Cell culture and treatment
In our study, SH-SY5Y cells were obtained from the ATCC (Manassas, Va., USA). MPP+ was got from the Sigma Company (St. Louis, MO, USA), and was utilized to induce disease model at the dosage of 100 µM for 24 hours. The complete Dulbecco’s Modified Eagle medium (DMEM) medium used in our study is composed of basal DMEM medium (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (Gibco), as well as 1% penicillin/streptomycin (Gibco). Here, the treated and un-treated SH-SY5Y cells were cultured in the complete DMEM medium. The NEAT1 siRNA, siRNA negative control, miR-5047 mimics, mimics negative control, miR-5047 inhibitor, inhibitor negative control, and YY1-associated factor 2 (YAF2) siRNA were synthesized by GenScript Company (Nanjing, China). All above gene segments, the NEAT1 overexpression plasmid, as well as empty plasmid were transfected into SH-SY5Y cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA).
Detection Of Proteins Expression
The expression levels of pro-caspase-1, cleaved caspase-1, YAF2, NLRP1 and adaptor apoptosis-associated speck-like (ASC) were determined using Western blot. Collected SH-SY5Y cells were washed with 0.01 M PBS solution for twice. Then, the cells were broken using RIPA lysis solution (Solarbio, Beijing, China) for protein extraction. Next, 20 µg protein for each group were separated by 12% SDS-PAGE gels for 2.5 h, and were transferred into polyvinylidene fluoride membranes. After that, membranes were blocked with 5% fresh non-fat milk for 1 hour followed by were maintained with the antibodies overnight at 4°C. The pro-caspase-1, anti-ASC, anti-NLRP1, anti- anti-cleaved caspase-1, and anti-β-actin (internal reference) were purchased from Abcam (Cambridge, MA). All primary antibodies were diluted at a ratio of 1:2000 when we used. Next day, membranes were maintained with secondary antibodies for 1 hour. Finally, an ECL kit was utilized to analyze the protein bands.
Detection Of Theneat1, Mir-5047 And Yaf2 Mrna Levels
The levels of NEAT1, miR-5047, and YAF2 mRNA were measured using qRT-PCR assay. SH-SY5Y cells were collected, and were washed with PBS for twice. Then, the total RNA was isolated using TRIzol reagent (Invitrogen), and was reversed transcription into complementary DNA usage the First-Strand Synthesis Kit (Invitrogen). Next step, the qPCR experiments were accomplished using SYBR Green PCR Master Mix Kit (Invitrogen). All steps were carried out strictly according to the instructions. In this study, the expression of NEAT1 and YAF2 mRNA normalized to GAPDH, and U6 served as the internal reference for miR-5047. The levels of NEAT1, miR-5047, and YAF2 mRNA were calculated through -2ΔΔCt method.
Determination Of The Apoptotic Rate In Sh-sy5y Cells
The apoptotic rate of SH-SY5Y cells was measured using TUNEL staining. SH-SY5Y cells at a density of 5 ⋅ 104 cells/well were inoculated into 24-well plates, and were treated with MPP+ or transfected for 24 hours. After that, the cells were fixed with 4% formaldehyde fixative buffer (Solarbio) for 1 hour, and were washed with PBS solution. After incubation with 3% H2O2 solution for 15 min, the treated and untreated SH-SY5Y cells were maintained with 0.1% Triton X-100 in PBS solution for 10 min. Subsequently, the apoptotic cells were marked by TUNEL solution (Solarbio) in accordance with the instruction. After incubation with DAPI solution in the dark for 5 min to mark nucleuses, the number of apoptotic cells (TUNEL-positive cells) were counted using Image J software.
Detection of the interaction of miR-5047 with NEAT1 and YAF2 3’-UTR
Dual-luciferase activity assay was fulfilled to determine the combination of NEAT1 and miR-5047, and the combination of miR-5047 and YAF2 3’-UTR. Here, the wild-type (WT) and mutated (Mut) gene sequences of NEAT1 and YAF2 3’-UTR containing the binding sites with miR-5047 were sub-cloned into the luciferase reporter plasmid. The recombinant plasmids of NEAT1-WT, NEAT1-Mut, YAF2 3’-UTR-WT, and YAF2 3’-UTR-Mut were transfected with miR-5047 mimics or mimics negative control into 293T cells. The luciferase activity was determined usage Dual-Luciferase Reporter Assay System (Promega, WI, USA).
Detection Of The Il-1β And Il-18 Levels In Supernatant
The levels of IL-1β and IL-18 in the supernatant of SH-SY5Y cells were measured through ELISA assay according to the protocols of human IL-1β ELISA kit and human IL-18 ELISA kit (R&D Systems).
Statistical analysis
The results in our study were analyzed using the SPSS 20.0 software (IBM., Armonk, NY), and the images were marked through GraphPad Prism 6.0. The data were presented as mean ± SD. All experiments were repeated for three time at least, and the value of P < 0.05 was recognized statistically significant. Student’s t-test and ANOVA analysis were utilized for the analysis between two groups and among multiple groups.