All experiments were authorized by the ethics committee of the Zhengzhou Central hospital. All the experiments were performed according to the ethical standards in the 1964 Declaration of Helsinki and its later amendments and were reviewed and approved by the Zhengzhou Central Hospital.

Human brain tissues were obtained from Zhengzhou Central Hospital. Nine male and 21 female patients (mean age: 80.8 ± 7.2) were diagnosed with AD and included in the study. The control groups were 11 male and 19 female non-dementia patients with an average age of 79.7 ± 10.3. Autopsied and histopathologically confirmed AD and control frozen frontal cortex samples were obtained from Zhengzhou Central Hospital. Before the operation, the written informed consent was obtained by individual patient to use the excised brain tissue and approved by the Institutional Review Board of the Zhengzhou Central Hospital, which in accordance with the National Institutes of Health guidelines as well.

The APPswe/PS1dE9 (APP/PS1) transgenic AD model mice (6-7 months old) used in this study were obtained from the Chinese Academy of Medical Science. All mice were housed in a 12/12 h light/dark cycle with free access to food and water. This type of mice is bred in a C57BL/6J genetic background and over-expresses human amyloid precursor protein. Mice were divided into three groups, wild type (WT), APP/PS1, and APP/PS1 + miR-361-3p groups (n = 15 in each group, 45 mice in total). Before stereotaxic injection, mice were anesthetized by isoflurane. There were no signs of peritonitis observed in our study. Stereotaxic injection of Agomir (100 μM, 1.5 μL) was performed on both side’s hippocampus regions (anterior/posterior = ± 1.9, dorsal/ventral = ± 2.4, medial/lateral = ± 1.9) by a Hamilton micro-syringe at the rate of 0.2 μL/min. Behavioral tests were performed two weeks after the injection. After the behavioral test, all mice were sacrificed due to excessive anesthesia. Death was confirmed when no breath and heartbeat could be detected. The hippocampi were dissected immediately and stored at -80 ℃ for biochemical analyses.

Human neuroblastoma cell line SH-SY5Y and 293 cell lines were purchased from ATCC, (catalog number: CRL-2266). Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) in a 37 °C incubator with 5% CO_2, containing 10% fetal bovine serum.

For transfection, cells were cultured in 25 cm² flasks. When the cell density achieved 70% ~ 80%, cells were transfected with 3 μg pAPPswe, which was obtained from GenePharma (GenePharma, Suzhou, China) using 10 μL of Lipofectamine 2000 Reagent (Invitrogen, Waltham, MA, USA).

M-361-3p mimics, inhibitors, and agomir were purchased from GenePharma (GenePharma, Suzhou, China). The mimics and inhibitor were transfected with Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), with a concentration of 50 nM/well.

WT or seed-mutated (MUT) BACE1 3ʹ-UTR sequences were chemically synthesized (Sangon, Shanghai, China) according to the seed sequence of miR-361-3p. 293 cells were seeded into 96-well plate and transfected with 100 ng of WT BACE1 3ʹ-UTR or MUT BACE1 3ʹ-UTR, using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). After 72h, a dual-luciferase reporter assay system was used to detected the luciferase activity according to the manufacturer’s recommendations (Promega, WI, USA). Renilla luciferase activity was used to normalize the luciferase activity.

RNA was extracted from the human tissue, 293, and SH-SY5Y cells by TRIzol (Takara, Dalian, China). The purity and concentration were measured by a ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The reverse transcription of RNA was achieved using the Prime Script^™ RT Reagent Kit (Takara, Dalian, China). The reactions were carried out with SYBR® Green Master Mix (Takara). The primers were as followed: miR-361-3p, F: 5′-UCCCCCAGGUGUGAUUCUGAUUU-3′, R: 5′-GCAAATCAGAATCACACCTG-3′; human BACE1, F: 5′-AGGGCTTGCACCTGTAGGAC-3′, R: 5′-GCCTGAGTATGACGCCAGTA-3′; human β-actin, F: 5’-GGACTTCGAGCAAGAGATGG-3’, and R: 5’-AGGAAGGAAGGCTGGAAGA-3’. Cycling conditions were as follows: Predenaturation at 95 °C for 5 min, denaturation for 10 sec at 95 °C, and annealing for 30 sec at 60 °C, with 40 cycles. The 2^-ΔΔCt method was used to analyze the data.

Human tissues, SH-SY5Y cells, or mice hippocampi were lysed in RIPA buffer (Biyotime biotechnology, Shanghai, China). 5× Laemmli SDS sample buffer was added into the supernatant of centrifuged lysate. Samples were boiled at 95 °C for 5 min. The boiled samples were loaded onto 10% SDS-PAGE, and transferred onto nitrocellulose filter membranes (Pall Corp. USA). Membranes were blocked in 5% nonfat milk for 2 hours at room temperature, and after that, incubated with the primary antibodies (BACE1, APP-β, cleaved caspase 3, Bcl-2, Bax, Proteintech Group, USA) with 1 : 1000 at 4 °C overnight. After washing, HRP-conjugated secondary antibodies (Proteintech Group, USA) were used for a 2 hours incubation at room temperature. After washing, the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and a Kodak medical X-ray film (Denville Scientific Inc, USA) were used to detect the signals of the protein-antibody complexes. The protein was quantified by the Multi Gauge software V3.0 from Fuji Film (Santa Clara, CA, USA).

SH-SY5Y cells were seeded into 6-well plates one day before transfection. The cells were harvested 3 days after the treatment. After washing with phosphate-buffered saline, cell apoptosis was assessed by an Annexin V-APC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, United States). Cells were collected, centrifuged, and re-suspended in 500 μl of binding buffer at a density of 10⁶ cells/ml. After that, 100 μl suspended buffer was incubated with 5 μl 7-AAD and 5 μl Annexin V-FITC avoiding light for 15 min at room temperature. Flow cytometry was performed for the apoptosis analysis.

The Morris water maze test was performed two weeks after the injection. The water tank was filled with water (25 ± 1 °C), and a hidden platform and the tank had four quadrants. The mice were trained for five consecutive days, four times each day. Every mouse was released into the water by facing the wall in one of four quadrants. Each mouse was allowed 60 seconds at most to find the hidden platform. If the mouse failed to reach the platform, it would directed to the platform and stayed there for 15 seconds. After training, exploratory tests were performed. In the test, the platform was removed; each mouse was allowed 60 seconds for free swimming. The recorded data were analyzed by computerized software (Wilmette, IL, USA).

GraphPad Prism 7 was used in this study. All data were presented as the mean ± SEM. Each experiment was repeated for three times. Two-way ANOVA and Student’s t-test were used for analysis. P < 0.05 indicates a statistically significant difference.

The protein level of BACE1 and APP-β in the human AD frontal cortex tissues were found significantly increased as compared to the control (Fig. 1A).The mRNA expression of miR-361-3p was decreased (Fig. 1B) and BACE1 was increased (Fig. 1C) in the human AD frontal cortex tissues as compared to the control. The results showed negative correlation between the expression level of miR-361-3p and BACE1 (Fig. 1D).

Figure 1.

Down-regulated miR-361-3p was correlated with high-regulated BACE1 in AD patients’ brains. A. The protein level of BACE1 in AD patients’ brains (n = 30) and normal (n = 30); B, C. The mRNA levels of miR-361-3p and BACE1 in AD and normal were determined by RT-qPCR. D. The correlation between miR-361-3p and BACE1 level was analyzed. The error bar indicates the SEM. ***P < 0.001.

The target scan was used to screen potential target genes of miR-361-3p, and BACE1 was emphasized. To further study whether miR-361-3p directly target BACE1, we designed the 3ʹ-UTR sequence of BACE1 (WT) and mutated BACE1 3ʹ-UTR sequences (MUT) (Fig. 2). pGL3-control plasmid or pRL-TK plasmid were co-transfected with miR-361-3p mimics or NC in 293 cells, respectively. Luciferase activity was measured 48h after the transfection. The result showed that miR-361-3p mimics markedly reduced the activity of the WT plasmid, but not MUT plasmid. Thus, it is concluded that miR-361-3p directly target BACE1 (Fig. 2).

Figure 2.

BACE1 was a target of miR-361-3p. A Construction schematic of WT or mutant BACE1 3'-UTR vectors. B. Relative luciferase activity was analyzed in 293 cells. The error bars indicate SEM. ***P < 0.001.

SH-SY5Y/APPswe cells were successfully constructed. The results showed decreased expression of miR-361-3p (Fig. 3A), but the expression of BACE1 and APP-β were notably increased in SH-SY5Y/APPswe cell, confirmed by RT-PCR and Western Blotting (Fig. 3A, B). Cell apoptosis was significantly increased in SH-SY5Y/APPswe cell compared with SH-SY5Y cells (Fig. 3C). Compared with SH-SY5Y cells, the expression levels of apoptosis-related proteins caspase 3 and bax in SH-SY5Y/APPswe cells were significantly increased, while the expression of Bcl-2 was decreased (Fig. 3D). miR-361-3p mimics and inhibitor can successfully increase and inhibit its expression, which confirmed by RT-PCR (Fig. 3E). After SH-SY5Y/APPswe cells were transfected with miR-361-3p mimics, the expression of BACE1 and APP-β were decreased significantly. On the contrary, after SH-SY5Y/APPswe cell were transfected with miR-361-3p inhibitor, the expression of BACE1 and APP-β were increased significantly (Fig. 3F, G). These results indicated that miR-361-3p regulated the expression of BACE1 and the accumulation of APP-β in APPswe transfected SH-SY5Y cells.

Figure 3.

MiR-361-3p regulated the expression of BACE1 and the accumulation of APP-β in SH-SY5Y cells. A. The relative miR-361-3p, BACE1, and APP-β mRNA expression were determined by RT-PCR; B. The protein levels of BACE1 and APP-β were detected by western blot; C. The apoptotic rate of SH-SY5Y and SH-SY5Y/APPswe cells. D. The protein levels of cleaved caspase3, Bcl-2, and Bax; E. The expression of miR-361-3p in SH-SY5Y/APPswe cells transfected with mimics and inhibitor was confirmed by RT-PCR; F. The relative miR-361-3p, BACE1, and APP-β mRNA expression were determined by RT-PCR; G. The protein levels of BACE1 and APP-β were detected by western blot. The error bar indicates the SEM. **P < 0.01, ***P < 0.001.

After SH-SY5Y/ APPswe cells were transfected with miR-361-3p mimics, the cell apoptosis was notably decreased. MiR-361-3p inhibitor transfection showed the opposite result (Fig. 4A). The apoptosis related-proteins cleaved caspase 3 and Bax were markedly decreased, and Bcl-2 was increased in SH-SY5Y/APPswe cell. MiR-361-3p inhibitor transfection showed the opposite results (Fig. 4B).

Figure 4.

MiR-361-3p suppressed apoptosis of SH-SY5Y/APPswe cell. A. The apoptotic rate in SH-SY5Y cells; B. The protein levels of cleaved caspase3, Bcl-2, and Bax. The error bar indicates the SEM. **P < 0.01, ***P < 0.001.

The Morris water maze test was performed to assess the effect of miR-361-3p on mice spatial learning and memory. As shown in Fig. 5A, APP/PS1 mice showed increased latency and decreased the frequency of crossing platform locations compared to WT mice. Injection of miR-361-3p in APP/PS1 mice decreased the latency and increased the frequency of crossing platform location as compared to that in APP/PS1 mice. The body weight showed no significant difference between groups of mice. These results showed impaired spatial learning and memory in APP/PS1 mice, while injections of miR-361-3p restored these impairments. APP/PS1 mice were sacrificed, and their hippocampi were dissected. The expression of miR-361-3p was decreased in APP/PS1 mice. MiR-361-3p was successfully overexpressed in APP/PS1 mice, which was confirmed by RT-PCR (Fig. 5B). The protein level of BACE1 and APP-β was markedly increased in APP/PS1 mice, and this was reversed by injection of miR-361-3p into the mice’s hippocampi (Fig. 5C). These results indicated that miR-361-3p exerted effects on spatial learning and memory improvement in APP/PS1 mice and BACE1 was involved in this pathological process.

Figure 5.

Overexpress miR-361-3p improved cognition in APP/PS1 mice. A. The escape latency to the hidden platform during training, frequency of crossing platform location and body weight were analyzed; B. The mRNA level of miR-361-3p; C. The protein levels of BACE1 and APP-β were detected by western blot. The error bar indicates the SEM. *P < 0.05, **P < 0.01, ***P < 0.001.