The miR-339-5p/ACSL4 Pathway Drives Ferroptosis in Macrophages Exposed to Silica Nanoparticles

The widespread application of silica nanoparticles (SiNPs) in industrial fields has raised increasing concerns regarding their potential biological toxicity. However, the cellular mechanisms underlying SiNPs-related injury remain incompletely understood. This study was designed to investigate whether ferroptosis contributes to SiNPs-mediated cytotoxicity in macrophages and to explore the potential regulatory role of microRNAs.

RAW264.7 cells were exposed to SiNPs to establish an in vitro toxicity model. Mitochondrial ultrastructure and cellular uptake were examined. Ferroptosis-related markers, including antioxidant capacity, lipid peroxidation, ferrous iron accumulation, and the expression of glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), and cyclooxygenase-2 (COX2), were assessed. The involvement of apoptosis and necroptosis was evaluated using specific pathway inhibitors. Transcriptomic sequencing was performed to identify differentially expressed miRNAs, followed by functional validation through miR-339-5p mimic transfection, and the direct interaction between miR-339-5p and ACSL4 was verified by dual luciferase reporter assay.

SiNPs were efficiently internalized by RAW264.7 cells and induced pronounced mitochondrial structural damage, characterized by mitochondrial shrinkage and loss of cristae. SiNPs exposure markedly impaired cellular antioxidant capacity and promoted the accumulation of lipid peroxides and intracellular Fe²⁺. This coincided with a reduction in GPX4 expression, whereas ACSL4 and COX2 levels increased. Ferroptosis was identified as the predominant mode of SiNPs-induced cytotoxicity, while apoptosis and necroptosis played minor roles. Transcriptomic analysis identified miR-339-5p as a potential regulator associated with ferroptosis-related pathways, and the dual luciferase reporter assay confirmed that miR-339-5p directly targets Acsl4. Overexpression of miR-339-5p significantly attenuated the ferroptosis response induced by SiNPs.

SiNPs induce ferroptosis as the predominant mode of cell death in macrophages through mechanisms involving oxidative stress and lipid peroxidation. miR-339-5p acts as a negative regulator of this process by directly targeting ACSL4, alleviating ferroptotic injury and providing new insights into the molecular mechanisms underlying the toxicity of SiNPs.