ABSTRACT
Background Occupational and environmental particulate matter may cause fibrosis, accompanied by RNA m6A modification changes. Neodymium oxide (Nd2O3) can cause mouse lung fibrosis, which contains a large number of fibroblasts. Objective To investigate m6A modification of tumor necrosis factor receptor-associated protein 6/nuclear factor-κB (TRAF6/NF-κB) signaling pathway in fibrosis of human embryonic lung fibroblasts induced by Nd2O3, and identify the key m6A modification sites of TRAF6. Methods Designed concentrations of Nd2O3 (0, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg∙L−1) were infected with HELF cells for 24 and 48 h, and cell viability was detected to determine exposure time and dose. Measurements included indicators of fibrosis [hydroxyproline (HYP) and transforming growth factor-β1 (TGF-β1)], m6A methylation level, methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), reading proteins (YTHDC2 and YTHDF2), fibrosis-associated genes (collagen-І, vimentin, and α-SMA), and proteins related to signaling pathway (TRAF6, NFKB1, P65, and P-P65). The enrichment of m6A in TRAF6 mRNA was measured by methylated RNA immunoprecipitation-quantitative real-time PCR (MeRIP-qPCR). Results The results of cell viability indicated that 6.25, 12.5, 25 mg∙L−1 Nd2O3 and 48 h exposure time were used for subsequent experiments. After 48 h exposure, compared with the control group, the HYP level in the 25 mg∙L−1 Nd2O3 group was increased, and the levels of TGF-β1 in the 6.25, 12.5, and 25 mg∙L−1 Nd2O3 groups were increased (P<0.05); the overall m6A methylation levels of HELF cells in the 12.5 and 25 mg∙L−1 Nd2O3 groups were increased (P<0.05). At mRNA level, compared with the control group, the mRNA expression levels of methyltransferases METTL3 and METTL14 (6.25, 12.5, and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the mRNA expression level of reading protein YTHDF2 (6.25, 12.5, and 25 mg∙L−1 Nd2O3) was increased (P<0.05), while the mRNA expression level of YTHDC2 (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the mRNA expression levels of demethylases FTO (12.5 and 25 mg∙L−1 Nd2O3) and ALKBH5 (25 mg∙L−1 Nd2O3) were decreased (P<0.05); the mRNA expression levels of fibrosis-related genes vimentin, α-SMA, and collagen-Ⅰ (6.25, 12.5, and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the mRNA expression levels of pathway-related genes TRAF6 (25 mg∙L−1 Nd2O3) and NFKB1 (12.5 and 25 mg∙L−1 Nd2O3) were increased (P<0.05). At protein level, compared with the control group, the expression levels of methyltransferases METTL3 (25 mg∙L−1 Nd2O3) and METTL14 (12.5 and 25 mg∙L−1 Nd2O3) were increased (P<0.05); the expression level of reading protein YTHDF2 (12.5 and 25 mg∙L−1 Nd2O3) was increased, while the expression level of YTHDC2 (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the expression level of demethylase FTO (25 mg∙L−1 Nd2O3) was decreased (P<0.05); the expression level of fibrosis-associated protein vimentin was increased at 25 mg∙L−1 Nd2O3, and the expression levels of α-SMA and collagen-Ⅰ were increased at 12.5 and 25 mg∙L−1 Nd2O3 (P<0.05); the expression levels of TRAF6 and P-P65 were increased at 25 mg∙L−1 Nd2O3 (P<0.05). The MeRIP-qPCR results showed that compared with the control group, the concentrations of m6A in all Nd2O3 groups were significantly increased (P<0.05). Conclusions Upon exposure of HELF cells to Nd2O3, the alterations in fibrosis-related indexes increase the expression of some m6A methylases and decrease the expression of demethylases, thereby increasing the m6A methylase level, and may promote the progression of fibrosis by activating the TRAF6/NF-κB signaling pathway.