Sixty adult male mice weighing 25-30 g were used in this study. All animals were placed in a room where temperature and humidity were controlled (18-25 °C and 50-60%, respectively), with a light/dark cycle of 12 hours, water and food were freely available. This study was approved by the Ethics Committee of the Shaanxi Provincial People's Hospital.
Mice were injected intraperitoneally with pilocarpine (300 mg/Kg) and scopolamine (1 mg/Kg) was injected 20 minutes before the injection of pilocarpine to reduce the peripheral effects of pilocarpine . The severity of seizures was graded using the Racine scale: class 1, facial convulsions; class 2, head nodding; class 3, forelimb clonus; class 4, bilateral forelimb clonus and rearing; and class 5, rearing and falling. One hour after the onset of grade 4-5 seizures, diazepam (10 mg/Kg) was used to terminate seizure. If the animals didn’t develop 4-5 grade seizures after 30 minutes of pilocarpine injection, they will be excluded. The control mice were injected with the same amount of normal saline.
Mouse astrocytes (MAs) were purchased from Shanghai Institute of Cell Biology (Shanghai, China). All of the cells were cultured with 10% FBS (Hyclone, Logan, UT, USA) at 37 °C in a humidified 5% CO2 atmosphere.
The siRNAs of Snhg3, Stat1, Fn14 and negative control siRNAs were synthesized by Invitrogen Carlsbad (CA, USA), recombinant protein and neutralizing antibody of Tweak were purchased from GenePharma Co. (Shanghai, China). For the construction of overexpression vectors, full-length cDNA of Snhg3 and full-length CDS of Stat1 were inserted into the pcDNA3.1 empty vector. When the cell density reached about 70% confluence, the vectors or siRNAs were transfected into astrocytes by using the Lipofectamine ® 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).
Each group of cells was seeded in a 96-well plate and cultured at 37 °C, 5% CO2 for 12, 24, 48, and 72 hours, respectively. Then, CCK-8 solution (10 μL) was added at each time point, after which the cells were further cultured for 4 hours, and cell proliferation was expressed by the OD value at 450 nm.
The transfected cells were resuspended in 200 μL serum-free DMEM and seeded in a 24-well transwell upper chamber with a density of 5 x 104 cells per well. A total of 1 mL DMEM containing 10% FBS was added to the lower chamber. After incubation for 48 hours in an incubator at 37 °C, the cells in the upper chamber were removed, and the cells invading in the lower chamber were fixed with formaldehyde.
Tweak and Snhg3 interference in vivo
After induction of TLE for 30 days, the Tweak and Snhg3 siRNAs (1 mg/Kg) or NC siRNAs (1 mg/Kg) were injected into the right lateral ventricle (0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral to bregma) of mice once daily for 3 d .
Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from the fresh tissues or cultures by using TRIZOL (Thermo Fisher Scientific, Waltham, MA, USA), and reversely transcribed into cDNAs according to the manufacturer’s instructions. The RNA expression was detected by using a SYBR ExScript qPCR kit (Takara, Dalian, China) with the following conditions: 95 ℃ for 3 min, followed by denaturation at 94 ℃ for 15 s, annealing at 55 ℃ for 25 s and extension at 72 ℃ for 15 s for 35 cycles. Each test was performed three times independently. Relative quantification was calculated with the 2 -ΔΔCT formula. 18S RNA was used as internal control.
RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to isolate proteins from fresh tissues or cultures. The protein samples were separated by polyacrylamide gels and then electro-transferred onto polyvinylidene fluoride membranes (Millipore, Boston, MA, USA). Primary antibodies used in the study including anti-Snhg3 (1:400 dilution; Abcam, Cambridge, UK), anti-Tweak (1:400 dilution; Abcam, Cambridge, UK), anti-Stat1 (1:400 dilution; Abcam, Cambridge, UK), anti-p-Stat1 (1:400 dilution; Abcam, Cambridge, UK), anti-Fn14 (1:400 dilution; Abcam, Cambridge, UK), anti-β-Actin (1:400 dilution; Abcam, Cambridge, UK), anti-OX42 (1:400 dilution; Abcam, Cambridge, UK). After incubation with antibodies at 4 °C overnight, membranes were incubated with HRP-conjugated secondary antibodies at 37 °C for 1 h. Detect the protein abundances by a ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA, USA).
Enzyme linked immunosorbent assay (ELISA)
The secretions of IL-1β, IL-6, TNF-α in cerebrospinal fluid and cell culture supernatant were detected by ELISA kits (Sigma, St. Louis, MO, USA) following the manufacturer’s instructions.
Morris water maze test
The water maze test was based on previous studies . If the mice did not find the platform within 120 seconds, their escape latency was recorded as 120 seconds and placed them on the platform allowed to rest for 30 seconds. On the 30th day, the spatial probe test without the platform was evaluated and the number of times the mouse was placed through the platform in 120 seconds was recorded.
Prediction of binding manners between Snhg3 and Stat1
We analyzed the promoter of Snhg3 by CHIPBase (http://rna.sysu.edu.cn/chipbase/), predicted the binding sites of Stat1 and Snhg3.
Luciferase reporter gene assay
Inserting different segments cloned from Snhg3 into pGL3-basic vector (Promega, Madison, WI, USA) and the Stat1 sequence was connected upstream. These vectors were transfected into the mouse astrocytes after correct sequencing, and samples were collected 48 hours later. Luciferase activity was detected using the double luciferase reporter assay system.
Chromatin immunoprecipitation (ChIP) assay
We first cross-linked the cells with formaldehyde and then quenched reaction by glycine fixative, next lysed the cells, and disrupted them with sonication. ChIP-IT Expression Chromatin Immunoprecipitation Kits (Active motif, Carlsbad, CA, USA) was used in CHIP assay. Incubate with the above complex using the Stat1 antibody and normal serum IgG overnight at 4 ℃. Twenty percent chromatin that was not incubated with the antibody was used as the input. Finally, the complex with beads were eluted according to the instructions and semi-quantitative analysis was performed.
Data analysis was performed using SPSS version 22.0, and every experiment was repeated at least three times. All data were expressed as mean ± standard error of the mean (SEM). Statistical differences between the two groups were compared using Student's t test or two-way ANOVA, and the difference was considered significant, P < 0.05.