A total of 60 samples of peripheral blood were collected from the median cubital veins of patients with AD and from normal age-matched individuals (>65 years old) Haikou People’s Hospital (Hainan, China). The patients with AD were aged between 65 and 86 years (male, n=17; female n=13), and the normal control individuals were aged between 65 and 74 years (male, n=19; female, n=11). Individuals with significant illness, including diabetes, heart disease, stroke or cancer, were excluded from the present study. All the samples were collected according to the legislation and ethical boards of Haikou People’s Hospital. Written informed consent was obtained from the patients. The samples were stored at −80°C until use.

Primary hippocampal neurons were obtained from the hippocampi of SAMR1 mice at embryonic day 15. Briefly, the hippocampi were mechanically dissociated and treated with 0.2% trypsin (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at 37°C in phosphate-buffered saline (PBS). The hippocampal cells were collected by centrifugation at 118 × g for 5 min, and then 2×10⁵ cells/ml were washed in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA), containing 10% fetal bovine serum (FBS; Invitrogen Life Technologies), and resuspended in DMEM supplemented with 10% FBS and 100 kU/l penicillin and streptomycin (Sigma-Aldrich). The cells were cultured in a humidified atmosphere of 95% air and 5% CO₂. Ectopic expression of miR-29c in cells was introduced by transfection with miR-29c mimics or inhibitors using Lipofectamine 2000 (Invitrogen Life Technologies).

The SAMR1 and SAMP8 mice were obtained from the Animal Center of Beijing University Medical Department (Beijing, China) and housed in standard conditions (12 h light/dark; 25±1°C; 50% humidity). Male 8-month-old SAMP8 (n=6) and SAMR1 (n=6) mice were used. The anesthetized (40 mg/kg 1% pentobarbital sodium; Sigma-Aldrich) SAMP8 mice were positioned in a stereotaxic apparatus (model 68016; RWD Life Science, Shenzhen, China), and 1.5 μl PBS, containing either 0.5 nmol miR-29c mimic or a scrambled control (Intvitrogen Life Technologies, Carlsbad, CA, USA), was injected for 8 min into the third ventricle. The control SAMR1 mice received an equal volume of the vehicle (PBS only). The present study was approved by the ethics committee of Southern Medical University (Guangzhou, China), in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (11).

The total RNA was extracted from the indicated tissues or cells using RNA Extraction reagent, according to the manufacturer’s instructions (CWBio, Beijing, China). The mRNA expression of BACE1 was detected using a SYBR green qPCR assay (CWBio). RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to reverse transcribe the RNA. The expression of β-actin was used as an endogenous control. The specific primers used were as follows: BACE1, forward 5′-AATTCGAAATGGCCCAAGCCCTGCCCT-3′ and reverse 5′-AGGGATCCGGGCCTCCTCACTTCAGCAG-3′, and β-actin, forward 5′-CATTAAGGAGAAGCTGTGCT-3′ and reverse 5′-GTTGAAGGTAGTTTCGTGGA-3′. A MiScript SYBR-Green PCR kit (Ribobio, Co., Ltd., Guangzhou, China) was used for qPCR to detect the expression levels of miR-29c. The specific primer sets for miRNA-29c and U6 were purchased from GeneCopoeia (Rockville, MD, USA). The expression of U6 was used as an endogenous control. A total of 2 μl cDNA (50 ng/μl) was used to analyze the expression levels on a CFX96 Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR conditions were as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec, and finally 72°C for 10 min. The data were then analyzed using the 2^−ΔΔCT method (12).

Mouse Aβ immunoassay kits (Invitrogen Life Technologies) were used to determine the levels of Aβ in the primary cultured hippocampal cells and the brain tissues of the mice, according to the manufacturer’s instructions. Briefly, the media in the cultured cells or the supernatants of the indicated tissues were used to measure the total protein quantities of each sample. The samples and the Aβ antibody were incubated overnight at 4°C, prior to incubation with horseradish peroxidase (HRP)-labeled anti-rabbit antibody for 30 min at room temperature. The wells were then developed using 100 μl tetramethylbenzidine reagent in the dark, and the absorbance was measured at 450 nm (Synergy™ Mx; BioTek Instrument, Inc., Winooski, VT, USA).

The total protein was extracted from the indicated cells or tissues using cold radioimmunoprecipitation lysis buffer (CWBio). A bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA) was used to determine the protein concentrations. The protein (60 μg) was subsequently separated on a 10% SDS-PAGE gel (Wuhan Boster Biological Technology, Ltd., Wuhan, China) and transferred onto a nitrocellulose membrane (Wuhan Boster Biological Technology, Ltd.). The membrane was blocked in 5% nonfat dried milk in PBS for 4 h, and was incubated with the following primary antibodies overnight at 4°C: Rabbit anti-protein kinase A (PKA; 1:1,00; cat. no. 4781; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-BACE (1:500; cat. no. 5606; Cell Signaling Technology, Inc.), rabbit anti-cAMP response element binding protein (CREB; 1:500; cat. no. 4820; Cell Signaling Technology, Inc.), rabbit anti-phosphorlyated (p) CREB (1:500; cat. no. 9198; Cell Signaling Technology, Inc.) and mouse anti-β-actin (1:3,000; cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.). The membranes were washed with Tris-buffered saline containing 0.1% Tween and incubated with HRP-labeled goat anti-rabbit (cat. no. A12004-1; 1:2,000) and goat anti-mouse (cat. no. A12003-1; 1:3,000) secondary antibodies (Epigentek, Farmingdale, NY, USA) for 2 h at room temperature. Enhanced chemiluminescence reagent (Wuhan Boster Biological Technology, Ltd.) was used to detect the signal on the membrane. The expression data were analyzed by densitometry using Image-Pro plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and normalized against that of the internal control.

Wild-type (wt) and mutant (mut) 3′-UTRs of BACE1 were constructed, which were inserted into the dual luciferase reporter vector (Promega Corporation, Madison, WI, USA). Briefly, to generate the wt-BACE1-3′-UTR, the 3′-UTR was amplified and cloned into the XbaI (Thermo Fisher Scientific) site of the pGL3-control vector (Promega Corporation), downstream of the luciferase gene. The mut-BACE1-3′-UTR was generated from wt-BACE1-3′-UTR by site-directed mutagenesis, by Genecopeoia (Guangzhou, China). For the luciferase assay, 100,000 cells were cultured to ~70% confluence in 24-well plates. Following culture, the cells were co-transfected with the miR-29c mimic and either the wt or mut 3′-UTR of the BACE1 dual luciferase reporter vector. Following incubation for 5 h with the transfection reagent/DNA complex, the medium was replaced with fresh medium, containing 10% FBS. At 48 h post-transfection, a Dual Luciferase Reporter Gene Assay kit (BioVision, Milpitas, CA, USA) was used to determine the luciferase activities in each group on a luminometer (Elecsys 2010; Roche Diagnostics, Basel, Switzerland). The activity of Renilla luciferase was normalized against that of firefly luciferase.

For all the groups, 5,000-6,000 cells/well were seeded into a 96-well plate. Following treatment, the plates were incubated for 0, 12, 24, 48 or 72 h at 37°C with 5% CO₂. To assess cell proliferation, an MTT assay was performed, according to the manufacturer’s instructions. MTT reagent (20 μl; 5 mg/ml; Wuhan Boster Biological Technology, Ltd.) in 200 μl FBS-free medium was added to each well and incubated for 4 h at 37°C. The medium was then removed and 100 μl dimethyl sulfoxide (Wuhan Boster Biological Technology, Ltd.) was added. The absorbance was detected at 490 nm using a microplate reader (Elecsys 2010). The assay was repeated three times in triplicate wells.

The Y-maze apparatus (model RD1102-YM-M; Mobiledatum Co., Ltd., Shanghai, China) was constructed from wood, painted in black, and had three arms at 120° angles. Each arm was 50 cm long, 15 cm high, 5 cm wide. The mice were initially placed at the end of one arm and allowed to move freely for 10 min. The series of arm entries was recorded using a video camera (model HDR-PJ790E; Sony, Tokyo, Japan). Spontaneous alternation was defined as: Successive entries into the three arms in overlapping triplet sets. The alternation percentages were determined as the ratios of actual alternations to maximum alternations, multiplied by 100. The percentages of alternation behaviors were recorded by an observer, in a blinded manner, followed by statistical analysis.

Statistical analyses were performed using Graphpad Prism 5 software (Graphpad Software, Inc., San Diego, CA, USA) and the data are expressed as the mean ± standard deviation. An unpaired two-tailed Student’s t-test was used to analyze the differences between the samples. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-29 were detected using SYBR green qPCR analysis. In the sample of 30 patients with AD and 30 normal individuals, no significant differences were observed in the expression levels of miR-29a and miR-29b (Fig. 1A and B), however, the results demonstrated that miR-29c was significantly decreased in 27 (90%) of the peripheral blood samples from the patients with AD, compared with the paired normal blood samples (Fig. 1C). However. In addition, an ELISA was used to measure the protein expression levels of BACE1. As shown in Fig. 1D, the protein expression levels of BACE1 were significantly increased in the peripheral blood from patients with AD, compared with the normal control individuals. In addition, the expression of miR-29c was negatively correlated with the protein expression of BACE1 in the peripheral blood of the patients with AD (Fig. 1E).

To investigate whether miR-29c targets the 3′UTR of BACE1, the present study cloned the 3′UTR of BACE1 downstream to a luciferase reporter gene (wt-BACE1). The mutant version (mut-BACE1), with binding site mutagenesis, was also constructed. To determine whether miR-29c regulated the expression of BACE1 at the transcriptional or translational level, the wt-BACE1 vector and either the pre-miR-29c mimic or scramble control (anti-miR-29c)were co-transfected into primary cultured hippocampal cells. As shown in Fig. 2A, the transfection efficiency was satisfactory for further investigation. The luciferase activity of the pre-miR-29c transfected cells was significantly reduced, compared with the scramble control cells (Fig. 2B). Additionally, The results demonstrated that the overexpression of miR-29c significantly reduced the mRNA and protein expression levels of BACE1, whereas downregulation of miR-29c increased the mRNA and protein expression levels of BACE1 (Fig. 2C). In addition, the protein expression of Aβ in the culture medium was determined. The protein expression of Aβ was decreased by the upregulation of miR-29c and increased by downregulation of miR-29c (Fig. 2D). These results suggested that miR-29c regulated the expression of BACE1 (Fig. 2E) at the transcriptional level by directly targeting its 3′UTR.

To determine whether miR-29c regulated hippocampal neuron proliferation, an MTT assay was performed by transfecting either pre-miR-29c or anti-miR-29c into the primary cultured hippocampal cells. Pre-miR-29c transfection exhibited a significant promotion of cell proliferation, compared with the control cells, whereas transfection with anti-miR-29c led to a significant inhibition on cell proliferation (Fig. 3A). To further investigate the molecular mechanism underlying miR-29c induction on cell growth, the expression levels of molecules associated with PKA signaling were detected. As shown in Fig. 3B, the protein expression of PKA was increased by the upregulation of miR-29c and decreased by the downregulation of miR-29c. Additionally, the expression of pCREB was induced significantly following transfection with pre-miR-29c, and was reduced following transfection with anti-miR-29c (Fig. 3C). These results suggested that miR-29c promoted hippocampal neuron cell proliferation by activating PKA signaling.

The present study subsequently examined the effects of miR-29c in vivo, by injecting miR-29c mimics into the hippocampi of SAMP8 mice. The expression levels of miR-29c in the SAMP8 mice were significantly lower compared with those in the SAMR1 mice. When the miR-29c mimic was injected into the hippocampi of SAMP8 mice, the expression of miR-29c was significantly increased compared with the vehicle-injected control mice (Fig. 4A). Similar to the effects in vitro, the upregulation of miR-29c significantly decreased the protein expression levels of BACE1 and Aβ in vivo (Fig. 4B and C). The present study also examined the learning and memory behaviors of the SAMP8 mice 2 months following transfection with miR-29c. As shown in Fig. 4D, overexpression of miR-29c in the hippocampus promoted the learning and memory behaviors of the SAMP8 mice. Furthermore, PAK signaling was also examined, and the protein expression levels of PKA and pCREB were induced significantly by the upregulation of miR-29c (Fig. 4E and F).