Experiments were performed in 30 adult male C57B/6J mice (weighing 20-30 g, 8 weeks old), which were obtained from the Model Animal Research Center of Nanjing University. The mice were kept in a controlled environment with a temperature of 22-25°C, a 12:12 h light/dark cycle, with free access to water and food. Manipulations were conducted during the light phase of the day. Efforts were made to minimize animal suffering and to reduce the number of animals used. The present study was performed in accordance with the guidelines of the Committee on Care and Use of Experimental Animals Resources and with the approval of Ethical Committee for The First Hospital of Yulin.

The mice were randomly divided into 3 different groups with 10 mice in each group, including the control group (n=10), the dimethyl sulfoxide (DMSO) control group (n=10) and the rotenone-induced PD group (n=10). The duration of the experiment was 1 month.

Rotenone (Sigma Chemical Co.) was first dissolved in DMSO, which was completed with sunflower oil. Rotenone was administered orally once daily (0.1 ml/10 g, 30 mg/kg) for 30 days to establish an in vivo model of PD, as previously described (16). The mice in the DMSO control group were orally administered once daily with DMSO for 30 days. The mice in the control group received no treatments. Afterwards, the mice in each group were submitted to a swimming test and traction test.

The swimming test was conducted to determine the motor disability of the mice in each group using a round glass swimming tank (length, 40 cm; width, 25 cm; height, 16 cm), which was filled with water (at a temperature of 22-25°C) to a depth of 12 cm. The mice were scored using the following scale: 0, No swimming with the head above the water; 1, occasional swim with mice floating using the hind paws; 2, alternations between swim-floating and passively floating; and 3, continuous swimming, as previously described (17).

The traction test was performed to determine the muscle strength and equilibrium of the mice. The forepaws of the mice were placed on a rope (diameter, 5 mm) which was approximately horizontally 70 cm at a distance from the ground. The hind limb placements of the mice were scored on a scale of 1 to 3, with the lowest score indicating the most severe deficits. The score was determined using the following criteria: 1 or 2, No or one hind limb seizing the rope; and 3, both hind limbs seizing the rope. Mice were allowed to hang upside down and stay on the rope for 30 sec, as previously described (18).

Animal health and behavior was monitored once every 2 days with the following principles: Soft and smooth hair, even breath and no scabs, etc. During the process, no mouse had died. After the swimming test and traction test, the animals were euthanized by barbiturate overdose (pentobarbital sodium, 150 mg/kg; i.p.) and were immediately decapitated. Death was verified by the absence of breathing, heartbeat or neurofeedback, etc. SN tissues were then extracted from the mice and fresh-frozen at −80°C which were then subjected to the following experiments.

Total RNA was extracted from the SH-SY5Y cells (please see below) and SN tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Reverse transcription and TaqMan microRNA assay were carried out using the Hairpin-it™ miRNA qPCR Quantitation kit (GenePharma Co.). Thereafter, qPCR was performed using the SYBR-Green kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) on the ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 15 sec, 58°C for 30 sec and 72°C for 30 sec. The primer sequences were as follows: Sirtuin 1 (SIRT1) forward, 5′-CTG GGG TGT CTG TTT CAT GTG G-3′ and reverse, 5′-GCT TGA GGA TCT GGA AGA TCT GG-3′; GAPDH forward, 5′-CCA CTC CTC CAC CTT TGA CG and reverse, 5′-CCA CCA CCC TGT TGC TGT AG-3′; forkhead box protein O1 (FOXO1) forward, 5′-TCC CAG TGA GCA GTA AAT C-3′ and reverse, 5′-CCA GCA GTT GAA CAA GTC-3′; p53 forward, 5′-GCT GGT TAG GTA GAG GGA GTT G-3′ and reverse, 5′-GTG TGG GAT GGG GTG AGA TTT C-3′; miR-384-5p, 5′-CGC GTA TGA ACA ATT TCT AGG AAT-3′; and U6, 5′-TTG GTG AAG CGT TCC ATA TTT T-3′. The relative expression levels of miRNA and mRNAs were determined using the 2^−ΔΔCq method (19). The expression of miR-384-5p was normalized to U6, while the expression of SIRT1, p53 and FOXO1 was normalized to GAPDH.

The binding sites between miR-384-5p and SIRT1 were analyzed by TargetScan release 7.1 (http://www.targetscan.org/vert_71/).

Proteins were isolated from the SH-SY5Y cells (please see below) or SN tissues by radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology). The protein concentrationin each sample was detected by BCA assay (Beyotime Institute of Biotechnology). The same amount of proteins from each sample (15 µg) were separated by 8-10% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking with 5% non-fat milk at room temperature for 1 h, the PVDF membranes were incubated with respective primary antibodies for α-synuclein rabbit mAb (dilution, 1:1,000, cat. no. 4179), SIRT1 rabbit mAb (dilution, 1:1,000, cat. no. 9475), p53 rabbit mAb (dilution, 1:1,000, cat. no. 2527), FOXO1 rabbit mAb (dilution, 1:1,000, cat. no. 2880), and GAPDH rabbit mAb (dilution, 1:1,000, cat. no. 5174) overnight at 4°C and anti-rabbit IgG, HRP-linked secondary antibodies (dilution, 1:2,000, cat. no. 7074) at room temperature for 2 h, successively. The above-mentioned antibodies were obtained from Cell Signaling Technology. ECL system (Bio-Rad Laboratories Inc.) was used to detect antibody bound proteins. Densitometric analysis was performed using Image J 1.8.0 software (National Institutes of Health).

SH-SY5Y cells human dopaminergic neuroblastoma were obtained from the American Type Culture Collection (cat. no. ATCC^® CRL-2266™; ATCC). STR profiling was used for the authentication of the SH-SY5Y cells. The SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium-Ham's Nutrient Mixture F-12 (DMEM/F12; Invitrogen; Thermo Fisher Scientific) which was supplemented with 10% fetal calf serum (FCS), and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO₂ at 37°C. The cells were randomly divided into 3 groups, including the control group, DMSO group and rotenone-induced PD group. The SH-SY5Y cells were exposed to rotenone (20 µM) for 24 h, as previously described (20) before being harvested for use in subsequent experiments.

The SH-SY5Y cells (2×10⁵ cells/well) were seeded into 6-well plates and cultured till 90% confluence prior to cell transfection. miR-NC inhibitor and miR-384-5p inhibitor were obtained from GenePharma. Briefly, miR-384-5p inhibitor (2.5 µl) or miR-NC inhibitor (2.5 µl) and Lipofectamine 2000 (5 µl, Invitrogen; Thermo Fisher Scientific, Inc.) were added into 250 µl DMEM. Subsequently, the mixture was mixed thoroughly and added into the 6-well plates to a final work concentration of 20 nmol/l. The above mixture was incubated for 48 h prior to use in the subsequent experiments.

The 293 cells (ATCC) and SH-SY5Y cells transfected with miR-384-5p inhibitor or miR-NC inhibitor were seeded into 96-well plates (1×10³ cells/well) and cultured for 24 h. The SIRT1 3′UTR was amplified from the cDNA of 293 cells and inserted into pLight Switch Prom (Switchgear Genomics). Subsequently, pLightSIRT1-WT-3′UTR reporter construct or pLight-SIRT1-MUT-3′UTR was transfected into the 293 cells and SH-SY5Y cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h after cell transfection, Firefly luciferase activity was determined by Light Switch Luciferase assay (Switchgear Genomics) through normalization to Renilla luciferase activity.

The Annexin V-FITC/PI Staining Apoptosis Detection kit was used for the detection of cell apoptosis. SH-SY5Y cells were collected and washed with PBS. Thereafter, the SH-SY5Y cells (3×10⁵) were resuspended and incubated with Annexin V-FIFC (5 µl) and PI (10 µl) at 37°C for 15 min. The cell apoptotic rate was analyzed using a flow cytometer (BD Biosciences).

Following incubation at 37°C for 48 h, the SH-SY5Y cells were fixed with 4% formaldehyde for 15 min at 4°C, followed by permeabilization with 0.2% Triton X-100 for 10 min at room temperature. The primary antibody for α-synuclein rabbit mAb (dilution, 1:200, cat. no. 4179) was incu bated at 4°C overnight. Anti-rabbit IgG (Alexa Fluor 488 conjugate, cat. no. 4412; dilution, 1:500) acted as a secondary antibody to detect fluorescence and was incubated at room temperature for 2 h. Subsequently, the cells were incubated with DAPI (cat. no. 4083; 0.5 µg/ml) for 10 min at room temperature for cell nuclei staining. The above-mentioned antibodies were obtained from Cell Signaling Technology. Images were captured under a confocal microscope (TCS SP 5 II, Leica). Fluorescence intensity was analyzed using MicroBrightField Stereo-Investigator (MBF Bioscience).

GraphPad Prism 7 software (GraphPad Software, Inc.) was applied for the analysis of the data. The results are expressed as the means ± SEM. Comparisons between 2 groups were analyzed using a Student's t-test, while comparisons among 3 groups were analyzed by one-way analysis of variance followed by Newman-Keuls post-hoc analysis. The correlation between miR-384-5p and SIRT1 was analyzed by Pearson's correlation analysis. A value of P<0.05 was considered to indicate a statistically significant difference.

In the in vivo model of PD in this study, the severity of parkinsonian signs was first evaluated through swimming scores and traction scores at day 1 following rotenone induction, as displayed in Fig. 1A and B. The results demonstrated that there were no significant differences in swimming scores between the control group and DMSO group, whereas in the group of rotenone-exposed PD mice, the swimming scores became significantly lower than those of the DMSO group (Fig. 1A).

Furthermore, the data presented in Fig. 1B demonstrated that there were no significant differences in traction scores between the control group and DMSO group, whereas the induction of PD with rotenone led to a significant decrease in the score of the traction test below the DMSO values.

In addition, the protein expression levels of α-synuclein were examined in each group. As shown in Fig. 1C and D, no significant were observed between the control group and DMSO group; however, in the group of rotenone-exposed PD mice, the protein expression levels of α-synuclein became significantly higher than those of the DMSO group. Taken together, these aforementioned results verify that the constructed PD model was successful.

Subsequently, the differences in the expression levels of miR-384-5p in the mice among the 3 groups were examined by RT-qPCR. The results revealed that compared with the control group, there were no obvious differences in miR-384-5p levels in the DMSO group; however, in the group of mice with rote-none-induced PD, the expression levels of miR-384-5p became significantly higher than those of the mice in the DMSO group (Fig. 2A).

The following experiment was performed in an aim to predict the potential targets for miR-384-5p. As exhibited in Fig. 2B, using the online software TargetScan, it was discovered that the 3′UTR of SIRT1 had the complementary site for miR-384-5p; moreover, the mutant sequence of the 3′UTR of SIRT1 was obtained.

Subsequently, the interaction between miR-384-5p and SIRT1 was confirmed by dual luciferase reporter assay. The results demonstrated that transfection with miR-384-5p inhibitor significantly induced the relative luciferase activity of 293 cells transfected with luciferase activity plasmid containing SIRT 3′UTR-WT, but not SIRT 3′UTR-MUT (Fig. 2C).

Thereafter, the differences in the mRNA and protein expression levels of SIRT1 in mice with rotenone-induced PD mice among the 3 groups were examined by RT-qPCR and western blot analysis, respectively. The results of RT-qPCR revealed that, compared with the control group, there were no obvious differences in the SIRT1 mRNA levels between the control and the DMSO group; however, in the group of mice with rotenone-induced PD, the mRNA levels of SIRT1 became significantly lower than those of the mice in the DMSO group (Fig. 3A). Moreover, the results of western blot analysis revealed similar results to those obtained with RT-qPCR (Fig. 3B and C).

Of note, the results of Pearson's correlation analysis demonstrated that there was a significant negative correlation between miR-384-5p and SIRT1 expression in the mice with rotenone-induced PD (Fig. 3D).

Based on the above-mentioned findings, the molecules that are associated with SIRT1 in mice with rotenone-induced PD were then investigated. A previous study found that p53 and FOXO1 were associated with SIRT1 in MPP⁺-induced SH-SY5Y cells (21). Therefore, this study also examined the association between SIRT1 and P53/FOXO1 in mice with rotenone-induced PD.

The differences in the mRNA and protein expression levels of p53/FOXO1 were determined among the 3 groups by RT-qPCR and western blot analysis, respectively. The results of RT-qPCR revealed that compared with the control group, there were no obvious differences in the p53/FOXO1 mRNA levels in the DMSO group. However, in the group of mice with rotenone-induced PD, the mRNA levels of p53/FOXO1 became significantly higher than those of the mice in the DMSO group (Fig. 4A and B). Moreover, the results of western blot analysis revealed similar results to those obtained with RT-qPCR (Fig. 4C-E).

In the in vitro model of PD in this study, the protein expression level of α-synuclein was first evaluated in the SH-SY5Y cells in the 3 groups. The results presented in Fig. 5 demonstrated that there were no significant differences in α-synuclein protein levels between the control group and DMSO group. However, in the PD group of rotenone-exposed SH-SY5Y cells, the α-synuclein protein levels became significantly higher than those in the DMSO group.

The data presented in Fig. 6 demonstrated that there were no significant differences in the apoptotic rate of SH-SY5Y cells between the control group and DMSO group; however, the exposure of the cells in the PD group to rotenone led to a significant increase in the cell apoptotic rate compared with the DMSO group.

Subsequently, the differences in the expression levels of miR-384-5p in the SH-SY5Y cells among the 3 groups were examined by RT-qPCR. The results revealed that compared with the control group, there were no obvious differences in miR-384-5p levels in the DMSO group. Howev PD er, in the PD group of rotenone-exposed SH-SY5Y cells, the expression levels of miR-384-5p became significantly higher than those in the DMSO group (Fig. 7A).

Subsequently, the interaction between miR-384-5p and SIRT1 was confirmed by dual luciferase reporter assay, and the results demonstrated that transfection with miR-384-5p inhibitor significantly induced the relative luciferase activity of SH-SY5Y cells transfected with luciferase activity plasmid containing the SIRT 3′UTR-WT, but not SIRT 3′UTR-MUT (Fig. 7B).

Thereafter, the differences in the mRNA and protein expression levels of SIRT1 in the rotenone-exposed SH-SY5Y cells in the PD group in the 3 groups were examined by RT-qPCR and western blot analysis, respectively. The results of RT-qPCR revealed that compared with the control group, there were no obvious differences in SIRT1 mRNA levels in the DMSO group; however, in the PD group of rotenone-exposed SH-SY5Y cells, the mRNA levels of SIRT1 became significantly lower than those in the DMSO group (Fig. 8A). Moreover, the results of western blot analysis revealed similar results to those obtained with RT-qPCR (Fig. 8B and C).

Of note, the results of Pearson's correlation analysis demonstrated that there was a significant negative correlation between miR-384-5p and SIRT1 expression in the rotenone-exposed SH-SY5Y cells in the PD group (Fig. 8D).

The association between SIRT1 and p53/FOXO1 expression in rotenone-exposed SH-SY5Y cells was then examined. The differences in the mRNA and protein expression levels of p53/FOXO1 among the 3 groups were examined by RT-qPCR and western blot analysis, respectively. The results of RT-qPCR revealed that compared with the control group, there were no obvious differences in p53/FOXO1 mRNA levels in the DMSO group. However, in the PD group of rotenone-exposed SH-SY5Y cells, the mRNA levels of p53/FOXO1 became significantly higher than those in the DMSO group (Fig. 9A and B). Moreover, the results of western blot analysis revealed similar results to those of RT-qPCR (Fig. 9C-E).