In total, 18 male Sprague-Dawley rats (mean weight, 180-220 g; age, 6-7 weeks) were supplied by the Laboratory Animal Center of Guangzhou University of Chinese Medicine. The rats were housed in an environment with a constant room humidity (50-70%), a temperature of 25±1°C and a 12-h light/dark cycle (lights on at 7:00 am). Water and food were available ad libitum. The experiments were approved by the Care and Use of Experimental Animals Committee of Guangzhou University of Chinese Medicine and performed according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. Each cage housed 5 rats to prevent any effects of social isolation.

The VD model was induced in rats by global cerebral ischemia using 2-VO. The rats used in the experiments were anesthetized by intraperitoneal injection (0.15 ml/100 g body weight) of 100 mg/kg ketamine and 10 mg/kg xylazine. The surgery separated the bilateral common carotid arteries, which were permanently ligated with small-diameter nylon sutures. Rats in the control group underwent the same procedure, but without ligation of the common carotid arteries. The body temperature of the rats was monitored throughout the procedure and maintained at 37°C. After surgery, the rats were placed back in their cages and normal feeding was resumed.

To determine the effect of miR-134-5p on cognitive function and synaptic changes in VD rats, the ICV injection method was used to administer the miR-134-5p antagomir (5′-CCCCUCUGGUCAACCAGU CAC A-3′; Guangzhou RiboBio Co., Ltd.). The animals were randomly divided into three groups (n=6/group): The control group, the VD model group and the miR-134-5p antagomir group (2-VO + miR-134-5p antagomir). Each group of rats were continuously injected via ICV for 3 days. The miR-134-5p antagomir was dissolved in sterile double-distilled water (ddH₂O) before use and a volume of 5 μl was injected (200 pmol/rat) into both sides of the lateral ventricle. Rats in the control and VD model groups were injected with the same volume of sterile ddH₂O. For the injection, the rats were anesthetized using an intraperitoneal injection (0.15 ml/100 g body weight) of 100 mg/kg ketamine and 10 mg/kg xylazine and placed on a stereotactic device with their backs on the panel. The antagomir was immediately injected into the lateral ventricle. According to the rat brain atlas, two small holes were drilled carefully in the skull bilaterally using a surgical drill. The stereotaxic coordinates of the ICV injection were as follows: -0.8 mm anterior, 1.5 mm lateral and -4.5 mm depth.

To evaluate spatial memory and learning, MWM tests were conducted 24 h after the last ICV injection. The apparatus consisted of a black circular pool (diameter, 180 cm) filled with water to a depth of 40 cm. The maze was divided into four quadrants. The temperature of the water was 23±2°C. A submerged circular escape platform (diameter, 20 cm), 1.5 cm below the water surface, was placed in a random quadrant 25-30 cm from the edge of the pool. The platform remained in the same position throughout the experiment. On days 1-5, the rats swam freely for 90 sec to find the platform. The rats were given 4 trials/day to train their spatial learning ability (n=6/group). If a rat failed to reach the platform, it was gently guided to the platform and placed on it for 20 sec. Each rat required a 15-min rest before the next trial. A spatial probe test was conducted on the 6th day, during which the hidden platform was removed. Rats were allowed to swim freely for 90 sec to determine their memory retention. After each trial, the rats were dried. The cleanliness of the water was maintained throughout the trails.

Rats from each group were randomly selected and anesthetized using an intraperitoneal injection (0.15 ml/100 g body weight) of 100 mg/kg ketamine and 10 mg/kg xylazine. The rats were then sacrificed using CO₂ asphyxiation with a fill rate of 20% of the chamber volume/min with CO₂. Death was confirmed using a combination of criteria, including lack of pulse, breathing, corneal reflex, response to a firm toe pinch and graying of the mucous membranes. The brains were quickly removed and stored at -80°C. Total RNA was extracted using TRIzol^® (Thermo Fisher Scientific, Inc.) from cultured PC12 cells or tissue on ice for 20 min, and then isolated using chloroform and isopropyl alcohol. The concentration and purity of the extracted RNA was determined using a UV spectrophotometer. RT was carried out using the Prime Script™ RT reagent kit (Takara Biotechnology, Co., Ltd.), according to the manufacturer's protocol. The thermocycling conditions were 37°C for 15 min and 85°C for 4 sec. A specific RT primer was used for miR-134-5p (5′-GTCGTATCCAGTGCAGGGTCCGAGTATTCGCACTGGATACGA-3′) and random primers were used for RT of U6. The qPCR was performed using a SYBR Premix EX TaqⅡ kit (Takara Biotechnology, Co., Ltd.). Relative quantification of gene expression was performed using CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories Inc.). The thermocy-cling conditions were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 55°C for 30 sec and 72°C for 1 min, with a final extension at 55°C for 5 sec. The following primers were synthesized by Sangon Biotech Co., Ltd. and used for qPCR of rno-miR-134-5p: Forward, 5′-CGCGTGTGACTGGTTGACCA-3′ and reverse, 5′-AGTGCAGGGTCCGAGGTATT-3′. The U6 qPCR primers were purchased from Sangon Biotech Co., Ltd. (cat. no. MQP-0101). Differences in gene expression were analyzed using the 2^−ΔΔCq method (35). U6 was used as an internal control for the detection of miRNA. Each experiment was repeated three times.

The target genes of Rattus norvegicus (rno)-miR-134-5p were predicted using bioinformatics tools, including TargetScan (version 7.2; www.targetscan.org/mmu_71), miRBase (version 22.1; www.mirbase.org) and miRWalk (version Nov/2018 Release; http://mirwalk.umm.uni-heidelberg.de). The predicted gene targets were screened using Venn diagrams and the DAVID Bioinformatics Resources 6.8 (david.ncifcrf.gov) for gene function analysis. The target genes of rno-miR-134-5p were verified using western blotting and dual-luciferase reporter gene analysis.

After 4 weeks of 2-VO and following the MWM test, rats in each group were randomly selected and anesthetized. Rats were immediately perfused transcardially with cold normal saline followed by ~100 ml of cold 4% para-formaldehyde to prefix the brain tissue. The brains were washed with cold saline and fixed in 4% paraformaldehyde at 4̊C overnight. Paraffin sections of 4 μm were used for H&E, TUNEL and immunohistochemical staining. Sections from the cortex and hippocampus were stained using an H&E staining kit (Beijing Solarbio Science & Technology). The sections were visualized using a light microscope (Leica dMI400; Leica Microsystems GmbH) and photographed. Each experiment was repeated three times.

TUNEL staining was used to identify apop-totic cells in the cortex, following the manufacturer's protocol (one-step TUNEL apoptosis assay kit; Beyotime Institute of Biotechnology). Nuclei were stained with DAPI (1:100; Beijing Solarbio Science & Technology) for 10 min at 37°C in a humidified atmosphere in the dark. Images of apoptotic cells were captured using a confocal laser scanning microscope (LSM 800; Carl Zeiss AG) at x200 magnification. Each experiment was repeated three times.

Immunohistochemical staining was performed using a Histostain^TM-Plus kit (Beijing Biosynthesis Biotech Co., Ltd.) according to the manufacturer's instructions. The dehydrated and transparent tissue block was placed in dissolved paraffin, and after the tissue block was completely immersed in paraffin, it was embedded and sliced. Paraffin sections were incubated with anti-Syn1 primary antibody (1:100; cat. no. ab8; Abcam) at 4°C overnight, and incubated with a biotinylated secondary antibody for 20 min at 37°C. The staining was examined and images were captured using a light microscope (Leica dMI400; Leica Microsystems GmbH) at x400 magnification. In total, five fields of view were randomly selected on each section to measure the expression level of Syn1. Each experiment was repeated three times.

Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) from cultured PC12 cells or tissue (pulverized frozen fresh cortex) on ice for 20 min. The supernatants were obtained by centrifugation at 12,000 x g at 4°C for 20 min. Protein concentrations were quantified using a bicinchoninic acid protein assay (Fdbio Science). Equal amounts of protein (20-50 μg/lane) were separated using 10% SDS-PAGE gels and transferred onto nitrocellulose membranes after electrophoresis. After blocking with 5% skimmed milk in TBS containing 0.01% Tween-20 (TBST) at room temperature for 2 h, the blots were incubated with anti-Foxp2 (1:1,000; cat. no. ab16046; Abcam), anti-Snap25 (1:1,000; cat no. A0986; ABclonal Biotech Co., Ltd.), anti-Syn1 (1:1,000; cat. no. ab8; Abcam) and anti-β-actin (1:5,000; cat. no. ab8227; Abcam) primary antibodies at 4°C overnight. Following washing with TBST, the blots were incubated with a secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1,000; cat. no HAF008, R&D Systems, Inc.) for 1 h at room temperature and washed with TBST. Protein bands were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Protein bands were analyzed using ImageJ software (version 1.4; National Institutes of Health). The expression of the target protein was calculated by comparing the gray value of each group with the corresponding internal reference gene. Each experiment was repeated three times.

PC12 rat pheochromocytoma cells are frequently used as a model in neuronal research (36). PC12 cells were purchased from Procell Life Science & Technology Co., Ltd. and were cultured at 37°C in a 5% CO₂ incubator in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.), 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% solution penicillin/streptomycin (Thermo Fisher Scientific, Inc.). The medium was replaced every 3 days. PC12 cells were plated into 6- or 24-well plates at a density of 9×10⁵ or 1.5×10⁵ cells/well, respectively.

PC12 cells were seeded in 24-well plates at a density of 1x10⁵ cells/well and were subsequently transfected with miR-134-5p mimic, miR-134-5p inhibitor, miR-134-5p mimic-NC or miR-134-5p inhibitor-NC (Guangzhou RiboBio Co., Ltd.), and co-transfected with pLUC-Foxp2-wild-type (WT) 3′-untranslated region (UTR) or pLUC-Foxp2-mutant (MUT) 3′-UTR plasmids (Shenzhen Huaan Ping Kang Bio Technology Co., Inc.) using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. In the pLUC-Foxp2 cloning vector, miRNA target sites, predicted using TargetScan software (version 7.2; http://www.targetscan.Org/vert_71/), were inserted after the Renilla luciferase region. The final concentration of miR-134-5p mimic and miRNA mimic negative control transfected into PC12 cells was 50 nM, and the final concentration of miR-134-5p inhibitor and miRNA inhibitor negative control was 100 nM; 200 ng plasmid was used for transfection. PC12 cells were lysed and the luciferase activity was quantified using the Dual-Luciferase^® Reporter assay kit (Promega Corporation), according to the manufacturer's protocol, 48 h after transfection. Each experiment was repeated three times.

PC12 cells were seeded into 6-well plates at a density of 9x10⁵ cells/well 1 day before transfection in order to reach a confluence of 60-80%. Transfection of miR-134-5p mimic (5′-UGUGACUGGUUGACCAGAGGGG-3′ and 5′-ACACUGACCAACUGGUCUCCCC-3′), miR-134-5p mimic-NC (cat. No miR1N0000001-1-5; sequence unavailable; Guangzhou RiboBio Co., Ltd.), miR-134-5p inhibitor (5′-CCCCUCUGGUCAACCAGUCACA-3′), miR-134-5p inhibitor-NC (cat. no miR2N0000001-1-5; sequence unavailable; Guangzhou RiboBio Co., Ltd.) or siRNA-Foxp2 (siR-Foxp2-001, AGCAGCAACAACTACAAG A; siR-Foxp2-002, CAAAGCTTCACCGCCAATA; siR-Foxp2-003, CGACATTCAGACAAATACA) was performed using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The final concentration of mimic, mimic-NC and siRNAs was 50 nM, and the final concentration of inhibitor and inhibitor-NC was 100 nM. Cells were lysed with RIPA buffer 48 h after transfection for western blot analysis. Each experiment was repeated three times.

Using ChIP assays, the molecular interaction between Foxp2 and the Syn1 promoter binding site was investigated. Nerve growth factor (NGF; 50 ng/ml) was used to induce PC12 cells for 1 week. According to the manufacturer's instructions (Magna ChIP^TM A; EMD Millipore), the culture medium was removed and 1% formaldehyde was added for crosslinking for 10 min at 37°C; 2 ml of 0.125 M glycine was added to quench unreacted formaldehyde. The cells were washed using ice-cold PBS twice and then collected and lysed in SDS Lysis Buffer (EMD Millipore). Chromatin was extracted from the nuclei. The chromatin was then sheared to 200-1,000 bp fragments by ultrasonication at 4°C for 40 min, and the effect of ultrasonic crushing was examined by agarose gel electrophoresis. The Foxp2 antibody (Abcam) was applied to immunoprecipitated DNA by using magnetic protein G beads. Following reverse crosslinking, ChIP-DNA complexes were extracted, washed and eluted using 100 μl ChIP Elution Buffer (EMD Millipore) with 1 μl protease K (EMD Millipore). DNA was purified by Spin Filter. The ChIP-DNA fragments were directly used in qPCR assays with specific primers for the promoter region of the Syn1. GAPDH was used as an internal control for the detection of mRNA. The primers used were as follows: Syn1-1: Forward, 5′-GGACCCCTAAGTTCCTTCCTCCA-3′ and reverse, 5′-AGACACAAACATTGGCAAAGGTGG-3′; Syn1-2: Forward, 5′-CTCCCAAATCCGCATGGGGT-3′ and reverse, 5′-GTCTCCTCTTGGCTTTGGGGATAGT-3′; Syn1-3: Forward, 5′-CTGAGGCAGTATCAGGGCACAG-3′ and reverse, 5′-CTGCCTTCTCAGCGCAGCC-3′; and GAPDH: Forward, 5′-GTCCATGCCATCACTGCCACT C-3′ and reverse, 5′-CGCCTGCTTCACCACCTTCTTG-3′. ChIP was analyzed by qPCR using the CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). Each experiment was repeated three times.

All data are presented as the mean ± standard deviation. Data were analyzed using Excel 2017 (Microsoft Corporation) and GraphPad Prism7 (version 7.0; GraphPad Software, Inc.). Comparisons between two groups were performed using a t-test. Comparison among multiple groups were analyzed using one-way ANOVA followed by Tukey's multiple comparisons test for post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

A rat model of dementia was produced using 2-VO for 4 weeks. In order to verify whether miR-134-5p could affect the spatial learning and memory ability of VD rats, MWM experiments were conducted after 3 days of lateral ventricular injection of the miR-134-5p antagomir (n=6/group). During MWM training, the escape latency of rats decreased significantly, indicating that the platform could be found with training. In consecutive trials, the VD rats were impaired while training to find the submerged escape platform, as indicated by longer escape latencies compared with the control group. The escape latency of the miR-134-5p antagomir group was shorter compared with that of the VD model group; this was also the case on the 5th day (Fig. 1A-C). In the MWM test, the number of rats crossing over the platform position, and the percentage of time in the target quadrant relative to the total escape latency time in the pool, was significantly decreased in VD model rats and significantly increased in the miR-134-5p antagomir group compared with the control group (Fig. 1D and E). Representative movement traces of the MWM test on the 6th day revealed that the number of VD model rats crossing the platform was significantly lower compared with that of rats in the control group. In addition, the number of miR-134-5p antagomir rats crossing the platform was significantly higher compared with in the VD model group (Fig. 1F). Using the MWM experiment, it was demonstrated that rats exhibited spatial learning impairment following 4 weeks of chronic ischemia. Behavioral tests also indicated that inhibiting miR-134-5p could relieve cognitive dysfunction, as well as enhance learning and memory ability, in VD rats.

To determine whether miR-134-5p is upregulated in the brains of VD rats, RT-qPCR analysis was performed (n=3/group). The results demonstrated that the expression of miR-134-5p in the cortical tissue of VD model rats was increased significantly compared with the control group (Fig. 1G). No significant differences were found in the hippocampus of VD model rats compared with the control group (Fig. 1H). These results revealed that miR-134-5p is implicated in chronic ischemia-induced VD. These results also indicated that miR-134-5p is differentially expressed in the cortex, but not in the hippocampus, of VD rats.

Histopathological changes in the cortex and the cornu ammonis 1 (CA1) region of the hippocampus were observed following staining with H&E at 4 weeks after 2-VO, and are shown in Fig. 2 (n=3/group). In the cortex of VD rats, neuronal cell loss, shrinkage, dark staining of neurons and marked vacuolar changes were observed in the cortex. Additionally, the space between neurons was enlarged. The nucleus was pyknotic and the structure was not clear. Inhibition of miR-134-5p attenuated chronic hypoperfusion-induced neuronal cell injury. The structure of the nucleus in the hippocampal CA1 region of VD rats was not sufficiently clear compared with the control group; however, no significant damage in hippocampal CA1 neurons was observed in VD rats. Neurons undergoing apoptosis were detected using TUNEL staining (n=3/group). As shown in Fig. 3A and B, there were more TUNEL-positive neurons in the cortex of VD rats compared with the hippocampal CA1 region. By contrast to VD rats, the miR-134-5p antagomir group had markedly fewer TUNEL-positive neurons. These results suggested that cortical injury was more prominent compared with that of the hippocampus in early VD rats following chronic ischemia for 4 weeks.

Bioinformatics analysis predicted that the miR-134-5p sequence GUCAGUG was associated with the Foxp2 gene sequence CAGUCAC (Fig. 4A). To assess the direct effect of miR-134-5p on Foxp2 gene expression, dual-luciferase reporters carrying the miR-134-5p target site in pLUC-Foxp2 were constructed (Fig. 4B). This analysis demonstrated that an miR-134-5p mimic significantly decreased the luciferase activity of Foxp2. By contrast, the miR-134-5p mimic did not reduce luciferase activity when the miR-134-5p seed sequence at the 3'UTR of Foxp2 was mutated (Fig. 4C). To determine whether the miRNA mimic and inhibitor transfections were successful, RT-qPCR analysis was performed (n=3/group). The results revealed that the expression of miR-134-5p was significantly increased in the mimic group compared with that in the mimic-NC group, and significantly reduced in the inhibitor group compared with that in the inhibitor-NC group (Fig. 4D). Western blot analysis identified Foxp2 as a potential target of miR-134-5p. The expression of Foxp2 was reduced in the mimic group compared with the mimic-NC group and increased in the inhibitor group compared with the inhibitor-NC group (Fig. 4E). These results indicated that miR-134-5p regulates Foxp2 directly.

Western blotting verified that the expression levels of the representative synaptic-associated proteins, Syn1 and Snap25, were significantly reduced in the cortex of VD model rats compared with the control group, and were markedly increased in the cortex of miR-134-5p antagomir rats compared with VD model rats (Fig. 5A). According to the results of immunohistochemical staining (n=3/group), the expression of Syn1 was significantly reduced in the cortex of VD rats compared with the control group. The miR-134-5p antagomir significantly enhanced Syn1 expression compared with the VD model group (Fig. 5B). To determine whether Foxp2 expression in the cortex was associated with synaptic reduction in the development of early VD and the targeting of miR-134-5p to Foxp2 in vivo, western blot analysis was performed to detect Foxp2 expression (n=3/group). The results indicated that the expression of Foxp2 in the cortex of VD rats was significantly decreased compared with that in the control group, and was significantly higher in the cortex of miR-134-5p antagomir rats compared with VD model rats (Fig. 5C). In order to further elucidate the effect of miR-134-5p on loss of synaptic proteins, miR-134-5p was transfected into PC12 cells. The results of the RT-qPCR analysis demonstrated that the miRNA mimic and inhibitor transfections were successful (n=3/group). The expression of miR-134-5p was significantly increased in the mimic group compared with the mimic-NC group, and significantly reduced in the inhibitor group compared with the inhibitor-NC group (Fig. 5D). Western blotting revealed that the expression levels of Snap25 and Syn1 were decreased in the miR-134-5p mimic group and increased in the miR-134-5p inhibitor group, compared with the respective miRNA NC groups (Fig. 5E). These results indicated concomitant synaptic protein loss in VD rats, and the downregulation of miR-134-5p relieved synaptic protein loss in the cortex of VD rats. In addition, these results indicated that miR-134-5p regulated the loss of synaptic proteins in vivo and in vitro, and the targeting of miR-134-5p to Foxp2 was further verified in vivo.

To investigate whether miR-134-5p regulates the expression of synaptic-associated proteins by targeting Foxp2, siR-Foxp2-001, siR-Foxp2-002 or siR-Foxp2-003 were transfected into PC12 cells to determine the effect of Foxp2 on the expression of Syn1 and Snap25. Western blot analysis revealed that siR-Foxp2-002 significantly suppressed the expression of Foxp2 at the protein level compared with siR-NC (Fig. 6A). The expression of synaptic-associated proteins, such as Syn1 and Snap25, was decreased in the siR-Foxp2-002 group compared with the siR-NC group (Fig. 6B). These data confirmed that the downregulation of Foxp2 significantly inhibited the expression of the synapse-associated proteins Syn1 and Snap25, thereby affecting the morphology and function of synapses. Due to the direct targeting effect of miR-134-5p on Foxp2, these results also indicated that miR-134-5p affects the expression of synaptic-associated proteins by regulating Foxp2, rather than by directly regulating synaptic-associated proteins.

To investigate whether Syn1 is a downstream target of Foxp2, ChIP assays were performed. The model group was PC12 cells induced with NGF (50 ng/ml) for 1 week. The control group received no treatment. Verified using an RT-qPCR assay, the level of DNA fragment in the model group was significantly increased compared with the control group (Fig. 6C). ChIP analysis revealed the binding of Foxp2 to the Syn1 promoter at −400/−600 bp upstream of the transcription start site in PC12 cells induced by NGF. These results indicated that Foxp2 regulates synaptic changes through its interaction with the Syn1 promoter.