Tanshinone IIA improves Alzheimer’s disease via RNA nuclear-enriched abundant transcript 1/microRNA-291a-3p/member RAS oncogene family Rab22a axis

Figure 3 Localization of the long non-coding RNA nuclear-enriched abundant transcript 1 in the cytoplasm, and its interaction with the microRNA miR-291a-3p. A: Nucleocytoplasmic separation was performed to detect the localization of the long non-coding RNA nuclear-enriched abundant transcript 1 (NEAT1) in neural stem cells (NSCs); B: Reverse transcription quantitative polymerase chain reaction validation of the NEAT1 overexpression vector; C: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide results of the effect of NEAT1 overexpression on the cell viability of amyloid-beta 1-42 (Aβ1-42)-induced NSCs that received tanshinone IIA (Tan-IIA) pretreatment; D: Flow cytometry results of the effect of NEAT1 overexpression on the apoptosis of Aβ1-42-induced NSCs that received Tan-IIA pretreatment; E: 2’,7’-dichlorofluorescin diacetate analysis of the effect of NEAT1 overexpression on the reactive oxygen species ratio of Aβ1-42-induced NSCs that received Tan-IIA pretreatment; F–L: Enzyme-linked immunosorbent assay results for the effect of NEAT1 overexpression on oxidative stress factors (malondialdehyde; nitric oxide; superoxide dismutase; glutathione) and neuroinflammation markers (tumor necrosis factor-alpha; interleukin 1β; interleukin 6) in Aβ1-42-induced NSCs that received Tan-IIA pretreatment; M: Starbase and Lncbase analysis, confirming miR-291a-3p as a potential target of NEAT1; N and O: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of miR-291a-3p expression in the Alzheimer’s disease mouse model and Aβ1-42-induced NSCs; P: RT-qPCR analysis of the expression of synthesized miR-291a-3p mimic/inhibitor; Q: Dual-luciferase assay confirming binding between NEAT1 and miR-291a-3p; R: RNA pull-down assay showing miR-291a-3p enrichment in bio-NEAT1; S and T: RT-qPCR analysis of the expression levels of miR-291a-3p (S) and NEAT1 (T) in NSCs. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. PI: Propidium iodide; FITC: Fluorescein isothiocyanate; Aβ1-42: Amyloid-beta 1-42; Tan-IIA: Tanshinone IIA; ROS: Reactive oxygen species; MDA: Malondialdehyde; NO: Nitric oxide; SOD: Superoxide dismutase; GSH: Glutathione; TNF-α: Tumor necrosis factor-alpha; IL-1β: Interleukin 1β; IL-6: Interleukin 6; NEAT1: Nuclear-enriched abundant transcript 1.