Animals
The Sprague Dawley (SD) rats (80-100 g, 6 weeks) were purchased from Shanghai Jiesijie Experimental Animal Co. Ltd. were used for experiments. Rats were raised in an air-conditioned room (about 22-26 ℃) and the cycle of light-dark was maintained at 12:12. Food and water were free available. Newborn SD rats (within 24 hours after birth) were used for in vitro experiments.
Pilocarpine-induced SE
A model of SE was established using pilocarpine and lithium chloride (Sigma, USA). Rats received an injection of lithium chloride (127.3mg/kg, i.p.), 20-24 hours before pilocarpine (30 mg/kg, i.p.) injection. Scopolamine (1 mg/kg, i.p.; Sigma) was administered 30 minutes before pilocarpine to prevent peripheral effects. A modified Racine scale was used to score behavioral seizures[25]. The SE model is considered to be successfully ignited when the rats reached IV-V score within 30 minutes and show a persistent state. For the rats without grade IV-V seizures, they were additional injectional of pilocarpine (10 mg/kg, i.p.) until reached grade IV or V seizures. There were no more than 60mg/kg per animal injected. The seizure lasted for 60 minutes and diazepam (10mg/kg) was intraperitoneal administered to terminate the seizure.
High Throughput Sequencing
The RNA high throughput sequencing of hippocampal tissues was performed on Illumina Novaseq 6000 sequencing platform (Illumina, USA). Gene Ontology (GO) categories analysis derived from Gene Ontology (http://www.geneontology.org) were used to analyze differentially expressed mRNA. The KEGG pathway database (http://www.genome.jp/kegg) was used to conduct the functional analysis. Differentially expressed lncRNAs were identified and screened by coding potential calculator (CPC) software. P < 0.05 was defined as statistical significance.
Cell Culture and Treatment
Primary microglia from the neonatal rat were cultured as previously report[26]. DMEM-F12 (Gibco, USA) was used to cultivate isolated microglia. In a humidified atmosphere of 5% CO2/95% air, the primary microglia were incubated at 37°C. Microglia were treated with NMDA (20 μM) at 85% confluence. After 48 hours of treatment, total RNA and protein were extracted.
siRNA and Cell Transfection
The negative control (NC) siRNA and lncRNA Mir155hg siRNA were designed by Shanghai Gene Pharma. Transfection of siRNAs was performed in accordance with manufacturer's instructions of Lipo3000 kit (Invitrogen, USA). Real-time PCR (RT-PCR) was used to assess knockdown efficiency 24 hours after transfection. The si-RNAs sequences are available in Table S1.
Western Blot
As previous described[25], proteins of Microglia and hippocampal were extracted using PMSF containing SDS lysis buffer (Beyotime, Shanghai, China). 10% SDS-PAGE electrophoresis was used to separate proteins and then transfer to PVDF membranes. After sealing with a blocking for one hour, the primary antibody was incubated (Table S4) overnight at 4°C. The secondary antibody was incubated at room temperature for one hour. Chemiluminescence (ECL) was employed to detect the signals. And Western blotting signal was quantified by ImageJ. Internal control was performed using GAPDH.
RNA FISH
Fluorescence in situ hybridization (FISH)was used to locate the subcellular location of lncRNA Mir155hg. Mir155hg lncRNA probe mix was designed and synthesized by RiboBio (Guangzhou, China). A situ hybridization kit (RiboBio, Guangzhou) was used for FISH analysis. In brief, a solution of 4% paraformaldehyde was applied to microglia plated on coverslips for 30 minutes. Washing with PBS removed Triton X*100 and coverslips were sealed with rubber cement. Then, probes were hybridized with coverslips overnight at 37°C. The final step was to mount coverslips with an anti-fade fluorescence mounting medium containing DAPI and observe them under a fluorescence microscope (OLYMPUS, Japan).
RT-PCR
As our previous study shown[25], total RNAs were extracted from microglia and hippocampal tissues using Trizol kit (Takara, Japan). Primer from the ScriptTM RT kit was used to synthesize cDNA. Primers were created by Life Technologies Software. RT-PCR was performed on an Applied Biosystem 7300 (Thermo Fisher Scientific). Normalized mRNA expression levels were compared to GAPDH. The primer sequences are shown in Table S3.
Immunofluorescence and Nissl Staining
Based on the methods of the previous study[27], rat brain tissues were transparentized and immersed in wax after fixing in 4% paraformaldehyde for 48 hours. Afterward, the tissues were sectioned and dewaxed with xylene. Paraffin-embedded sections were stained with Nissl stain and Iba1 antibody. Following dehydration with neutral glue, sections were cleaned and fixed. Finally, the hippocampal CA1 and CA3 regions were observed using an optical microscope (Olympus, Japan).
Morris Water Maze (MWM)
The MWM was performed as we previous study described[25]. Adaptive swimming training was carried out at 4-5 weeks after SE modeling. The training phase of covert platform test lasted for four days. In space detection test, the rats were put in the water from entry points after the platform was removed. Within the 60 seconds, the swimming time of rats in the target quadrant and times to cross the original platform position were recorded.
Electroencephalogram (EEG)
As described in our previous study[25], EEG recordings are being collected using stainless-steel screws implanted in the skull over the frontal and parietal cortexes. Implant coordinates were as follows: anterior-posterior (AP) = − 2.2mm, lateral (L) = ± 3.2mm. Recording and reference electrodes were implanted in these holes using stainless steel screws. One week after electrode implantation, rats began modeling.
Dual-Luciferase Reporter Assay
The binding site between target miR-155 and Socs1 mRNA was predicted by RNA22[28]. Mutant 3′UTR or wild-type sequences of Socs1 containing predicted miR-155-5p binding sites were inserted into the vector. A combination of Lipofectamine 8000 transfection with the recombinant vectors, miR-155-5p mimics and inhibitor (Table S2) were transfected into the 293T cells. Luciferase assay kit (Beyotime, Shanghai, China) was used to determine the firefly luciferase activity.
Statistical Analysis
The data were presented as mean ± SD. We performed all analyses using GraphPad Prism8. The differences between the different groups were analyzed with ANOVA and Student's t test. Significant differences were defined as P < 0.05. Three times were repeated in each experiment.