetic Beads (Thermo Fisher Scientific, United States) following the manufacturer’s instructions. The enrichment of miRNAs in the capture fractions was evaluated by qRT-PCR analysis. Probe sequences used were listed in (Additional file 4: Table 4).
RNA-binding protein immunoprecipitation (RIP)
RIP assay was performed by using a Magna RIP RNA Binding Protein Immunoprecipitation Kit (Millipore, United States), according to the manufacturer’s instructions. Briefly, PBMCs were harvested and lysed in RIP lysis buffer on ice for 30 min. After centrifugation, the supernatant was incubated with 30μl of Protein G agarose beads (Roche, United States) and antibodies. After overnight incubation, the immune complexes were centrifuged then washed six times with washing buffer. The beads bound proteins were further analyzed using western blotting. The immunoprecipitated RNA was applied to qRT-PCR analysis.
Western blot analysis
Complete proteome from PBMCs was extracted after lysing in RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Sigma-Aldrich, United States) and then separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis. The samples were electro transferred into polyvinylidene fluoride membrane (Millipore, United States). Post blocking with 5% fat free milk, the membranes were treated with prepared primary antibodies: anti-c-myb (Abcam, England), rabbit IL2Rα antibody (CST, United States), rabbit GAPDH antibody (CST, United States) was used as control. Membranes were washed, followed by treating with secondary antibody anti-rabbit antibody (CST, United States). The signal of the blot was examined by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, United States) with ChemiDocTM Touch Imaging System (Bio-Rad, United States). The integrated density values were calculated using Quantity One software (Bio-Rad, United States).
Flow cytometry analysis
The flow cytometric analysis was performed on freshly isolated PBMCs as a previous study showed that CD25 expression is affected by freezing–thaw procedures. PBMCs were stained with fluorochrome-conjugated antibodies to identify blood CD4+ and CD8+ T cell subsets by flow cytometry. The staining procedure was conducted blinded to genotype and performed simultaneously for each pair. Prior to staining, FcR Blocking Reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the PBMCs to prevent non-specific binding. We used the following monoclonal antibodies specific for: CD3 (Biolegend, clone, UCHT1), CD4 (Biolegend, United States), CD8 (Biolegend, United States), CD25 (Biolegend, United States), IFN-γ (Biolegend, United States), IL-4 (Biolegend, United States) FOXP3 (Biolegend, United States), IL17A (Biolegend, United States). All antibodies were purchased from BioLegend (San Diego, CA, United States). The stained PBMCs were washed 3 times in cold PBS/2%FCS/0.1% NaN3 and data were acquired on a FACS Canto II 8 color flow cytometer (BD Biosciences, San Jose, CA, United States) aiming for 30,000 acquisitions.
Statistical analysis
Data were analyzed and visualized with GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, United States). Quantitative values are expressed as means±standard deviation (SD) of at least three independent repetitions. Statistical differences among groups were tested with unpaired two-tailed student’s t test. A P-value less than 0.05 was chosen to indicate statistical significance.