A total of 36 healthy male Sprague Dawley rats (9–10 weeks-old, 220–240 g) were provided by the Animal Experimental Center of Guangxi Medical University (Nanning, China). Animals were housed in groups of five under the following conditions: 22–26°C room temperature, 50–60% humidity, under a 12 h light/dark cycle, with lights on at 8:00 a.m. The rats were given free access to food and water. Animals were handled according to the guidelines on animal experimentation of the Council for International Organizations of Medical Sciences (World Health Organization, Geneva, Switzerland), and the Guangxi Medical University Animal Care and Use Committee approved the animal protocols.

Epileptic rats were randomly divided into 2 groups of 12 rats, including the epilepsy group and the miR-34c agomir group. Additionally, 12 rats were used as the sham group, receiving treatment with an equal amount of saline solution administered into the abdominal cavity.

The rats were treated with 60 mg/kg PTZ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) by intraperitoneal (i.p.) injection. All of the rats were observed for 20 min following PTZ injection and behavioral alterations were recorded and graded according to the Racine grading system as follows: 0, No reaction; grade I, face and ears twitching; grade II, nodding, neck and back spasms; grade III, unilateral forelimb clonus; grade IV, bilateral forelimb clonus and standing; and grade V, systemic seizure and loss of balance. Rats with grade IV or V seizures were selected as successful epilepsy models. These rats were secured in stereotaxic apparatus (Shanghai Alcott Biotech Co., Ltd., Shanghai, China) following anesthetization with 10% chloral hydrate (3.5 ml/kg; i.p.), on the 2nd day following treatment with PTZ. The skin was cut along the middle of the head and the anterior fontanel was exposed. The right ventricle was positioned (AP=−0.9 mm, R=−1.3 mm, V=−3.5 mm) in the stereotaxic atlas according to Paxinos et al (14). A 10 µl micro syringe was fixed on the micro injection pump pointing to the hole of the right ventricle drilled on the skull with a syringe needle. The speed of injection was set at 3 min/µl. Rats in the miR-34c agomir group were injected with Rattus norvegicus-miR-34c agomir (cat. no. miR40004723-1-10, Guangzhou RiboBio Co., Ltd., Guangzhou, China), while rats in the epilepsy group were injected with an equivalent amount of scramble miR (cat. no. miR04101-1-10, Guangzhou RiboBio Co., Ltd.) into the right ventricle. The needle remained in place for 10 min subsequent to the injection. The rats received a repeated i.p. injection of 35 mg/kg PTZ every other 48 h from the 3rd day following surgery, with a total of 14 injections, to rekindle the seizures.

The Morris water maze test was used to evaluate the cognitive function of the rats. The Morris water maze included a round pool (diameter, 120 cm; height, 50 cm; depth, 25 cm). Additionally, there was a platform with a diameter of 10 cm, located 2 cm below the water level. The water in the maze was kept at a temperature of 22±1°C. A digital camera was connected to the computer monitor screen above the pool, and the data were acquired and processed using the SLY-WMS Morris water maze system (version 2.1, Panlab, S.L.U. Barcelona, Spain).

The place navigation test lasted for 5 days, and training occurred at 9:00 a.m. every day. The rats were placed into the water in each the four quadrants, sequentially in a clockwise manner. The time that the rats took to find the platform was observed and recorded (escape latency). If a rat failed to find the platform within 90 sec, they were guided to and kept on the platform for 10 sec and the escape latency was recorded as 90 sec.

The spatial probe test was used to evaluate the ability of the rats to remember spatial locations, subsequent to the rats having learned how to find the platform. On the 6th day, the platform was removed and the rats were observed for 90 sec in the water. The frequency with which the rat travelled to the original position of the platform (crossing time), and the time and distance percentages that they remained in the target quadrant, were recorded.

After 1 h finishing the final water maze test, the rats were anesthetized by injecting 10% chloral hydrate (300 mg/kg) intraperitoneally, and were perfused by normal saline intracardially. The hippocampal tissue was isolated on ice for PCR and western blotting. The expression of miR-34c was detected by RT-qPCR to verify the effect of the agomir. The miR-34c primers were designed and synthesized by Takara Biotechnology Co., Ltd. (cat. no. MQA174, Takara, Dalian, China). Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The concentration and purity of the RNA were measured using the Scientific NanoDrop Thermo 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). The amplification of miR-34c cDNA was performed using an Mir-X^™ miRNA First-Strand Synthesis kit (Takara Biotechnology Co., Ltd.). The reaction system was at a volume of 10 µl, and the cDNA was stored at −20°C. Using an Mir-X miRNA RT-qPCR SYBR kit (Takara, Biotechnology Co., Ltd.), the PCR was performed using the Light Cycler 480 Real-time PCR system (denaturation at 95°C for 10 sec; 40 cycles of amplification at 95°C for 5 sec and 60°C for 20 sec). U6 (forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse 5′-CGCTTCACGAATTTGCGTGTCAT-3′) was used as an internal control. The relative amount of miR-34c in the hippocampal tissue was analyzed using the relative quantification 2^−ΔΔCq method (15).

Potential target genes of miR-34c were predicted using MicroCosm (https://www.microcosm.com/), miRanda (http://34.236.212.39/microrna/home.do), and miRDB (http://www.mirdb.org/). The Gene Ontology (GO; www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome.jp/keg/) databases were used to obtain information regarding the functions of the predicted target genes. GO and KEGG analysis was performed on the predicted target genes of miR-34c to investigate the regulatory mechanism of the overexpressed miR-34c in response to cognitive impairment in epilepsy.

After perfusion with normal saline intracardially, the rats were sacrificed and brain tissue was removed, postfixed overnight in the same solution at 4°C, embedded in paraffin wax, and sectioned coronally at 3 µm for immunofluorescence staining. In brief, the slices were immune-labeled using primary antibody specific to glutamate receptor ionotropic, NMDA 2B (NR2B; 1:1,000; cat. no. 14544, Cell Signaling Technology, Inc., Danvers, MA, USA) diluted in PBS overnight at 4°C. Following washing with PBS three times, the slices were incubated with the secondary antibody (goat anti-rabbit immunoglobulin G; 1:1,000; cat. no. A8275, Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Fluorescence images were acquired using an Olympus fluorescence microscope (Olympus Corporation, Tokyo, Japan) and an image capture system.

Expression of NR2B protein in whole cell lysate, and phosphorylated (p)-NADPH-dependent diflavin oxidoreductase 1 (NR1) and p-glutamate receptor 1 (GluR1) protein at the postsynaptic density (PSD), were detected using western blotting. The total protein was extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China), and was detected using a bicinchoninic acid protein assay kit. Isolated protein harvested from 150 g fresh brain tissue was heat-denatured at 100°C for 5 min, subjected to electrophoresis on a 10% SDS-PAGE gel for 2.5 h and transferred to a 0.45 mm polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA) using the semi-dry transfer apparatus. The membranes were blocked in 5% skimmed milk for 1 h at 24°C and incubated on ice overnight with the following primary antibodies: Anti-NR2B (1:1,000), with anti-GAPDH (1:5,000; cat. no. 2118, Cell Signaling Technology, Inc.) as an internal control; anti-NR1 (1:1,000; cat. no. 5704, Cell Signaling Technology, Inc.), with anti-β-actin (1:5,000; cat. no. 8457, Cell Signaling Technology, Inc.) as an internal control; and anti-GluR1 (1:1,000; cat. No. 8850, Cell Signaling Technology, Inc.), with anti-β-actin (1:5,000) as an internal control. The membranes were incubated with an Alexa Fluor^® secondary anti-rabbit antibody (1:5,000; cat. no. 8889, Cell Signaling Technology, Inc.) for 1 h following washing at room temperature. The bands were scanned using the LI-COR Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA), and analyzed using LI-COR Odyssey software (version 3.0; LI-COR Biosciences).

Data are presented as the mean ± standard deviation. SPSS statistical software (version 16.0; SPSS, Inc., Chicago, IL, USA) was used to analyze the data and single factor analysis of variance followed by the Least-Significant Difference post-hoc test, which was performed to compare the groups. P<0.05 was considered to indicate a statistically significant difference.

The place navigation test may be used to analyze learning ability in animals. The results of the present study demonstrated that the escape latency of each group decreased as the number of training days increased. The miR-34c agomir group exhibited increased escape latencies at each time point compared with the sham and epilepsy groups (Fig. 1A). As presented in Fig. 1B, compared with the sham group, the escape latency on the 5th day was significantly increased in the epilepsy and miR-34c agomir groups (both P<0.05), indicating a decreased learning ability following epileptic seizures. Additionally, rats in the miR-34c agomir group exhibited an impairment in studying ability.

On the 6th day, the platform was removed to perform the spatial probe test. As presented in Fig. 1C, the epilepsy and miR-34c agomir groups exhibited significantly decreased crossing times compared with the sham group (both P<0.05); however, no significant differences were observed between the epilepsy and agomir groups. As presented in Fig. 1D, no differences were observed in mean speed among the three groups on the 6th day. The time and distance for which the rats remained in the target quadrant in the epilepsy and miR-34c agomir groups were decreased compared with the sham group (all P<0.05; Fig. 1E and F). In the miR-34c agomir group, the time and distance for which the rats remained in the target quadrant were significantly decreased compared with the epilepsy group (both P<0.05). The results of the present study demonstrated that treatment with the miR-34c agomir led to memory impairment. The track plot from the 6th day is presented in Fig. 1G.

As presented in Fig. 2, compared with the sham group, the miR-34c expression in the epilepsy group was significantly upregulated (P<0.05), demonstrating that miR-34c was increased following epilepsy induced by PTZ. In addition, the expression of miR-34c in the miR-34c agomir group was also significantly increased compared with the sham group (P<0.05).

Following KEGG pathway enrichment analysis, the predicted target genes of miR-34c were primarily enriched in six KEGG pathways, including LTP, dopaminergic synapse, calcium signaling pathway and amphetamine addiction. It is known that LTP serves an important role in cognitive function (Fig. 3).

As demonstrated by counting positive cells in the cortex, rats in the epilepsy and miR-34c agomir groups exhibited decreased NR2B expression compared with the sham group, and the number of NR2B-positive cells in miR-34c agomir group was decreased compared with the sham group (Fig. 4).

As presented in Fig. 5A, the expression of NR2B in the epilepsy group was significantly downregulated compared with the sham group (P<0.05), indicating that NR2B expression in the hippocampus was decreased following epilepsy. The expression of NR2B protein in the miR-34c agomir group was also decreased compared with the sham group (P<0.05), which demonstrated that the levels of NR2B in the hippocampus were consistent with those in the cortex.

Compared with the sham group, the levels of p-GluR1 and p-NR1 in the PSD of the hippocampus in the epilepsy group were significantly decreased (P<0.05). In addition, the expression of p-GluR1 and p-NR1 in the PSD of the hippocampus in the miR-34c agomir group were also decreased compared with the sham group (P<0.05) (Fig. 5B and C). The results of the western blot analysis are presented in Fig. 5D.