Ethical approval
All experimental procedures for animal usage were in accordance with the guidelines for care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Harbin Medical University.
Animal models
Ten- to twelve-week-old wistar rats, weighting 250-300g, were selected, which obtained from Animal Research Center of Harbin Medical University (Harbin, China).
To develop the HSP animal model, rats were sensitized by injection (intraperitoneal, i.p.) of 1ml saline containing 1 mg ovalbumin (OVA; Sigma) and 10 mg alum (Sigma) once per week for 2 weeks. Subsequently, rats each received OVA physiological saline liquor (15 mg/kg) by external jugular vein injection, and also received OVA physiological saline liquor (0.3%; 1 ml; 5 injection points) through intradermal injection. Meanwhile, rats in the control group received equal amounts of physiological saline. The method for HSP model rats was previously described [11].
Twenty-four hours after HSP model establishment, all rats were anesthetized by injecting with dose (40mg/kg) of pentobarbital sodium (Sigma). Subsequently, Blood samples were collected from the posterior orbital venous plexus of the inner canthus of rats for further analysis.
Isolation and culture of rat monocyte-derived macrophages
Isolation and culture of rat monocyte-derived macrophages were performed as previously described [12]. Peripheral blood mononuclear cells (PBMCs) were isolated using a Ficoll (Solarbio Life Sciences, Beijing, China) density gradient. Monocytes were obtained from PBMCs using anti-CD14 magnetic beads (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s instruction. CD14+ monocytes
were differentiated to macrophages during 7 days of culture in RPMI 1640 (Sigma-Aldrich, St. Louis, MO), containing 10% fetal bovine serum (Sigma-Aldrich) and 50 ng/ml macrophage colony-stimulating factor (Sigma-Aldrich) for 7 days. Half of the culture medium was changed every 3 days unless otherwise indicated.
Flow cytometry and M1/M2 macrophage-related cytokines detection
The phenotypes of rat monocyte-derived macrophages were qualified using flow cytometry analysis according to a previous study [13]. Briefly, macrophages were stained with the following fluorochrome-labeled antibodies: anti-CD80-PE (eBioscience, Thermo Fisher Scientific, Inc., Waltham, MA) and anti-
CD206-MR5D3 (Novus Biologicals Inc., Littleton, CO). After staining for surface markers, macrophages were fixed and analyzed with an Accuri C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and the results were analyzed using FlowJo software (Treestar, Inc., Ashland, OR). The concentrations of IL-12, IFN-γ, IL-4, and TGF-β in rat monocyte-derived macrophages from control or Henoch Schonlein purpura rat were measured using their commercial ELISA kits (R&D Systems,
Minneapolis, MN) according to the manufacturer’s instructions.
Real-time polymerase chain reaction analysis
Total RNA was extracted from rat monocyte-derived macrophages using RNeasy Plus Mini Kit (Invitrogen Co, USA) according to the manufacturer’s instructions, and cDNA was generated through reverse transcription using the PrimeScript RT Master Mix kit (TaKaRa Bio Inc, Japan). Equal amounts of cDNA were diluted and amplified through real-time polymerase chain reaction (PCR) using All-in-one qPCR Mix (TaKaRa Bio Inc) in a 20 µl reaction volume containing 10 µl of 2×All-in-one qPCR Mix (TaKaRa Bio Inc), 1 µl of 2 mmol/L forward primer, 1 µl of 2 mmol/L reverse primer, 1 µl of cDNA, and 6 µl of nuclease-free water. After an initial denaturation step for 10 min at 95°C, the conditions for cycling were 40 cycles of 10 s at 95°C, 20 s at 60°C, and 15 s at 72°C. For the normalization of each sample, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, TaKaRa Bio Inc) primers were used to measure the amount of GAPDH cDNA. The primers used are listed below (Table 1). Relative expression levels of mRNA, lncRNA, and miRNA was determined using the 2−ΔΔCt method [14]. Relative MEG8 and miR-181a-5p expression were normalized to 18S and the mRNA level of SHP2 was normalized to GAPDH.
Western blotting analysis
The protein levels in rat monocyte-derived macrophages were detected by Western blotting analysis. Briefly, the cells from different groups were lysed by Radio-Immunoprecipitation Assay lysis buffer (Beyotime Bio Inc, Shanghai, China). The protein concentrations were determined by BCA protein estimation kit (Beyotime Bio Inc). Then, equal amounts of protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Beyotime Bio Inc), and transferred to polyvinylidene difluoride membranes (Beyotime Bio Inc). After blocking with 5% non-fat dry milk, the membranes were incubated with primary antibodies at 4°C overnight. Then the corresponding secondary antibodies conjugated with horseradish peroxidase were added. ECL detection reagent (7 Sea Biotech, Shanghai, China) was used to visualize the blots. The results were quantified with Gel-Pro-Analyzer (Media Cybernetics, USA).
Plasmid construction and cell transfection
To overexpress MEG8 in rat monocyte-derived macrophages, the synthetic MEG8 sequence was subcloned into the pcDNA3.1 vector (Invitrogen), generating a pcDNA3.1-MEG8 vector. An empty pcDNA3.1 vector used as a control. Cells were transfected with these plasmids using Lipofectamine 2000 (Invitrogen) according to
the manufacturer’s instructions. To knockdown MEG8 and SHP2, MEG8 small interfering RNA (si-MEG8), si-SHP2, and the corresponding scramble controls were
purchased from (GenePharma, Shanghai, China) and transfected into cells using Lipofectamine RNAiMAX Reagent (Invitrogen). hsa-miR-181a-5p mimic, mimic negative control (NC), hsa-miR-181a-5p inhibitor, and NC inhibitor (GenePharma) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cells were harvested 48 hour post-transfection. The overexpression and knockdown efficiency was determined using RT-PCR analysis [15].
Luciferase activity assay
The fragments of MEG8 and 3’-UTR of SHP2 containing the predicted wild-type (WT) binding sites of miR-181a-5p or mutated miR-181a-5p binding sites (MUT) were amplified using PCR and inserted into a pMIR-REPORT luciferase reporter vector (Ambion, Austin, TX), named as MEG8-WT, MEG8-MUT, SHP2-WT, and SHP2-MUT. When the luciferase reporter assay is performed, cells were cotransfected with 200 ng constructed luciferase reporter vectors, 25 ng pRL-TK (expressing Renilla luciferase as the internal control) and 20μM miR-181a-5p mimic or mimic NC using Lipofectamine 2000. A luciferase reporter assay system (Promega Corporation, Fitchburg, WI) was applied to analyze the luciferase activities at 24 hr posttransfection.
Statistical analysis
All statistical analyses were performed using SPSS version 20.0 software (SPSS, Inc., Chicago). The unpaired Student t test was used to analyze differences between only two value sets. One-way analysis of variance was used to analyze differences among three or more groups. The data are presented as the mean ± standard deviation from at least three independent experiments. P < 0.05 was considered statistically significant.