Figures & data
Table 1. The sequences of the miRNAs used in this study
Table 2. The sequences of the primers used in this study
Figure 1. In SH-SY5Y cells treated with MPP+, NEAT1 expression is up-regulated while miR-124-3p is down-regulated
A, B. SH-SY5Y cells were treated with MPP+ at different concentrations (0, 250, 500, 1000 or 2000 μM), and the expression levels of NEAT1 and miR-124-3p in SH-SY5Y cells were detected by qRT-PCR. C, D. SH-SY5Y cells were treated with 1 mM MPP+ for different times (0, 12, 24, 48, 72 h), and the expression levels of NEAT1 and miR-124-3p were detected by qRT-PCR. All experiments were performed in triplicate. *P < 0.05, ** P < 0.01, and *** P < 0.001.
Figure 2. NEAT1 targets miR-124-3p in SH-SY5Y cells
A. StarBase database was adopted to predict the recognition sequence of miR-124-3p in NEAT1. B. The luciferase activity of HEK-293 cells co-transfected with NEAT1-WT or NEAT1-Mut and miR-124-3p mimics or NC mimics was examined, to validate the predicted binding site between miR-124-3p and NEAT1. C-D. The interaction between NEAT1 and miR-124-3p in SH-SY5Y cells was verified by RIP and RNA pull-down assays. E. The expression levels of NEAT1 and miR-124-3p in SH-SY5Y cells transfected with pc-NC, si-NC, pc-NEAT1 and si-NEAT1 (si-NEAT1-1, si-NEAT1-2 and si-NEAT1-3) were detected by qRT-PCR. All experiments were performed in triplicate. *P < 0.05, ** P < 0.01, and *** P < 0.001.
Figure 3. NEAT1 participates in MPP+-induced injury of SH-SY5Y cells through the regulation of miR-124-3p
SH-SY5Y cells were divided into four groups after transfection: si-NC group, si-NEAT group, si-NEAT + NC inhibitor group, and si-NEAT1 + miR-124-3p inhibitors group. A. The expression of miR-124-3p in SH-SY5Y cells was detected by qRT-PCR after transfection. B. MTT assay was used to detect the viability of SH-SY5Y cells. C. LDH cytotoxicity detection kit was used to determine the injury of SH-SY5Y cells. D-F. The levels of TNF-α, IL-1β, and IL-6 were detected by ELISA. All experiments were performed in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 4. PDE4B acts as a target of miR-124-3p
A. Bioinformatics was used to predict the binding sites between miR-124-3p and 3ʹ-UTR of PDE4B. B. Dual-luciferase reporter assay verified the targeted binding relationship between miR-124-3p and PDE4B. C-D. After the expression of NEAT1 and miR-124-3p were selectively regulated, qRT-PCR and western blot were used to analyze the expression of PDE4B mRNA and protein. All experiments were performed in triplicate. **P < 0.01, and ***P < 0.001.
Figure 5. NEAT1 regulates the inflammatory response and injury of SH-SY5Y cells through miR-124-3p/PDE4B/mTOR axis
A, B. qRT-PCR and Western blot were used to detect the expression of miR-124-3p and PDE4B in SH-SY5Y cells, respectively, after the cells were transfected with NEAT1 overexpression plasmids, or co-transfected NEAT1 overexpression plasmids and miR-124-3p mimics. SH-SY5Y cells were then divided into four groups: NC mimics group, miR-124-3p mimics group, pc-NC group and pc-PED4B + miR-124-3p mimics group. C. MTT assay was used to detect the viability of SH-SY5Y cells. D. LDH release was determined by LDH cytotoxicity detection kit. E-G. The levels of TNF-α, IL-1β, and IL-6 were detected by ELISA. H. The expression levels of mTOR and p-mTOR protein were detected by Western blot. All experiments were performed in triplicate. **P < 0.01, and ***P < 0.001.
Figure 6. Graphic Abstract. NEAT1 functions as a molecular sponge to regulate miR-124-3p, PDE4B and mTOR signaling, to mediate the neurological inflammation and injury in PD
