Molecular mechanism for miR-350 in regulating of titanium dioxide nanoparticles in macrophage RAW264.7 cells

Abstract

This study investigated the role of microRNA(miRNA) in regulating the cytotoxicity of TiO2 nanoparticles (nano-TiO2) to RAW264.7 cells. RAW264.7 cells were treated with 0 and 100 μg/ml nano-TiO2 for 24 h (for miRNA analysis). The differentially expressed miRNAs were detected using Illumina HiSeq™ 2000 sequencing. Through the bio-informatics analysis, miR-350 was found to play an important role in multiple signaling pathways, including MAPK signaling pathway, NF-kappa B signaling pathway and Apoptosis. To characterize the miR-350 function, miR-350 mimic was transfected into RAW264.7 cells for 24 h. MTT and Flow Cytometry were performed to detect cell proliferation, apoptosis and cell cycle (repetition), respectively. QRT-PCR, Western Blot methods and Luciferase assays were applied to detect expression of putative target gene PIK3R3. The results showed that miRNA profiles were differentially dysregulated. The apoptosis rate of miR-350 mimic group was significantly higher than negative control group (p < .05). Cell proliferation and cell cycle had no significant differences between treatment and negative control group. Compared with negative control, the level of protein of PIK3R3 was significantly decreased (p < .05), and the expression of 3′UTR constructs of PIK3R3 was significantly decreased (p < .05) in miR-350 mimic group. The expression of miRNAs was changed after exposed to nano-TiO2, and biological function and target gene results showed miR-350 may promote RAW264.7 cell apoptosis through the negative regulation of PIK3R3 gene. Our results could provide a basis for further understanding of toxicity and possible mechanisms of nano-TiO2 exposure.