Brain-Derived Neurotrophic Factor Mediated Perfluorooctane Sulfonate Induced-Neurotoxicity via Epigenetics Regulation in SK-N-SH Cells

Abstract

Perfluorooctane sulfonate (PFOS), a new kind of persistent organic pollutant, is widely distributed in the environment and exists in various organisms, where it is also a neurotoxic compound. However, the potential mechanism of its neurotoxicity is still unclear. To examine the role of epigenetics in the neurotoxicity induced by PFOS, SK-N-SH cells were treated with different concentrations of PFOS or control medium (0.1% DMSO) for 48 h. The mRNA levels of DNA methyltransferases (DNMTs) and Brain-derived neurotrophic factor (BDNF), microRNA-16, microRNA-22, and microRNA-30a-5p were detected by Quantitative PCR (QPCR). Enzyme Linked Immunosorbent Assay (ELISA) was used to measure the protein levels of BDNF, and a western blot was applied to analyze the protein levels of DNMTs. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the BDNF promoter I and IV. Results of MTT assays indicated that treatment with PFOS could lead to a significant decrease of cell viability, and the treated cells became shrunk. In addition, PFOS exposure decreased the expression of BDNF at mRNA and protein levels, increased the expression of microRNA-16, microRNA-22, microRNA-30a-5p, and decreased the expression of DNMT1 at mRNA and protein levels, but increased the expression of DNMT3b at mRNA and protein levels. Our results also demonstrate that PFOS exposure changes the methylation status of BDNF promoter I and IV. The findings of the present study suggest that methylation regulation of BDNF gene promoter and increases of BDNF-related-microRNA might underlie the mechanisms of PFOS-induced neurotoxicity.

Keywords: BDNF; DNA methylation; DNMTs; PFOS; microRNA.

Figure 1

Effects of PFOS on SK-N-SH cell morphology. SK-N-SH cell morphology was observed after a 48-h exposure to various concentrations of PFOS. (A) Control; (B) 50 μM PFOS; (C) 100 μM PFOS; (D) 150 μM PFOS; (E) 200 μM PFOS; (F) 250 μM PFOS. (Bar: 50 μm).

Figure 2

Effects of PFOS on SK-N-SH cell viability. Cell viability was determined by MTT assay after 24 or 48-h exposure to various concentrations of PFOS (50, 100, 150, 200, 250 μM) or DMSO (control). Data are presented as mean ± SD of three separate experiments. * Compared with control, respectively: p < 0.05.

Figure 3

Effects of PFOS on the expression of brain-derived neurotrophic factor (BDNF), in SK-N-SH cells. (A) BDNF mRNA levels in SH-SY5Y cells. Each data point was normalized to the control (DMSO). (B) BDNF protein levels in SH-SY5Y cells. The data are presented as mean ± SD from three independent experiments. * Compared with control: p < 0.05.

Figure 4

Bisulfite-sequencing methylation analysis of individual CpG dinucleotides of the BDNF promoter I (A) and IV (B) from the SK-N-SH Cells. (A) Locations of these 30 CpG sites within promoter I are listed and the methylation status was assessed by bisulfite sequencing. (B) Locations of these 20 CpG sites within promoter IV are listed and the methylation status was assessed by bisulfite sequencing. (C) Methylation of promoter. White and black circles denote unmethylated and methylated CpG sites, respectively. Each row indicates a specific plasmid clone. Ten clones came from the same group. The underline in the image (A,B) represent CpG dinucleotides site.

Figure 5

Effects of PFOS on the expression of DNMTs in SK-N-SH cells. Cells were cultured for 48 h with various concentrations of PFOS (50, 100, 150, 200 μM) or DMSO (control). (A) DNMT1 mRNA levels in SK-N-SH cells; (B) DNMT1 protein levels in SK-N-SH cells; (C) DNMT3a mRNA levels in SK-N-SH cells; (D) DNMT3a protein levels in SK-N-SH cells; (E) DNMT3b mRNA levels in SK-N-SH cells; (F) DNMT3b protein levels in SK-N-SH cells; (G) Protein lane of DNMT1, DNMT3a, DNMT3b, and β-actin. Lane 1, control; lane 2, 50 μM PFOS; lane 3, 100 μM PFOS; lane 4, 150 μM PFOS. The data are presented as mean ± SD from three independent experiments. * Compared with control: p < 0.05.

Figure 6

Effects of PFOS on the expression of mRNAs in SK-N-SH cells. Cells were cultured for 48 h with various concentrations of PFOS (50, 100, 150, 200 μM) or DMSO (control). (A) mRNA levels of microRNA-16 in SK-N-SH cells; (B) mRNA levels of microRNA-30a-5p in SK-N-SH cells; (C) mRNA levels of microRNA-22 in SK-N-SH cells. Each data point was normalized to the control (DMSO), and the data are presented as mean ± SD from three independent experiments. * Compared with control: p < 0.05.