The sequence of LncRNA-BG was obtained from UCSC Genome Browser (http://genome.ucsc.edu/) and submitted to the CatRapid public database (http://s.tartaglialab.com/page/catrapid_group) and MEM Database（https://biit.cs.ut.ee/mem/）to predict its interacting proteins.
RNA pull down Experiment
To verify the interaction between RORγt and lncRNA-BG, the RNA pull-down were carried out. LncRNA-BG was synthesized in vitro ( Genscript, Nanjing, China) and biotin-labeled using PierceTM RNA3' End Desthiobiotinylation Kit. CD4+ T cell extracts were incubated with Biotin labeled lncRNA-BG at 4 °C for 1 h. Then streptavidin magnetic beads (New England Biolabs, USA) were added and incubated at room temperature for 1 h. Lysate proteins in each reaction were detected by Western blot using mouse-anti human RORγt primary antibody (BD sciences, SD, USA) and goat-anti mouse secondary antibody ( Boster, Wuhan, China ).
Dual luciferase reporter assay
The IL-17 promoter was cloned into the luciferase expression reporter plasmid to construct pGL3-IL-17 (Genscript, Nanjing, China). LncRNA-BG and RORγt were cloned into pcDNA3.1 to construct pcDNA3.1-BG and pcDNA3.1-RORγt (Genscript, Nanjing, China). After 293 cells were cultured in a 12-well plate for 24 hours (80% confluence), pGL3-IL-17,RORγt and lncRNA-BG were cotransfected into 293T cells in different combinations. The fluorescence signals were detected 48 h after transfection according to the manufacturer's instruction and relative fluorescence values ( Luciferase / Renilla ) were calculated.
Patients and health control
A total of 16 samples were recruited from Xiangya Hospital of Central South University, including 10 COPD patients and 6 healthy controls. These patients were diagnosed with COPD according to the criteria of the Respiratory Pathology Branch of the Chinese Medical Association and the Global Initiative for Chronic Obstructive Lung Disease (GOLD). All participants were provided written informed consent.
Collection and culture of CD4+ T cells
The peripheral blood samples collected by the subjects were preserved in tubes with pretreatment of heparin sodium. Peripheral blood mononuclear cells (PBMCs) were isolated in Ficoll-Hypaque solution by density gradient centrifugation. CD4+ T cells were then isolated using a positive selection magnetic isolation system (Miltenyi, Cologne, Germany) according to the manufacturer’s instructions. The isolated CD4+ T cells were assessed by flow cytometry. CD4+ T cells with purity above 90% can be used for further analysis. The isolated CD4+ T cells were routinely cultured in human T cell culture medium at 37 °C and 5% CO2.
Human T-lymphocytic leukemia cell line Hut78 with mature T-cell induction-assisted characteristics was obtained from the American type culture collection cell library (ATCC). Hut 78 was cultured in IMDM medium (HyClone, Logan, USA) containing 20% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2.
Th17 cell differentiation
Hut78 cells (2×106 Cells/mL) were cultured with 20 ng/mL of IL-6, 5 ng/mL of TGF-β, 25 ng/mL of IL-23, 5 μg/mL of anti-IFN-γ and 5 μg/mL of anti-IL-4 for 3 days under the activation of 2 μg/mL anti-CD3 and 5 μg/mL of anti-CD28. CD3, CD28, TGF-β and IL-23 were purchased from Sino Biological in Wuhan, China while Anti-IFN-γ and anti-IL-4 were purchased from Bioxcell in West Lebanon, USA .
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-17A in the supernatant of CD4+ T cells of patients with COPD and TH17 cells cultured in vitro culture were measured by using ELISA according to the manufacturer's instructions (Fankew, Shanghai, China). In short, the supernatant was added to a 96-well plate with 100 μl per well. Appropriate biotin-binding antibodies (Fankew, Shanghai, China) were added to each well, and incubated at room temperature for 2 hours. After washing five times, substrate solution was added to each well and incubated in darkness at room temperature for 30 minutes. The optical density (OD) of each well was detected at a wavelength of 450 nm. The concentration of IL-17A in the sample was calculated according to the standard curve.
Quantitative real-time PCR (qRT-PCR)
Total RNA in the cells was extracted with TRIzol reagent (TaKaRa, Beijing, China) according to the manufacturer's instructions. The concentration of the extracted RNA was detected by spectrophotometer. Through reverse transcription, RNA were detected using Reverse transcription kits (TaKaRa, Beijing, China) according to the procedure of product specification. The obtained cDNA was detected by qRT-PCR using SYBR®-Green and fluorescent quantitative PCR detection system (bimake, Houston, USA) according to the procedure of product specification. GAPDH was used as internal reference gene. The relative level changes of target genes were calculated by 2−ΔCt method.
To over express lncRNA-BG level in CD4+ T cells, lentivirus vectors containing pcDNA-BG (GenePharma, Shanghai, China) were transfected into hut78 cells. 2 ml of hut78 cell suspension was inoculated into six-well culture plate for 24 hours before transfection.10 μl lentiviruses was added to each well and then 1μl of polybrene (GenePharma, Shanghai, China) was added to improve transfection efficiency for 24 hours. To down-regulate lncRNA-BG level in CD4+ T cells, 10 μl of lentivirus vectors containing siRNA-BG (Jtsbio, Wuhan, China) and 1 μl of polybrene (Jtsbio, Wuhan, China) were added to each well for 24 hours. Empty lentivirus vectors were used as negative controls.
The differentiated T cells were incubated for 4 hours at 37°C and 5% CO2 with 0.1 mg/ml of monensin (Sigma, Saint Louis, USA), 1 μg/ml of ionomycin (Sigma, Saint Louis, USA), 500 ng/ml of sparfloxacin (Sigma, Saint Louis, USA).Then cells were stained with FITC-IL-17A monoclonal antibody (Multiscience, Hangzhou, China) and APC-CD4 monoclonal antibody (Multiscience, Hangzhou, China). The flow cytometry was performed in Cytek Athena system (Cytek, San Francisco, USA). Flow Jo software was used to analyze the results.
The induced T cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes, washed with PBS, permeated with PBS+0.2% Triton X-100 (PBTX) for 10 minutes, then blocked in 10% goat serum for 1 hour. The cells were incubated overnight at 4 °C with Rabbit anti-RORγt (GeneTex, Irvine, USA). The following day, the cells were washed with phosphate-buffered saline (PBS) containing 0.3% Triton X-100, blocked in 10% goat serum (GS) for 1 hour and then incubated at room temperature for 1 hour with the biotinylated universal secondary antibody and horseradish peroxidase-labelled streptomycin-avidin, The images were collected under fluorescence microscope after the slides were sealed with fluorescence quenching agent.
Statistical analysis was performed using SPSS 17.0. The data was expressed as a mean ± standard deviation. To compare the difference between two groups or multi-groups, t-test and one-way ANOVA were performed and LSD was used for post hoc test. P < 0.05 was considered significant.