Ethics statement
All the animal related experiments were permitted by the Ethics Committee of Liaocheng People’s Hospital and accomplished according to the Chinese Animal Welfare Act. Efforts were made to lessen the number of animals used and their aching.
Pilocarpine-induced epilepsy in rats
A total of 30 Sprague-Dawley rats (21 d, 80 ± 20 g) were purchased from the Charles River (Beijing, China). All rats were housed in a suitable environment on a 12-h light/dark cycle and allowed to drink and eat freely. After 5 weeks of feeding, the rats were prepared for further experiment. The rats were assigned into control group (n = 3) and epilepsy group (n = 27). To establish the epilepsy model, lithium chloride (125 mg/kg; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was intraperitoneally injected 16 h prior to atropine administration (1 mg/kg, intraperitoneal injection; Wuhan Boster Biological Technology Co., Ltd., Hubei, China). After that, pilocarpine (15 ng/kg; Dalian Meilun Biotechnology Co., Ltd., Liaoning, China) was intraperitoneally injected into rats to induce status epilepticus (SE). After 30 min, seizures were terminated by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg). Subsequently, the rat behavior was observed after pilocarpine injection. On the basis of the Racine's scale, the rats identified with stage IV or above were as successful models and selected for subsequent experiments.
Cell culture and treatment
Both human embryonic kidney 293T (HEK293T) cells and rat astrocytes CTX-TNA2 and DI TNC1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) [15], which were seeded in Dulbecco’s modified Eagles Medium containing 10% fetal bovine serum (FBS), 4 mM glutamine, 100 µg/mL streptomycin, 100 U/mL penicillin and 4.5 g/L glucose under an atmosphere of 5% CO2. CTX-TNA2 and DI TNC1 cells with stable growth were inoculated into 24 well plates and treated with interleukin (IL)-1β (10 ng/h) for 24 h for subsequent experiments. pCDNA3.1(+) plasmid was purchased from Kelei Bio. (Shanghai, China). NEAT1 mimic, miR-139 mimic and si-ROCK1were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The plasmid transfection was performed with the Lipofectamine 3000 transfection Kit (Invitrogen Inc., Carlsbad, CA, USA), and the results were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR).
Animal grouping and hippocampal tissues
Three rats without pilocarpine and atropine treatment were as normal group. Twenty seven epileptic rats were randomly divided into nine groups, three in each group: epilepsy group (epileptic rats without transfection), NEAT1 mimic group (epileptic rats transfected with NEAT1 mimic), NEAT1 control group (epileptic rats transfected with NEAT1 control), miR-139 mimic group (epileptic rats transfected with miR-139 mimic), miR-139 control group (epileptic rats transfected with miR-139 control), NMM group (epileptic rats transfected with NEAT1 mimic and miR-139 mimic), NM group (epileptic rats transfected with NEAT1 mimic), si-ROCK1 group (epileptic rats transfected with si-ROCK1), and ROCK1-NC group (epileptic rats transfected with ROCK1-NC). Epileptic rats were treated with intracerebroventricular injection and anesthetized with 1% pentobarbital sodium (40 mg/kg) intraperitoneally. The mouse head was fixed on the stereotaxic instrument, cut a 2-cm incision along the midline of the head to expose the "cross" bone structure of the anterior fontanelle. According to the rat stereoscopic map of Paxinos Watson’s, the skull was drilled 1 mm behind the anterior fontanelle and 1.5 mm beside it. The microinjector was inserted vertically along the drill hole into the dura mater for 4.0 mm to inject RNA segment slowly. Then, the hole was sealed with dental cement, the incision was sewed with surgical suture, and the rat was fed in the cage regularly.
Hematoxylin-eosin (HE) staining
The rats were injected with 1% pentobarbital sodium (40 mg/kg) intraperitoneally for anesthesia. The rats were fixed in the supine position to open the chest and expose the heart. The syringe was inserted into the ascending aorta through the left ventricular. The right atrial appendage of the rat was cut open to infuse 4% paraformaldehyde into the heart until the heart turned white. Following the removal of all blood, the rats stopped autonomous breathing and heartbeat. After the confirmation of rat death, the cranium was opened to extract the brain immediately [7]. The brains were fixed into Bouin's fixative solution for 24 h, dehydrated by gradient alcohol, put into dimethylbenzene, and embedded in paraffin. The hippocampal tissues were cut into the 5-µm thick sections. The sections were then put into dimethylbenzene to dewax for 5 min, soaked in gradient concentration alcohol. After rinsing, the sections were put into hematoxylin dye solution (Leagene Bio., Anhui, China) for 12 min, immersed in 1% hydrochloric acid alcohol for 10 s, then transferred to eosin solution (Leagene Bio., Anhui, China) for 4 min. After that, the sections were dehydrated and cleared again, sealed with gum and observed under a microscope.
Microarray analysis
LncRNA microarray analysis was performed by Affymetrix (Santa Clara, CA, USA). Briefly, lncRNA was separately extracted using the TRIzol Reagent (Invitrogen Inc., Carlsbad, CA, USA) and purified with Rat LncRNA Array V3.0 (Arraystar). LncRNA was then labeled with Cy3 (000213, Thermo Fisher Scientific Inc., Waltham, MA, USA) and hybridized at 48 °C, 0.644 g to the gene chip. After washing and staining, the slides were scanned with the GeneChipTM Scanner 3000 7G Software for 16 h (Thermo Fisher Scientific Inc., Waltham, MA, USA). Data analysis was conducted using Expression Console Software (version 1.3.1), including data normalization, aggregation, and quality control assessment. Robust multichip analysis was used to correct and normalize the original data. The differential expression of lncRNA was identified according to t-test analysis, P < 0.05 and |fold change| > 1.5 was defined as differential lncRNA, which was clustered by levels and plotted into heat map.
RNA isolation and quantitation
Total RNA from hippocampal tissues was isolated using TRIzol Reagent (Invitrogen Inc., Carlsbad, CA, USA). Reverse transcription was performed with PrimeScript™ reagent Kit (Takara Holdings Inc., Kyoto, Japan) for equal amounts of RNA to cDNA. The quantitative PCR was performed using the TaqMan™ Fast Advanced Master Mix on an QuantStudio 3 and 5 Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA) at the conditions of 95℃ for 10 min, followed by 40 cycles at 95℃ for 15 s and 55℃ for 20 s, followed by 72 °C for 30 s. Each sample was determined in triplicate. The relative expression was calculated with 2−ΔΔCt method. The primers are presented in Supplementary Table 1.
5-ethynyl-2′-deoxyuridine (EdU) assay
Cell-Light EdU Apollo643 In Vitro Kit (100T, Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China) was used to conduct EdU assay. Cells of logarithmic growth stage were inoculated into a 96-well plate (1 × 104 cells/well) with 100 µL, 50 µM EdU culture medium for 2 h. After washed by phosphate buffer saline (PBS) for 5 min, the cells were incubated with 50 µL PBS containing 4% paraformaldehyde for 30 min. After that, the cells were incubated with the corresponding staining solution in a shaker for 5 min, and added with 100 µL penetrant (PBS containing 0.5% TritonX-100) for another 10-min incubation. Immediately after staining, the cells were observed under the fluorescence inverted microscope (Cx31, Olympus Optical Co., Ltd, Tokyo, Japan). Five visual fields were selected to take photos, and all the experiments are carried out three times independently.
Flow cytometry
A total of 1 × 106 cells were centrifuged for precipitate and suspended with 0.3 mL PBS containing 10% calf serum. Then, the fluid was transferred into a 1.5-mL eppendorf tube and fixed with 0.7 mL absolute ethanol at -20℃ for more than 24 h. After that, the fluid was concentrated at 1610 g for 30 s with the supernatant removed. The cells were resuspended by 1 mL PBS with the supernatant removed again. The precipitated cells were suspended by 100 µL 1 mg/mL RNase A, and incubated with 400 µL 50 µg/mL PI devoid of light for 10 min. The cell apoptosis was detected using the flow cytometer CytoFLEX (BECKMAN COULTER, CA, USA).
Enzyme-linked immunosorbent assay (ELISA)
After 48 h of transfection, the cells were inoculated in a 24-well plate (1 × 106 cells/well) for 24 h. The supernatant of cell culture medium was collected and centrifuged at 4℃ for 1 min at 1800 g. Detection of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β expression in supernatant was conducted using the rat ELISA Kits (ab100772, ab100785, ab100768, Abcam). The specific antibody globulin was diluted to 10 ug /mL, which was added to the plate (0.3 mL/well) and incubated at 4℃ overnight. The cells were incubated with 0.2 mL diluted plasma at 37℃ for 1 h, 0.2 mL diluted enzyme labeled antibody solution at 37℃ for 1 h and 0.2 mL substrate at room temperature for 30 min. Finally, each well was added with 0.05 mL H2SO4 to terminate the reaction. The optical density (OD) value was determined with the enzyme marker (OPD = 492 nm).
Dual luciferase reporter gene assay
The 3’UTR sequences of NEAT1 binding to miR-139 and miR-139 binding to ROCK1 were predicted by online prediction software Starbase (http://starbase.sysu.edu.cn/). The 3’UTR of wild type (WT) and mutant (MUT) of NEAT1/miR-139 and miR-139/ROCK1 were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China) and inserted into pMIR-REPORTTM (Thermo Fisher Scientific Inc., Waltham, MA, USA) Luciferase Report vector [16]. Using the Lipofectamine 3000 transfection Kit (Invitrogen Inc., Carlsbad, CA, USA), WT and MUT plasmids were co-transfected with NEAT1/miR-139 and miR-139/ROCK1 into cells, respectively. After 24 h, the cells were lysed. Luciferase activity intensity (intensity = RLU1/RLU2, RLU1 is the luciferase activity of firefly, RLU1 is the luciferase activity of renilla) was detected by dual luciferase reporter assay system (Promega Corporation, WI, USA) [16].
Western blot analysis
The hippocampal tissues were homogenized and then lysed (RIPA lysis buffer, pullair Gene Technology Co., Ltd.). After 30 min, the lysate was transferred to a 1.5-mL centrifuge tube with a pipette, and concentrated at 4℃ at 25764 g for 5 min. The supernatant was treated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and the gel obtained was transferred to the polyvinylidene fluoride membrane (EMD Millipore, Thermo Fisher Scientific Inc., Waltham, MA, USA). The membranes were blocked with 5% skim milk at room temperature and then incubated with primary antibodies against RhoA-GTP (1:1000, ab41435, Abcam), RhoA (1:5000, ab187027, Abcam), ROCK1 (1:3000, ab45171, Abcam), and β-actin (1:200, ab115777, Abcam) for 16 h. Subsequently, the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam). The intensities of the protein bands were quantified with ImageJ software 1.8.0 (National Institute of Health, Md., USA).
Statistical analysis
All statistical analyses were performed using SPSS 21.0 (IBM Corp. Armonk, NY, USA). Data were in normal distribution according to Kolmogorov-Smirnov method and described as mean ± standard deviation. Differences among multiple groups were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA. Tukey’s multiple comparisons test was used for the pairwise comparison after ANOVA analysis. p was obtained by two-tailed test and p < 0.05 was considered statistically significant.