2.1 Animals and materials
A total of 20 adult male Sprague Dawley (SD; 250–280 g) rats were purchased from the Animal Centre of Guangzhou University of Chinese Medicine (Guangzhou, China). Adult male SD rats were randomly divided into sham-operated (control) and bilateral common carotid artery occlusion (2-VO) model groups. The experiments were approved by the care and use of experimental animals committee of Guangzhou University of Chinese Medicine. All rats were allowed to acclimatize for one week before surgery. The animals were conditioned to the same environment during the study. Food and water were available. Room humidity (50–70%) and temperature (21‑25°C) were maintained consistently, and the laboratory was maintained on a 12-h light/dark cycle.
2.2 2-VO model preparation
Under anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine administered via intraperitoneal injection), the rats were subjected to 2 h of 2-VO surgery using an intraluminal suture as described in a previous report (Soria et al., 2013). The rats were fixed and allowed to breathe spontaneously during the surgery. The common carotid arteries were exposed and separated from the vagus nerves carefully by making a neck midline incision. Both common carotid arteries were permanently tied off with two 3.0 sutures. Sham animals underwent the same surgical procedure except for the occlusion of the two carotids. Body temperature was monitored using a rectal probe and maintained between 36.5°C and 37.5°C until the animal recovered from anesthesia. The animals were allowed to regain full consciousness in a nursery cage, which was heated (~ 30°C) and oxygenated before returning to their normal cage for four weeks. After the Morris water maze test, the frontal lobe was removed for experiments.
2.3 Extraction of total RNA and real-time quantitative polymerase chain reaction (RT‐qPCR)
According to the manufacturer’s protocol, total RNA (600 ng) was extracted from the frontal lobes of the different groups using TRIzol reagent (Invitrogen; ThermoFisher Scientific, Inc., Waltham, MA, USA). The concentration and purity of RNA were determined using a spectrophotometer (Nanodrop 2000; Thermo Fisher Scientific, Inc., Wilmington, USA). The specific stem‑loop primer of miR-161 was used in the RT-PCR, and the U6 gene was used as the reference. The following primers were used: miR-161, RT primer 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTGCAAA-3', forward 5'-CGTCTGGAGGGCCTTGC-3', and reverse 5'-AGTGCAGGGTCCGAGGTATT-3'. U6 forward, 5'-CTCGCTTCGGCAGCACATAT-3' and reverse, 5'-TTGCGTGTCATCCTTGCG-3'. EGLN2, forward 5'-GCTTTGGTGGGCAGGATGGTG-3', and reverse 5'-GTCGCTGGCACTCCTTGGTAAC − 3', the β-actin gene was used as the reference. GPX4, forward 5'-GCTTTGGTGGGCAGGATGGTG-3', and reverse 5'-GTCGCTGGCACTCCTTGGTAAC − 3', the β-actin gene was used as the reference. Reverse transcription was performed using the PrimeScript RT reagent kit (Takara, Biotechnology, Co., Ltd., Dalian, China) according to the manufacturer's protocol, at 37°C for 15 min and 85°C for 30 sec. A Light Cycler 480 SYBR Green I Master (Roche Diagnostics, GmbH, Mannheim, Germany) was used with the following amplification process: denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec, and extension at 72°C for 1 min. PCR products were detected using the CFX96 RT-qPCR instrument (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each experiment was repeated three times, and the data were presented as the mean ± standard error of the mean (SEM).
2.4 Morris water maze test
Spatial learning and memory were tested after 24 h of 2-VO using a modification of the procedure described by Morris(Bromley-Brits K et al., 2011). A circular pool (150 cm in diameter, 60 cm in height) was filled to a depth of 30 cm with water at a temperature of 22 ± 1°C. The pool was divided evenly into four quadrants, NE, NW, SE, and SW. A platform (20 cm in diameter) was placed 1 cm below the pool surface midway between the center and rim of the pool in the NE quadrant throughout the entire experiment. On day one, the rat was placed into the SW quadrant of the pool, and the time taken by the rat to find the escape platform was recorded. If the rat failed within 200 sec, it was placed on the platform for 15 sec and removed from the pool. From day two, the rat was given four trials a day for five consecutive days between 9:00 and 17:00 with an intertrial interval of 15 min, during which the rat was subjected to the acquisition of spatial learning. Later, probe trials were run in the same way as the four trials on day five, during which the platform was removed from the pool, and the rats were allowed to swim freely. The capability of the rats to detect the memory of the platform was recorded. After each trial, the rats were taken out, dried, and put into a separate cage.
2.5 Dual-luciferase reporter system analysis
Bioinformatics software (TargetScan, www.targetscan.org; miRBase, www.mirbase.org) were used to predict the target gene of miR-161, and a dual-luciferase reporter system confirmed the targeting. First, psiCHECK-2-EGLN2-wild-type (WT) and psiCHECK-2-EGLN2-mutant (MUT) plasmids (cat. nos. G0172509 and G0172509-2; Shenzhen Huaan Ping Kang Bio Technology, Inc., Shenzhen, China) were constructed. Using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Wilmington, USA), PC12 cells [American Type culture collection, (ATCC), Manassas, VA, USA] were transfected with psiCHECK-2-EGLN2 cloning vectors (200 ng), miR-161 mimics (100 nM), or miR-161 negative control (NC; 100 nM) for 6 h in 24-well plates (5×104 cells per well). After 24 h of transfection, the fluorescent value of each well was quantified with the Dual‑Luciferase Reporter assay kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's protocol. In the psiCHECK-2-EGLN2 plasmid, the target site of Rattus norvegicus (rno)-miR-161 was inserted into the downstream sequence of Renilla luciferase (hRluc). Firefly luciferase was used as the internal reference and for normalization. PC12 cells transfected with psiCHECK-2-EGLN2 vector were used as the positive control. Cells transfected with miR-161 NC and psiCHECK-2-EGLN2 were regarded as the NC. Each experiment was repeated three times, and the data are presented as the mean ± SEM.
2.6 Transfection of miR-161 in PC12 cells
PC12 cells were grown in 6 well-plates (5×105 cells per well) and divided into control, miR-161 mimic, miR-161 inhibitor, siRNA-EGLN2, and NC groups. Each group was transfected with the respective miRNA or siRNA and then incubated with serum-free medium for 24 h, except the control group, which was incubated with RPMI-1640 medium containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Wilmington, USA). PC12 cells were digested with 0.25% Trypsin and resuspended in 500 µL of binding buffer.
2.7 Transfection of siRNA‑EGLN2 in PC12 cells
The siRNA-EGLN2 inserts, which specifically bound and degraded the EGLN2 mRNA, was purchased from Guangzhou RiboBio Co., Ltd. Guangzhou, China. Using Lipofectamine 2000, PC12 cells were transfected with 100 nM of siRNA-EGLN2 for 24h in 6-well plates (5×105 cells per well). After 24 h of transfection, RT-qPCR was performed to select the most efficient segment. The siRNA-EGLN2 sequences were: 5'-GCATGCGGTACTATGGTAT-3'.
2.8 Western blotting analysis
The frontal lobes and PC12 cells were collected. Cell debris was removed by centrifuging at 12,000g for 10 min at 4°C, and the supernatants were harvested for further evaluation and stored at ‑80°C. The bicinchoninic acid method was used to detect protein concentration. Total protein (30 µg) was separated by SDS-PAGE, using a 10% gel, and transferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). After blocking with 5% BSA for 2 h at room temperature, the membranes were incubated with primary antibodies (EGLN2, cat. no. ab71598, 1:1000; GPX4, cat. no. ab140595, 1:1000; β-actin, cat. no. ab8245, 1:5000; Abcam, Inc., Cambridge, UK) at 4℃ overnight. After rinsing with Tris-buffered saline with 0.01% Tween-20, the PVDF membranes were incubated with the secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit, cat. no. ab6721; HRP-conjugated goat anti-mouse, cat. no. ab6789; 1:5,000; Abcam, Inc., Cambridge, UK] for 2 h at room temperature. Enhanced chemiluminescence (ECL) western blotting substrate (Pierce; Thermo Fisher Scientific, Inc., Wilmington, USA) was applied to visualize the protein bands. Image acquisition was performed using a Tanon-6200 gel imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). Image processing software (ImageJ; Version 14.8; National Institutes of Health) was used to analyze the images. Each experiment was repeated three times, and the data are presented as the mean ± SEM.
2.9 Bioinformatics data
The data from the GEO database (GSE122063), which included a gene expression profile dataset of homo sapiens, was analysised by The R Programming Language. The basic information of GSE122063 data is as follows:Homo sapiens organism, each sample (VD = 8, Controls = 11) was run, at a minimum, in duplicate with a dye swap (Cy3/Cy5) on Agilent Human 8x60k v2 microarrays. EdgR (version 3.32.1) and limma (version: 3.46.0) packages in bioconductor were utilized to analyze GPX4 expression difference. Protein-protein interaction networks functional enrichment analysis software (STRING, https://cn.string-db.org/) was used to predict interrelationship of EGLN2 and GPX4.
2.10 Statistical Analysis
The experimental data were presented as the mean ± SEM. SPSS 22.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The figures were produced using GraphPad Prism (Version 6.0; GraphPad Software, Inc., La Jolla, CA, USA). The Student's t-test was used to analyze the difference between the two groups. The comparison between multiple groups was analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test as the post hoc test. P < 0.05 was considered to indicate a statistically significant difference. Each experiment was repeated three times.