Study of Key Biological Pathways and Important microRNAs Involved in Silver Nanoparticles Induced Cytotoxicity Based on microRNA Sequencing Technology.

The aim of this study was to screen and research key biological pathways and important microRNAs (miRNAs) involved in silver nanoparticles (SNPs) induced cytotoxicity using miRNA sequencing technology. First, the influences of five SNPs with different sizes (∼5, ∼20, ∼50, ∼100 and ∼200 nm), named as SNP-5, SNP-20, SNP-50, SNP-100, and SNP-200, on human dermal fibroblasts (HDFs) proliferation were evaluated by MTT assay after 4, 8, 12, 24, 48 and 72 h. miRNA sequencing technology was then utilized to screen differentially expressed miRNAs in SNPs treated HDFs for 4 h. Bioinformatics analysis was performed to screen key biological pathways and important miRNAs in SNP-induced cytotoxicity. Finally, the functions of key biological pathways and expression level of important miRNAs were verified using different cellular/molecular biology experiments. MTT results showed that the effect of SNP-5 on cell proliferation was greatest at all-time points, while the effect of SNP-20 was minimal. miRNA sequencing results showed that 457, 76, 177, 461 and 341 miRNAs were found to be differentially expressed in HDFs after treatment with five SNPs. Biological pathway analysis for differentially expressed miRNAs revealed that MAPK signaling pathway played a key role in SNP-induced cytotoxicity and its functions were verified by cell cycle and cell apoptosis tests. Four important miRNAs (miR-424-5p, miR-340-5p, miR-30b-5p, and miR-132-3p) were screened and expressions of miR-424-5p and miR-340-5p were confirmed by qRT-PCR. In conclusion, results from this study suggested that the SNPs arrested cell cycle, promoted cell apoptosis and finally induced cytotoxicity mainly through the MAPK signaling pathway. The expression of two important miRNAs had strong correlation with cytotoxicity of differently sized SNPs.