PC12 and SK-N-SH cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China. PC12 cells were maintained in RPMI-1640 (31800-014; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; SH30084.03; Hyclone; GE Healthcare Life Sciences; Logan, UT, USA) 100 U/ml penicillin and 100 U/ml streptomycin. SK-N-SH cells were maintained in minimum essential medium (MEM, 41500034, Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. The two cell lines were cultured at 37°C under a humidified atmosphere of 5% CO₂. Aβ1–40 (GL Biochem, Ltd., Shanghai, China) was dissolved in sterile PBS and incubated for 5 days at 37°C to induce aggregation prior to treatment. 4×10³ cells/well PC12 and SK-N-SH cells were seeded in 6-well plates and then treated with 500 nM Aβ1–40 for 7 days at 37°C to establish the AD model.

The let-7a mimic and scrambled negative control miRNA (miR-NC) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences were as follows: let-7a mimic, 5′-UGAGGUAGUAGGUUGUAUAGUU-3′; miR-NC 5′-UUCUCCGAACGUGUCACGUTT-3′. Aβ1-40-treated PC12 and SK-N-SH cells were transfected with 100 pmol let-7a mimic or miR-NC using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. The cells were divided into four groups: Control, Aβ1-40, Aβ1–40 + let-7a mimic and Aβ1–40 + miR NC. Cells were collected for various assays at 24 or 48 h after the transfection.

At 24 h after the transfection, total RNA from PC12 cells and SK-N-SH cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. For mRNA analysis, cDNA was synthesized using a PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, Inc., Otsu, Japan) followed according to the manufacturer's protocol. For miRNA analysis, cDNA was synthesized using an miScript Reverse Transcription kit (Qiagen GmbH, Hilden, Germany) followed according to the manufacturer's protocol. Then, qPCR was performed using a SYBR Premix Ex Taq II kit (Takara Bio, Inc.) in a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions consisted of denaturation at 95°C for 10 min, 95°C for 10 sec, annealing at 60°C for 20 sec, extension at 72°C for 30 sec for 40 cycles. The primer sequences used are shown in Table I. In three repeated experiments, the mRNA and miRNA expression levels were normalized to internal controls β-actin and U6 using the 2^−∆∆Cq method (21), respectively.

An MTT assay was performed to evaluate cell viability at 48 h after the transfection. Briefly, 3×10³ PC12 and SK-N-SH cells/well were seeded in 96-well plates and exposed to the aforementioned treatments. Subsequently, 20 µl MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a concentration of 5 mg/ml was added into each well. Following incubation for 4 h at 37°C, the medium was discarded and 200 µl DMSO was added into each well. The formazan crystalline product was dissolved and optical density was measured at 490 nm using a microplate reader.

At 48 h after the transfection, Hoechst 33342 staining was performed to evaluate the morphological changes of apoptotic nuclei. 2×10⁶ PC12 and SK-N-SH cells/well were seeded into 6-well plates and subjected to the aforementioned treatments. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Subsequently, the cells were washed with PBS and incubated with 10 µg/ml Hoechst 33342 for 5 min at 37°C. Following washing with PBS three times for 3 min each, the cells were observed under a fluorescence microscope at a magnification of ×400. The apoptotic nuclei appeared condensed or fragmented compared with normal nuclei.

At 48 h after the transfection, the percentage of apoptosis in PC12 cells was quantified using the Annexin V-Fluorescein Isothiocyanate (FITC)/Propidium Iodide (PI) Apoptosis Detection kit (KGA106; KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's protocol. Briefly, 2×10⁶ cells from different groups were washed with PBS, stained with Annexin V-FITC and PI for 15 min in the dark at room temperature. Then samples (10,000 cells/sample) were analyzed using a flow cytometer (BD Accuri C6; BD Biosciences, San Jose, CA, USA).

At 48 h after the transfection, western blot assay was performed. The following primary antibodies were used: Anti-phosphoinositide 3-kinase (PI3K) (1:500; WL02240; Wanleibio, Co., Ltd., Shenyang, China), anti-Akt (1:500; WL0003; Wanleibio, Co., Ltd.), anti-phosphorylated (p)-Akt (1:500; WLP001; Wanleibio, Co., Ltd.), anti-mammalian target of rapamycin (mTOR; 1:200; sc-101738; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-mTOR (sc-8319; 1:200; Santa Cruz Biotechnology, Inc.), anti-microtubule-associated protein 1A/1B-light chain 3 (LC3; 1:500; WL01506; Wanleibio, Co., Ltd.), anti-beclin-1 (1:500; WL02237; Wanleibio, Co., Ltd.), anti-p62 (1:500; WL02385; Wanleibio, Co., Ltd.), anti-β-actin (1:500; WL01845; Wanleibio, Co., Ltd.). Briefly, cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentration was determined using the BCA method. Proteins from each sample (20 µg) were separated by 8, 10 or 13% SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% dried skimmed milk for 1 h at room temperature and incubated with primary antibodies at 4°C overnight. Subsequently, IgG conjugated goat anti-rabbit (1:5,000; WLA023; Wanleibio Co., Ltd.) or IgG conjugated goat anti-mouse (1:5,000; WLA024; Wanleibio Co., Ltd.) secondary antibodies were added for 30 min at room temperature. Blots were developed using enhanced chemiluminescence detection reagent (Beyotime Institute of Biotechnology). Scanning densitometry using a Gel-Pro Analyzer 4 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to assess the level of each target protein relative to β-actin in three repeated experiments.

Briefly, at 48 h after the transfection, 2×10⁶ PC12 cells from different treatment groups were plated to slides and fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS three times for 5 min each, the cells were permeabilized with 0.1% Triton X-100 for 30 min at room temperature, followed by blocking with 10% normal goat serum (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) for 15 min at room temperature. Cells were subsequently incubated with primary antibody LC3 (WL01506; Wanleibio, Co., Ltd.) at a 1:200 dilution at 4°C overnight. After washing with PBS three times, the cells were incubated with Cy3-conjugated secondary antibody (dilution, 1:200; A0516; Beyotime Institute of Biotechnology) for 30 min. The nuclei of cells were stained with DAPI for 5 min at room temperature. Slides were viewed by immunofluorescence microscopy (BX53; Olympus Corp., Tokyo, Japan) at ×400 magnification.

To inhibit autophagy, PC12 cells (2×10⁶ cells/well) were incubated with the autophagy inhibitor 3-methyladenine (3-MA; 1 mM; Sigma-Aldrich; Merck KGaA) at 37°C for 1 h prior to transfection with let-7a mimic. The cells were divided into five groups: Control, Aβ1-40, Aβ1–40 + let-7a mimic, Aβ1–40 + miR-NC and Aβ1–40 + let-7a mimic + 3-MA.

To induce activation of the PI3K/Akt/mTOR pathway, PC12 cells with different treatments were treated with 100 ng/ml insulin-like growth factor 1 (IGF-1; Bachem AG, Bubendorf, Switzerland prior to transfection for 48 h at 37°C. The cells were divided into five groups: Control, Aβ1-40, Aβ1–40 + let-7a mimic, Aβ1–40 + miR-NC and Aβ1–40 + let-7a mimic + IGF-1.

Data are expressed as the mean ± standard deviation. Differences among groups were evaluated by one-way analysis of variance followed by Bonferroni's multiple comparison test using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of let-7a in Aβ1-40-treated PC12 and SK-N-SH cells, the let-7a mimic was transfected into PC12 and SK-N-SH cells. The levels of let-7a in PC12 and SK-N-SH cells were subsequently detected by RT-qPCR. As shown in Fig. 1, in both cell types, the level of let-7a in the Aβ1–40 + let-7a mimic group was significantly higher compared with the Aβ1–40 group (P<0.001).

After transfection for 24 h, cell viability was assessed by MTT assay. As shown in Fig. 2, Aβ1–40 treatment resulted in a significant decrease in cell viability compared with the control group in both cell lines (P<0.001). Furthermore, overexpression of let-7a significantly decreased the cell viability in PC12 and SK-N-SH cells compared with the Aβ1–40 group (P<0.05 and P<0.01, respectively).

The effect of let-7a on Aβ1-40-induced apoptosis in PC12 and SK-N-SH cells was determined by Annexin V-FITC/PI staining. As shown in Fig. 3A and B, after transfection for 24 h the percentage of apoptosis induced by Aβ1–40 was significantly increased by overexpression of let-7a in the two cell lines, compared with controls (P<0.001). Furthermore, the morphological changes of apoptotic nuclei were assessed by Hoechst 33342 staining and are presented in Fig. 3C. There were more condensed or fragmented apoptotic nuclei in the Aβ1–40 group in PC12 and SK-N-SH cells compared with the control, and let-7a overexpression markedly increased this phenomenon compared with the Aβ1–40 group.

To elucidate the role of let-7a in the regulation of autophagy, the expression of LC3 among different groups of PC12 cells was determined by immunofluorescence staining. As shown in Fig. 4A, Aβ1–40 induced a marked increase in LC3 expression compared with control, and let-7a overexpression further increased LC3 expression induced by Aβ1–40 treatment. Furthermore, the levels of LC3 I, LC3 II, beclin-1 and p62 were assessed by western blotting (Fig. 4B-E). The ratio of LC3 II/I and the levels of beclin-1 and p62 were significantly upregulated by Aβ1–40 treatment in PC12 cells compared with the control (all P<0.05). These results were further increased by let-7a overexpression in PC12 cells (P<0.05, P<0.05 and P<0.001, respectively). Likewise, in SK-N-SH cells the ratio of LC3 II/I induced by Aβ1–40 was significantly upregulated by let-7a overexpression (Fig. 4F and G; P<0.01).

As shown in Fig. 5A-C, the mRNA expression levels of beclin-1, Atg-5 and Atg-7 were determined by RT-qPCR. The results indicated that the expression levels of beclin-1, Atg-5 and Atg-7 in Aβ1-40-treated PC12 cells were increased significantly compared with the control group (all P<0.001). Overexpression of let-7a led to higher levels of beclin-1, Atg-5 and Atg-7 expression compared with the Aβ1–40 group (P<0.001 for all). Furthermore, treatment with autophagy inhibitor 3-MA was able to restrain the morphological changes of apoptotic nuclei aggravated by let-7a overexpression in the context of Aβ1-40-treated PC12 cells (Fig. 5D). The apoptosis rate increased by let-7a overexpression was also significantly inhibited by 3-MA in Aβ1-40-treated PC12 cells (Fig. 5E; P<0.001). The results indicated that let-7a overexpression aggravated Aβ1-40-induced apoptosis, at least in part, via activating autophagy.

As the activation of PI3K/AKT/mTOR pathway has been reported to block autophagy (22), the role of let-7a in the regulation of PI3K/AKT/mTOR pathway was investigated in the current study in PC12 cells. The results of western blot analysis (Fig. 6) indicated significant decreases in the levels of PI3K, p-Akt and p-mTOR in the Aβ1–40 treatment group compared with the control group (all P<0.01), which were further decreased by let-7a overexpression (all P<0.05). Furthermore, the PI3K/AKT pathway was activated by treatment with IGF-1 and the changes in apoptosis and autophagy were evaluated in PC12 cells. As shown in Fig. 7A-C, the levels of PI3K and p-AKT were significantly upregulated by IGF-1 compared with the let-7a overexpression group (P<0.001 and P<0.05, respectively). The increased apoptosis rate induced by let-7a overexpression was restrained by IGF-1 in Aβ1-40-treated PC12 cells (P<0.001; Fig. 7D and E). The morphological changes of apoptotic nuclei aggravated by let-7a overexpression were alleviated by IGF-1 in Aβ1-40-treated PC12 cells (Fig. 7F). Furthermore, the ratio of LC3 II/I promoted by let-7a overexpression was significantly reduced by IGF-1 in Aβ1-40-treated PC12 cells (P<0.01; Fig. 7G and H). These results suggested that inhibition of the PI3K/Akt/mTOR signaling pathway is involved in the regulation of autophagy by let-7a overexpression, which strengthens Aβ1-40-induced neurotoxicity.