Changes of m6A modification in arsenic-induced oxidative stress of human embryonic lung fibroblasts

Abstract

BackgroundArsenic can be toxic to human by triggering oxidative stress, which is companied by epigenetic modifications.ObjectiveTo investigate the modification of N6-methyladenosine (m6A) in human embryonic lung fibroblasts (HELF) during oxidative stress induced by sodium arsenite (NaAsO2).MethodsHELF cells were treated by designed concentrations of NaAsO2 (0, 2.5, 5, 10, and 20 μmol·L−1) for 48 h. Cell viability was detected by 3-(4,5-dimethylthia zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) method; the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) as well as the content of malondialdehyde (MDA) were detected with corresponding kits; the level of m6A methylation in total RNA was detected by enzyme-linked immunosorbent assay; the mRNA expressions of m6A modified enzymes were detected by real-time fluorescence quantitative PCR, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms' tumor 1-associated protein (WTAP), fat mass and obesity-associated protein (FTO), alkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases 5 (ALKBH5), YTH domain containing protein 2 (YTHDC2), YTH domain family protein 2 (YTHDF2), and YTH domain family protein 3 (YTHDF3); the protein expressions of METTL3, FTO, YTHDC2, YTHDF3, and nuclear factor erythroid 2-related factor 2 (NRF2) were detected by Western blotting. The enrichment of m6A in NRF2 mRNA was detected by RNA methylated immunoprecipitation combined with real-time fluorescence quantitative PCR (MeRIP-qPCR).ResultsAfter the 0, 2.5, 5, 10, and 20 μmol·L−1 NaAsO2 treatment, the MTS results showed that compared with the control group, the cell viability of the 20 μmol·L−1 group decreased to 84% (P<0.05). The colorimetry results showed that compared with the control group, the activities of T-SOD in the 10 and 20 μmol·L−1 groups decreased (P<0.05); the activities of GSH-Px in the 2.5 and 10 μmol·L−1 groups decreased (P<0.05); the contents of MDA in the 10 and 20 μmol·L−1 groups increased. The results of enzyme-linked immunosorbent assay showed that the overall m6A methylation levels in the 0, 2.5, 5, 10, and 20 μmol·L−1 groups were (0.193 ± 0.023)%, (0.247 ± 0.021)%, (0.253 ± 0.006)%, (0.233 ± 0.006)%, and (0.262 ± 0.010)%, respectively, and compared with the control group, the m6A methylation levels in all the NaAsO2 treated groups increased (P<0.05). The real-time fluorescence quantitative PCR results showed that compared with the control group, the mRNA relative expression level of METTL3 decreased in the 2.5, 10, and 20 μmol·L−1 groups (P<0.05); the mRNA relative expression level of FTO decreased in the 20 μmol·L−1 group; the mRNA relative expression level of YTHDC2 increased in the 10 and 20 μmol·L−1 groups (P<0.05); the mRNA relative expression level of YTHDF3 increased in the 2.5, 10, and 20 μmol·L−1 groups (P<0.05). The Western blotting results showed that compared with the control group, the relative protein expression of METTL3 decreased in the 10 and 20 μmol·L−1 groups; the relative protein expression of FTO decreased in the 5 and 20 μmol·L−1 groups; the relative protein expression of YTHDC2 decreased in the 20 μmol·L−1 group (P<0.05); the relative nuclear protein expression of NRF2 decreased in the 10 and 20 μmol·L−1 groups (P<0.05). The MeRIP-qPCR results showed that m6A enrichment was significantly increased in the 20 μmol·L−1 NaAsO2 exposure group compared with the control group (P<0.05). After over-expression of FTO, the mRNA and protein relative expression levels of FTO and the relative expression level of nuclear protein of NRF2 in the FTO group were higher than those in the control group (P<0.05); the mRNA and protein relative expression levels of FTO in the NaAsO2 + FTO group and the nuclear protein expression level of NRF2 were higher than those in the NaAsO2 group (P<0.05).ConclusionIn the process of oxidative stress induced by NaAsO2, m6A methylation level, m6A modified enzymes, m6A modification of NRF2 mRNA, and NRF2 expression could change in HELF cells.

Keywords

• arsenic
• oxidative stress
• n6-methyladenosine
• human embryonic lung fibroblast
• nuclear factor erythroid 2-related factor 2