Fig. 4: miR-221-3p alleviates ferroptosis in NPCs by inhibiting IRF8-STAT1 axis. | Nature Communications

Fig. 4: miR-221-3p alleviates ferroptosis in NPCs by inhibiting IRF8-STAT1 axis.

From: Injectable ECM-mimetic dynamic hydrogels abolish ferroptosis-induced post-discectomy herniation through delivering nucleus pulposus progenitor cell-derived exosomes

Fig. 4

a RNA sequencing of NPCs following treatment with NPPC-Exo or miR-221-3p overexpressed NPPC-Exo (miR-Exo, 50 μg/mL). PCA analysis (b) volcano plot (c) and heatmap (d) demonstrate significant DEGs among different groups (n = 3 biologically independent samples). The significant DEGs in panel (b–d) were filtered with thresholds of a |log2(fold change, FC)| >2 and P value < 0.05 using the R package DESeq2. e Sequence alignments of miR-221-3p and its target site in the 3’UTR of Irf8. f Luciferase reporter assay conducted on HEK293T cells treated with miR-221-3p mimic, overexpressing either Irf8-wildtype 3’UTR (Irf8-WT) or Irf8-mutant 3’UTR (Irf8-Mut), n = 5 biologically independent samples. Western blot bands (g) and quantitative analysis (h) of IRF8 protein levels in NPCs treated with miR-Exo with or without anti-miR-221-3p (n = 6 biologically independent samples). Normal: NPCs with no treatment; Model: NPCs treated with Erastin; miR-Exo: NPCs treated with Erastin and miR-221-3p overexpressed NPPC-Exo (50 μg/mL); miR-Exo + anti-miR: NPCs treated with Erastin, miR-221-3p overexpressed NPPC-Exo (50 μg/mL), and anti-miR-221-3p. Western blot bands (i) and quantitative analysis (j) of COL2, GPX4, STAT1, and STAT1 phosphorylation protein levels in NPCs treated with miR-221-3p overexpressed NPPC-Exo at 50 μg/mL (n = 6 biologically independent samples). Western blot bands (k) and quantitative analysis (l) of COL2, STAT1, STAT1 phosphorylation, GPX4, and SLC7A11 protein levels in NPCs treated with miR-Exo with or without anti-miR-221-3p (n = 6 biologically independent samples). Comparisons were performed by two-tailed Student’s t test in panels (f, h, j, l). Data are presented as means ± SD. NPPC nucleus pulposus progenitor cell, NPPC-Exo NPPC-derived exosomes, miR-Exo miR-221-3p overexpressed NPPC-Exo, IRF8 interferon regulatory factor 8, COL2 Collagen II, ACSL4 acyl-coenzyme A synthetase long-chain family member 4, GPX4 Glutathione Peroxidase 4, STAT1 signal transducer and activator of transcription 1, SLC7A11 solute carrier family 7 member 11. Schematic illustrations were generated using BioRender (Wang, V., 2025; accessible at: https://BioRender.com/i62o243). Source data are provided as a Source Data file.

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