Rat primary cell cultures: Primary cultures for rat cortical neurons, rat cortical astrocytes, and rat cortical microglia were prepared as described previously(20-22). All cells were cultured at 37 °C in a humidified chamber of 95% air and 5% CO2. Primary neuron cultures. Primary neuron cultures were prepared from cerebral cortices of embryonic day (E)17 Sprague–Dawley rat embryos. In brief, cortices were dissected and dissociated using papain dissociation system (Worthington Biochemical Corporation, LK003150). Primary neuron were spread on plates coated with poly-d-lysine (Sigma, P7886) and cultured in DMEM (NBM, Life Technology, 11965-084) containing 25 mM glucose, 4 mM glutamine, 1 mM sodium pyruvate, and 5% FBS at a density of 2 × 105 cells ml−1 (1 ml for 12-well format, 0.5 ml for 24-well format). At 24 h after seeding, the medium was changed to Neurobasal medium (Invitrogen, 21103-049) supplemented with B-27 (Invitrogen, 17504044) and 0.5 mM glutamine. Primary neurons were cultured at 37 °C in a humidified chamber of 95% air and 5% CO2. Primary neuron cultures were used for experiments from 7 to 10 days after seeding. Rat cortical astrocytes Primary astrocyte cultures were prepared from cerebral cortices of 2-day-old neonatal Sprague–Dawley rats. In brief, dissociated cortical cells were suspended in DMEM (Life Technology, 11965-084) containing 25mM glucose, 4mM glutamine, 1mM sodium pyruvate, and 10% FBS and plated on uncoated 25cm2 flasks at a density of 6×105 cells cm− 2. Monolayers of type 1 astrocytes were obtained 12–14 days after plating. Non-astrocytic cells such as microglia and neurons were detached from the flasks by shaking and removed by changing the medium. The microglia were collected for further culture. Astrocytes were dissociated by trypsinization and then reseeded on uncoated T75 flasks. After the astrocytes reached 70–80% confluence, were used for experiments.
Rat brain endothelial cells: A rat brain microendothelial cell line, RBE.4 were maintained in EBM-2 containing EGM-2MV Single Quots kit onto collagen-coated 25 cm2 flasks at a density of 2×105 cells/cm2 incubated in a 5% CO2 incubator at 37°C.
RNA extraction and real-time PCR.
Total RNA was extracted from the primary cultured cells using RNeasy Plus Mini Kit (50) (QIAGEN, Cat. No. 74134) in accordance with the manufacturer’s instructions. Total RNA was reverse-transcribed with hairpin-loop primers designed to target the specific miRNA at a concentration of 600 ng/μL used for cDNA synthesis PrimeScriptTM 1st strand cDNA Synthesis Kit (TaKaRa Clontech, Cat#6110A). Real-Time Quantitative Reverse-Transcription Polymerase Chain Reaction was performed with 20 ng cDNA in a 20 μL volume on the ABI 7500 System. Semi-quantitative real-time PCR, using RT2 SYBR Green ROX qPCR Mastermix (QIAGEN, Cat. No. 330520), For each of the selected miRNAs, real-time PCR measurements were performed to obtain a mean CT value for each sample. The CT values of the different samples were compared using the 2-ΔΔCT method, and U6 expression levels were used as an internal reference. For mRNA assays total RNA was reverse- transcribed with oligoDT primers at a concentration of 600 ng/μL used for cDNA synthesis PrimeScriptTM 1st strand cDNA Synthesis Kit (TaKaRa Clontech, Cat#6110A). TaqMan mRNA assays (Applied Biosystems Inc., Carlsbad, CA, USA) were used to quantify SUMO-2 mRNA (Rn00821719-g1, invitrogen) expression levels, in accordance with the manufacturer’s protocol. and B2m (Rn00560865-m1, invitrogen) expression levels were used as an internal reference.
Transfection of miRNA-194-5p inhibitor or control miRNA.
The Rno-miR-194-5p Anti-miR-miRNA Inhibitor ID:AM10004 (Ambion, AM17000) or MiR-Con ID:AM10004 (Ambion, AM17000) was mixed with Lipofectamine RNAiMAX Reagent (Invitrogen, 13778-075). Each mixture was added into primary rat neuron (7 days in vitro) or rat cortical astrocytes as manufacturer’s instructions.
Oxygen-glucose deprivation (OGD) and reoxygenation
OGD experiments were performed using a specialized, humidified chamber (Heidolph, incubator 1000, Brinkmann Instruments) kept at 37 °C, which contained an anaerobic gas mixture (90% N2, 5% H2, and 5% CO2) as we descripted before (22). To initiate OGD, culture medium was replaced with deoxygenated, glucose-free DMEM (Life Technology, 11966-025). After 2 h challenge, cultures were removed from the anaerobic chamber, and the OGD solution in the cultures was replaced with maintenance medium. Cells were then allowed to recover for 18 h (for neurotoxicity assay) in a regular incubator.
Determination of cell viability
Neuronal injury was measured by standard cell cytotoxicity assays such as Cell Counting Kit 8 cytotoxicity assay (DOJINDO, CK04-13) and PI (Propidium Iodide Incorporation) staining. Cell viability was quantified using a Cell Counting kit-8 (CCK-8, Dojindo) according to the manufacturer’s instructions. CCK-8 solution (10 μl) was added to each well of the plate, and the cells were incubated at 37 ⁰C for 2 hours. The optical density at a wavelength of 450 nm was measured with microplate reader. The relative assessments of neuronal injury were normalized by comparison with control cell as 100% cell survival (CCK8). For the PI staining, Cell viability was assessed after staining of naive cell cultures with propidium iodide (PI) to distinguish between living and dead cells (0.001mg/mL for 10minutes with subsequent rinsing) and fixed with 4% paraformaldehyde in phosphate buffered saline. Five images per well were taken using microscope. Viable neurons not incorporating PI were counted in transmission images and quantified.
Western blot analysis
Pro-PREP Protein Extraction Solution (iNtRON Biotechnology, 17081) was used to collect samples. Each sample was loaded onto 4–20% Tris-glycine gels. After electrophoresis and transferring to nitrocellulose membranes, the membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 0.2% I-block (Tropix, T2015) for 90 min at room temperature. Membranes were then incubated overnight at 4 °C with the following primary antibodies, anti-β -actin (1:1,000, Sigma-Aldrich, A5441), SUMO2/3-specific antibody (1:1,000, Abcam, ab3742). After incubation with peroxidase-conjugated secondary antibodies, visualization was enhanced by chemiluminescence (GE Healthcare, NA931 (anti-mouse), NA934 (anti-rabbit)). Optical density was assessed using the NIH Image analysis software.
Luciferase reporter assays were performed to verify the direct binding of miR-194-5p to SUMO2 transcripts.
Construction of 3′-UTR reporter plasmids and luciferase assays synthesized double-strand oligonucleotides containing 20 bases upstream and 15 bases downstream of miR-194-5p seed sequences. We constructed pmiR-GLO-194-5p SUMO2-TS (targeting sequence) reporter vector, containing the 3′-UTR of SUMO2. The pmiRGLO-194-5p-SUMO2-WTS (with wide targeting sequence) also generated in this cloning step. The pmiRGLO-194-5p-SUMO2-MTS (with mutated targeting sequence) also generated in this cloning step, by reversing the seed targeting sequence of SUMO2. The 293T cells were co-transfected with either pmiRGLO-194-5p-SUMO2-MTS or pmiRGLO-194-5p-SUMO2-WTS with miR-Con or miR-194. Luciferase reporter assay cells were lysed in passive lysing buffer and then analyzed for the firefly and renilla luciferase activities using the commercial Dual-Luciferase reporter assay system on the GloMax-multi Detection Luminometer (Promega Corporation, E7031, Madison, WI, USA), and the firefly luciferase activity was normalized to the renilla luciferase activity. In all, 24 h after transfection, the firefly and Renilla luciferase activities were detected consecutively using Dual-Luciferase Kit (Promega Corporation). Relative protein levels were expressed as Firefly/Renilla luciferase ratios.
SUMO2 knockdown Lentiviral vector contraction and transduction in primary neurons.
The SUMO2 knockdown by shRNA infection Lentiviral vectors encoding shRNAs, Recombinant lentivirus (pLKD-CMV-eGFP-sumo2-shRNA) against rat SUMO2 and control vector (pLKD-CMV-eGFP-U6-shRNA) were designed and reconstructed by Obio Technology Co., Ltd, (Shanghai, China). The target sequences for rat SUMO2-shRNA are Seq1: 5′-GCGAGATCAGCATCTGCAT- 3′, Seq2: 5′-GCAGCTCATGGTACATGCT-3′. Four siRNA oligonucleotide sequences targeting sumo2 (NCBI accession Gene ID NM_133594) were Seq1: 5′-GTTTGTCAATGAGGCAGATCA-3′ (Y6228), and Seq2:5′-GGCATACACCACTTAGTAA-3′(Y6229), Seq3: 5′-GGATGAAGATACGATTGAT-3′ (Y6230) The NC sequence was 5′-TTCTCCGAACGTGTCACGT-3′ (Y004) whose knockdown effect has been demonstrated previously. The vector clones were constructed pcDNA-sumo2 and transformed into competent Escherichia coli. The sense and antisense oligomers were cloned into SUMO2 restriction sites of lentiviral vector (pLenti-U6-shRNA-CMV-EGFP) to drive the shRNA expression under the U6 promoter. The SUMO2 knockdown lentivirus was packaged with pLKD-CMV-eGFP plasmid and transfected into C6 cell line. The viral supernatant was harvested, filtered, and concentrated. For lentivirus infection, primary cultured rat neurons were seeded in 24-well plate. After neurons were cultured for 3 days, cells were infected with recombinant SUMO2 shRNA-lentivirus or or NC-shRNA-lentivirus control vector (as negative control) at a multiplicity of infection (MOI) of 20 in medium (Supplementary Fig. A). To determine the transduction efficiency in primary neurons, GFP expression was examined by fluorescence microscopy (ZEISS, German) at different multiplicities of infection (MOIs) on day 3 after infection. Primary cultured rat neurons were transfected with the NC or recombinant vectors. After 8 h infection, the medium was replaced by fresh complete medium for another 4 days prior to further experiments, neurons were harvested at 4 days after transfection. GFP-positive cells were confirmed under a fluorescence microscope (Supplementary Fig. B). Image acquisition was executed using an inverted fluorescence microscope (Olympus) equipped with a 60× oil immersion objective. Sustained SUMO2 downregulation levels were confirmed by western blot analysis neurons after transfection (Supplementary Fig. C).
All values are represented as mean± SD of at least three independent experiments. When only two groups were compared, Student’s t-test was used. Multiple comparisons were evaluated by one-way ANOVA followed by Tukey's tests. P-values of <0.05 were deemed to be significantly different.