Fig 2: Plek and Sdcbp2 might be the targets of miR-409-5p. (A) The wild-type (WT) and mutated plek or Sdcbp2 binding sites with miR-409-5p. (B) Relative Renilla/luciferase luminescence of a psiCHECK2 vector construct harboring Plek or mutant Plek cotransfected with miR-409-5p in the HEK 293T cells, with empty psiCHECK2 vector as control. (C) Relative Renilla/luciferase luminescence of a psiCHECK2 vector construct harboring Sdcbp2 or mutant Sdcbp2 cotransfected with miR-409-5p in the HEK 293T cells, with empty psiCHECK2 vector as control. The results were shown as the mean ± SD (*p < 0.05,**p < 0.01). The experiment was repeated independently for at least three times. (D,E) MiR-409-5p mimic (100 nM) or inhibitor (100 nM) was transfected into Neuro-2a cells. After 24 h, plek and sdcbp2 mRNA levels were assessed by RT-qPCR. (F,G) Western blot analysis of Plek and SDCBP2 protein level in primary cortical neurons transfected with miR-409-5p mimic (100 nM) or inhibitor (100 nM) for 72 h. ImageJ software was used to quantify the gray degree values of Plek and SDCBP2. All results were shown as the mean ± SD (*p < 0.05). The experiment was repeated independently for three times. Unpaired t-test was used to analyze differences.

Fig 3: Expression levels of Plek and Sdcbp2 in APP/PS1 cortex. (A,B) The relative expression levels of plek and sdcbp2 were examined by RT-qPCR analysis in 4- to 12-month-old APP/PS1 and WT mice cortices (4–6 mice for each age). Two-way ANOVA was used to analyze differences. *p < 0.05.

Fig 4: (A) Overexpression of Plek partially rescues miR-409-5p-induced neurite outgrowth inhibition. PC12 cells were cotransfected with miR-409-5p mimic or inhibitor (concentration ratio 1:5) with or without PEGFP-Plek before differentiation. After 48 h, cells were fixed and immunostained with anti-ß-tubulin III antibody. (B–D) Quantification of neurite length or branches was analyzed by ImageJ software. The results were shown as the mean ± SD (*p < 0.05, **p < 0.01). The experiment was repeated independently for three times. Red, ß-tubulin III; Green, GFP; Yellow, merged. Unpaired t-test was used to analyze differences.

Fig 5: Molecular docking and molecular dynamics simulation show the binding ability between DMAG and its core targets. a Detailed interactions of receptors (ITGA2B, ITGB3, TLR2, PLEK) and ligands (DMAG) by molecular docking. The yellow dotted line indicates the interaction between ligand and receptors. b The RMSD curves of receptors (ITGA2B, ITGB3, TLR2, PLEK) binding to ligands (DMAG) during 25 ns by molecular dynamics simulation