Participants
Twenty-three PD patients (male: 12, female: 11) with a mean age of 63.5 ± 1.0 years from the Brain Hospital affiliated with the Nanjing Medical University were included in this study. The participants with PD were diagnosed according to the PD diagnostic criteria released by the Movement Disorders and PD Group, Chinese Neurology Association. Participants who were diagnosed as having other comorbid conditions, such as diabetes, arthritis, and heart disease were excluded from the study. In addition to the PD cohort, 30 age- and gender-matched healthy volunteers (male: 15, female: 15) with a mean age of 65.0 ± 0.8 years were contacted via the physical examination center of our hospital and invited to participate in this study as a member of the control group. To be eligible for inclusion, all controls were required to be free of neurological conditions and have no history of diabetes, arthritis, or heart disease. The protocols of this research were approved by the appropriate Ethics Committees of Nanjing Brain Hospital (Ethic Reference No: 2017-KY010), and all participants involved gave their written informed consent to participate.
Animals and treatments
20 Eight-week-old C57/BL mice were provided by Nanjing Medical University (Nanjing, Jiangsu, China). They were acclimatized and kept at a temperature of 22 ± 2℃ and a humidity of 60 ± 2% with ad libitum feeding. All experimental procedures used in this study were approved by the Institutional Animal Care and Use Committee of our hospital. The C57/BL mice model received injections of miR-1976 mimic lentiviral vector to the neurons of the substantia nigra, and were killed 24 hours after. Meanwhile, the control 20 mice received saline injections only.
Cell culture and transfection
The dopaminergic neuron cell line (MES23.5) used in this study was cultured in DMEM supplemented with 15% FBS at 37℃ and 5% CO2. Then the MES23.5 cells were prepared for transfection.
Hsa-miR-1976 mimic and mimic negative control, as well as PINK1 shRNA and PINK1 inhibitor were purchased from Shengbo Biotechnology Co., Ltd (Shanghai, China). After confluence of the MES23.5 cells cultured in the DMEM medium reached about 50%, cells (1×106) were seeded into each well of a 6-well plate and were transfected with the above mimics or inhibitors by using the LipofectamineTM 2000 (Invitrogen, CA, USA) according to the manufacturer’s protocol. Then the cells were collected after 12 h, 24 h and 36 h for the next experiment.
Pretreatment of human blood samples
Blood samples (3 mL) were drawn from each subject and were aliquoted at 3500 rpm for 5 minutes. Plasma samples were then stored at -80℃ before analysis.
Total RNA isolation
Total RNA was isolated using the TRIzol protocol. In brief, the plasma samples were modified by TRIzol (Ambion, USA). Following a 10-minute incubation at ambient temperature, 200 ml of chloroform was added and mixed vigorously, before the mixture was centrifuged at 14000 rpm for 15 minutes at a temperature of 2-8℃. The upper aqueous phase was then transferred into a new cube and combined with 500 ml of ethanol to facilitate RNA extraction. The RNA precipitant obtained by centrifuge was washed with 75% ethyl alcohol, and the integrity of extracted total RNA was identified by microspectrophotometer.
miRNA isolation
The total RNA (100 mg) was added to 5 times the volume of lysis buffer, then combined with 1/10th the volume of miRNA Homogenate Additive and 1/3rd the volume of ethyl alcohol. The mixture was then centrifuged at 5000 rpm for 1 minute. Following centrifugation, the precipitate was subsequently combined with 700 μl of miRNA Washing Solution 1 and 500 μl of miRNA Washing Solution 2 and passed through the centrifugation process a further two times. Finally, 50 μl of Elution Solution was added onto the column for 2-minute incubation, and the samples were centrifuged for 1 minute at 10000 rpm. The flowthrough containing the miRNA was stored at -80℃ until used.
MicroRNA Microarray experiments
Firstly, the solution used for microarray hybridization was prepared by combining 15% formamide 2.4 μl, 0.2% SDS 3.2 μl, 3×SSC 2.4 μl,50×Denhardt,s 1.6 μl, and DEPC 6.4 μl. Following this process, the RNA described above was mixed into this solution for 3-minute degeneration at 95℃. Hybridization of the miRNA was performed on the mammals’ miRNA microarray chip (V3.0). Then the hybridized chips were washed and scanned on a microarray scanner (Agilent, Santa Clara, USA).
Real-time polymerase chain reaction (PCR)
Total RNA was extracted using the procedures described above for microarray hybridization. Both the primers of miRNAs (miR-1976, miR-153, miR-103a, miR-29a, miR-210, miR-375, miR-146a, and miR-101a) and the internal reference U6 reverse transcription primers were provided by Ribobio (Guangzhou, Guangdong, China). For real-time PCR, the cDNA from individual samples of total RNA was synthesized using the FastQuant RT kit (TIANGEN, Beijing, China). The real-time PCR was performed in a 20 μl reaction system comprising 10 μl SYBR Green (TaKaRa, Japan), 1 μl upstream/downstream primers,1 μl dNTPs,2 μl Taq, and 5 μl cDNA. The cycling conditions included 10 minutes of 95℃ predenaturation, followed by 40 cycles of 5-second denaturation at 95℃, 2 minutes annealing at 60℃, and 30 seconds elongation at 72℃. All the reaction was performed on an ABI Prime 7300 Quantitative PCR detector.
Luciferase assay
The 3’UTR fragment of PINK, which contains binding sites for miR-1976 (WT), and a 3’UTR fragment with a corresponding mutation (MUT) were designed according to the results of information prediction. The primers used in this assay were: miR-1976-F: CTC CTG CCC TCC TTG CTG T, U6-F:CCT AGC ACC ATG AAG ATC AAG AT. They were then plated into a 24-well plate and co-transfected with 40 nM of either miR-1976 mimic or a negative control, and 100 ng of either PINK 3’UTR WT or MUT reporter by using the Lipofectamine 300. Forty-eight hours later, the cells were collected and the luciferase activity was measured by using the dual luciferase assay system (Promega, USA).
Electron microscope
The MES23.5 cells were firstly cultured and then digested by pancreatin. After washing with PBS, cells were centrifuged for 10 minutes at 800-1000 rpm, after which, the supernatant was discarded and the left precipitate was resuspended at 1500 rpm for 10 minutes. The compacted cell mass was then fixed with 2.5% glutaraldehyde and 1% osmic acid. After embedding with Epon 812, cells were stained and observed using a transmission electron microscope (JEM-1010).
Apoptosis assay
The MES23.5 cells were seeded onto 64-well plates, pretreated with pancreatic enzymes in their logarithmic phase and washed twice with PBS. Each pellet was resuspended in 250 μl binding buffer and combined with both 5 μl Annexin V-FITC and propidium iodide for a 30-minute period of incubation. Following this, the flow cytometry was used for apoptosis at an excitation wavelength of 488 nm and emission wavelength of 515 nm.
Bioinformatics analysis of miR-1976
Focusing on the miR-1976, we predicted its putative mRNA targets using TargetScan and microrna.org, respectively. Firstly, we searched the target genes from these two databases (Target gene dataset A), then the common target genes were collected, and intersected with Target gene dataset B (different expression genes that negatively related with miR-1976 detected based on the miRNA microarray).
Gene ontology (GO) and pathway analysis
GO (http://geneontology.org) and Pathway analysis (https://www.kegg.jp) were performed to detect the coordinated changes in functionally related genes. GO analysis was carried out by using the database; and the pathway analysis of obtained predicted target genes was conducted by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Statistical analysis
The statistical analyses were conducted using SPSS 20.0 software (SPSS, Chicago, USA). All data were presented as mean ± SD and analyzed using independent samples t-tests. Categorical variables were assessed using the Chi-square test. For comparison of differences, P < 0.05 was regarded as a statistically significant difference.