Fumonisin B1 alters global m6A RNA methylation and epigenetically regulates Keap1-Nrf2 signaling in human hepatoma (HepG2) cells.

FB1 is a common contaminant of cereal grains that affects human and animal health. It has become increasingly evident that epigenetic changes are implicated in FB1 toxicity. N6-methyladenosine (m6A), the most abundant post-transcriptional RNA modification, is influenced by fluctuations in redox status. Since oxidative stress is a characteristic of FB1 exposure, we determined if there is cross-talk between oxidative stress and m6A in FB1-exposed HepG2 cells. Briefly, HepG2 cells were treated with FB1 (0, 5, 50, 100, 200 µM; 24 h) and ROS, LDH and m6A levels were quantified. qPCR was used to determine the expression of m6A modulators, Nrf2, Keap1 and miR-27b, while western blotting was used to quantify Keap1 and Nrf2 protein expression. Methylation status of Keap1 and Nrf2 promoters was assessed and RNA immunoprecipitation quantified m6A-Keap1 and m6A-Nrf2 levels. FB1 induced accumulation of intracellular ROS (p ≤ 0.001) and LDH leakage (p ≤ 0.001). Elevated m6A levels (p ≤ 0.05) were accompanied by an increase in m6A "writers" [METLL3 (p ≤ 0.01) and METLL14 (p ≤ 0.01)], and "readers" [YTHDF1 (p ≤ 0.01), YTHDF2 (p ≤ 0.01), YTHDF3 (p ≤ 0.001) and YTHDC2 (p ≤ 0.01)] and a decrease in m6A "erasers" [ALKBH5 (p ≤ 0.001) and FTO (p ≤ 0.001)]. Hypermethylation and hypomethylation occurred at Keap1 (p ≤ 0.001) and Nrf2 (p ≤ 0.001) promoters, respectively. MiR-27b was reduced (p ≤ 0.001); however, m6A-Keap1 (p ≤ 0.05) and m6A-Nrf2 (p ≤ 0.01) levels were upregulated. This resulted in the ultimate decrease in Keap1 (p ≤ 0.001) and increase in Nrf2 (p ≤ 0.001) expression. Our findings reveal that m6A RNA methylation can be modified by exposure to FB1, and a cross-talk between m6A and redox regulators does occur.