Comprehensive evaluation of microRNA expression profiling reveals the neural signaling specific cytotoxicity of superparamagnetic iron oxide nanoparticles (SPIONs) through N-methyl-D-aspartate receptor

Abstract

Though nanomaterials are considered as drug carriers or imaging reagents targeting the central nervous system their cytotoxicity effect on neuronal cells has not been well studied. In this study, we treated PC12 cells, a model neuronal cell line, with a nanomaterial that is widely accepted for medical use, superparamagnetic iron oxide nanoparticles (SPIONs). Our results suggest that, after treated with SPIONs, the expression pattern of the cellular miRNAs changed widely in PC12 cells. As potential miRNA targets, NMDAR, one of the candidate mRNAs that were selected using GO and KEGG pathway enrichment, was significantly down regulated by SPIONs treatment. We further illustrated that SPIONs may induce cell death through NMDAR suppression. This study revealed a NMDAR neurotoxic effect of SPIONs and provides a reliable approach for assessing the neurocytotoxic effects of nanomaterials based on the comprehensive annotation of miRNA profiling.

Fig 1. Morphology and uptake of SPIONS in PC12 cells.

A confocal image of the SPIONs is shown (A). Prussian blue staining was performed to measure the uptake of SPIONs (214ug/ml) in PC12 cells at 0 (B), 12 (C) and 24 h (D). The majority of cells can incorporate SPIONs at 24 h (D).

Fig 2. miRNA high-throughput deep sequencing for PC12 cells treated with or without SPIONs.

Deep sequencing results show that the majority of the reads were 18–25nt, with the most abundant size of 22nt in both the control and SPIONs treated group (A). The abundance of many miRNAs is less than 0.1% (B). Correlation analysis between the convergence of miRNAs in SPIONs treated or non-treated cells shows that the relative amounts of some miRNAs deviated strikingly, although most miRNAs were regulated somewhat (C).

Fig 3. Assessment of neuronal cytotoxicity of SPIONs.

Based on their miRNA target profile, 5883 mRNAs were predicted to be regulated in cells following treatment with SPIONs. Approximately 31% of the mRNA candidates were significantly regulated by nanomaterial treatment. This gene set was enriched for gene ontology (GO) functions (A). We also sorted the mRNAs into KEGG pathways through DAVID. The “Amyotrophic Lateral Sclerosis (ALS) pathway” (C) was of the most highly affected KEGG pathway with p <10⁻⁷(B).

Fig 4. SPIONs induce neuronal death through the NMDAR pathway.

A selected group of the genes predicted to be regulated by SPION treatment are shown (A). The expression of NMDAR2A and NMDAR2D were suppressed, while NMDAR2C showed a slight elevation expression pattern, which was verified by western blotting (B). The expression of apoptosis-related proteins, capase-12 and Cyt-c were also predicted to be unregulated and were verified to be increased following treatment with SPION (C). NF: Neurofilament. NF-LP: Neurofilament-light peptide.