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

Abstract

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.

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Availability of data and material

All datasets generated in this study are available from the corresponding author on reasonable request.

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Funding

The authors acknowledge the National Research Foundation (NRF-Grant No. 120820) of South Africa and College of Health Science (University of KwaZulu-Natal) for funding this study.

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TA, TG, and AC conceptualised and designed the study. TA conducted all laboratory experiments, analysed the data and wrote the manuscript. TG and AC revised the manuscript. All authors have read the manuscript prior to submission.

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Correspondence to Anil A. Chuturgoon.

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Ethic was received from the University of KwaZulu-Natal’s Biomedical Research Ethics Committee. Ethics No. BE322/19.

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Arumugam, T., Ghazi, T. & Chuturgoon, A.A. Fumonisin B1 alters global m6A RNA methylation and epigenetically regulates Keap1-Nrf2 signaling in human hepatoma (HepG2) cells. Arch Toxicol 95, 1367–1378 (2021). https://doi.org/10.1007/s00204-021-02986-5

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Keywords

  • Fumonisin B1
  • Epigenetics
  • m6A RNA methylation
  • Oxidative stress
  • Keap1
  • Nrf2