Acquisition and culture of human neuroblastoma SH-SY5Y cells
Human neuroblastoma SH-SY5Y cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were stored in Dulbecco’s modified Eagle medium (DMEM) medium (Gibco, Grand Island, NY, USA) supplemented with 1% penicillin/streptomycinin, 10% fetal bovine serum (FBS, Gibco) and incubated in a humidified atmosphere containing 5% CO2 at 37℃.
Dual-luciferase reporter verification
The association between lncRNA SOX2-OT and miR-942-5p was identified by bioinformatics software (Starbase). In addition, TargetScan determined the relationship between miR-942-5p and NAIF1. We then used a dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China) and the QuikChange Site-Directed Mutagenesis Kit (Stratagene, San Diego, CA) to generate NAIF1-WT, NAIF1-MUT, SOX2-OT-WT and SOX2-OT-MUT according to the manufacturer’s instructions. Finally, the luciferase activity was analyzed by the dual luciferase reporter gene analysis system (Promega, USA).
Establishment of PD cell model in vitro
To explore the expression levels of miR-942-5p, lncRNA SOX2-OT and NAIF1 in the PD cell model. SH-SY5Y cells were treated with 0, 0.25, 0.5, 1 or 2 mM MPP+ (Sigma, St. Louis, MO, USA) for 24 h, or exposed to 1 mM MPP+ for 0, 6, 12, 24 or 48 h .
Cell transfection assay
SH-SY5Y cells were inoculated at a concentration of 5 × 104 cells/ml in 6-well plates and incubated overnight. The miR-942-5p inhibitors was used to down-regulate miR-942-5p expression in SH-SY5Y cells using an inhibitor-control as the negative control. SOX2-OT-siRNA was used for SOX2-OT down-regulation. The miR-942-5p mimic and NAIF1-plasmid were used to up-regulate miR-942-5p and NAIF1 expression in SH-SY5Y cells. Control-siRNA, SOX2-OT-siRNA, inhibitor control, miR-942-5p inhibitor, SOX2-OT-siRNA + inhibitor control or SOX2-OT-siRNA + miR-942-5p inhibitor, and mimic control, miR-942-5p mimic, control-plasmid, NAIF1-plasmid, miR-942-5p mimic + control-plasmid- or miR-942-5p mimic + NAIF1-plasmid were transfected into SH-SY5Y cells using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. After 48 h of cell culture at 37°C, the cells were collected to test the transfection efficiency using qRT-PCR, or further cultured in the presence of 1 mM MPP+ for 24 h.
RNA extraction and quantitative real-time PCR (qRT-PCR)
Total cellular RNA was isolated from SHSY5Y cells using TRIzol reagent (Invitrogen, USA), and reverse transcribed into first-strand cDNA using a cDNA Synthesis Kit (Invitrogen) according to the instructions provided by the manufacturer. The expression levels of miR-942-5p, lncRNA SOX2-OT and NAIF1 mRNA were quantified by the Prism 7000 real-time PCR system using Power SYBR Green Master mix (Vazyme, Piscataway, NJ, USA) according to the instructions provided by the manufacturer. The amplification conditions were as follows: denaturation at 94℃ for 35 cycles of 60 seconds, anneal at 60℃ for 60 seconds, then extend at 72℃ for 1 minute, and then at 72℃ for 10 minutes. U6 and GAPDH were used as inner control genes. Calculate the relative expression levels of miR-942-5p, lncRNA SOX2-OT and NAIF1 mRNA by the 2-ΔΔCt method .
3-(4,5-dimethylthiazol-2-yl)-2,5-dipheyltetrazolium bromide (MTT) assay
SH-SY5Y cells were inoculated into 96-well plates in triplicate, incubating overnight. Subsequently, the medium was removed, and after transfection of the cells at 37℃, the cells were treated with 1 mM MPP+. The cells were incubated with 10 μl MTT solution (Beyotime, Shanghai, China) for 4 hours. Subsequently, after removing the solution, adding 100 μl dimethyl sulfoxide (DMSO) to each well to dissolve the formazan product. Detection was achieved by monitoring the absorbance at 570 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). The optical density value was used to normalize the relative cell viability relative to the control group.
Flow cytometry (FCM) to detect apoptosis
SH-SY5Y cells were seeded into 6-well plates overnight, and collected by trypsinization following treatment. Washing the cells once with PBS buffer and subsequently re-suspended in 1 × binding buffer. A total of 100 µl of cell suspension was transferred into a 5 ml test tube and mixed with 5 μl fluorescein isothiocyanate (FITC)-Annexin V and 5 μl propidium iodide (PI) (BD Biosciences, San Diego, CA), respectively, according to the manufacturer’s specifications. We analyzed the induction of apoptosis with a FACSCalibur flow cytometer (BD Biosciences, USA) within one hour, and analyzed the data with FlowJo software (version 7.6.1; FlowJo LLC).
Western blot analysis
After 48 hours of transfection and cell culture, SH-SY5Y cells were treated with 1 mM MPP+ for 24 hours, washed three times with cold PBS, and immediately lysed with RIPA lysis buffer (Beyotime, Shanghai, China). The lysate was centrifuged at 12,000 rpm at 4℃ for 10 minutes, and the total protein level was measured by the BCA protein kit (Pierce, USA). Equal amounts of protein samples were separated using a sodium dodecyl sulfate (SDS) gel-polyacrylamide gel electrophoresis (PAGE), and then transfer it to the PVDF membranes. After sealing with 5% skim milk for 1 hour, the PVDF membranes were incubated with NAIF1 (1: 1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), cleaved-Caspase3 (1: 1,000; Cell Signaling Technology, Inc.), Caspase3 (1: 1,000; Cell Signaling Technology, Inc.), GAPDH (1:1,000; Cell Signaling Technology, Inc.) antibodies, immediately overnight at 4℃. The next day, the membranes were washed 3 times with PBST buffer and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (1:2,000; Cell Signaling Technology, Inc.) at 37℃ for 1 hour. We finally visualized the protein bands using ECL luminescent substrate (Pierce) according to the manufacturer's instructions. The experiments were repeated for 3 times at least.
Lactate dehydrogenase (LDH) activity assay
SH-SY5Y cells were cultured with 1 mM MPP+ for 24 hours, then the activity of LDH released into the culture medium was measured using a lactate dehydrogenase assay kit (Jiancheng Institute of Bioengineering, China) according to the manufacturer's instructions. A micro-plate reader (Bio-Rad, Hercules, CA, USA) was used to record the absorbance at 490 nm.
SH-SY5Y cells were treated with MPP+ for 24 hours, gathered and centrifuged to detect the expression levels of TNF-α and IL-1β using an ELISA kit (BioLegend, Inc., CA, USA) according to the instructions provided by the manufacturer. A microplate reader (Bio-Rad, Hercules, CA, USA) was employed to measure the absorbance at 450 nm.
Reactive oxygen species (ROS) release and superoxide dismutase (SOD) activity test
The treated cells were incubated with 10 μM DCFH-DA (Sigma) at 37°C for 45 minutes in the dark. A fluorescence microplate reader (Labsystems Oy, Helsinki, Finland) was used to quantify the fluorescence intensity, using an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
SH-SY5Y cells were gathered and lysed using cell lysis buffer (Beyotime, Shanghai, China). According to the manufacturer's instructions, SOD activity assay kit (China Jiancheng Institute of Biological Engineering) was used to determine the SOD activity.
The experimental data were indicated as the mean ± standard deviation (SD) of at least three independent experiments. SPSS 13.0 software was performed for statistical analysis. The difference between the two groups were determined by Student’s t-test, and the one-way analysis of variance followed by Bonferroni post-hoc test was applied to analyze the difference between multiple groups. The p-value below 0.05 (P <0.05) was identified as a significant difference.