Fusaric acid alters global N6- methyladenosine RNA methylation and PI3K/AKT signalling in U87MG cells.

Abstract

Mycotoxins are a global concern due to the extensive damage and loss they cause in the agricultural sector. Mycotoxins are transferred from animal feed into animal-derived consumables,such as eggs, milk and meat presenting a danger to humans. The picolinic acid derivative Fusaric acid (FA), produced by various species of Fusarium, poses a significant risk to both animal and human health because of its toxicological effects on various tissues. It is noted for its unusually potent phytotoxicity in plants and shows prevalence in causing hepatotoxicity, genotoxicity and nephrotoxicity in humans. However, the effects on arguably the most important organ in the human body, the brain, remains incompletely understood. The study set out to investigate the cytotoxic effects of FA on U87MG human glioblastoma cells by monitoring alterations in global m6A RNA methylation as well as gene and/or protein expression levels of the m6A complex and the PI3K/Akt pathway. Methods comprised of (i) culture of U87MG cells; (ii) MTT assay (IC50: 180 μg/ml FA, 24 hrs) which was then used for subsequent treatments; (iii) ELISA; (iv) qRT-PCR (quantify mRNA expression of METTL3, METTL14, YTHDF1, YTHDF2, YTHDF3, FTO, WTAP, YTHDC1, YTHDC2, ALKBH5, BDNF and CREB); (v) western blot (protein expression of BDNF, P-AKT, PCREB and PI3K). FA caused an upregulation (2.1059-fold; p = 0.0150) of global m6A RNA methylation in U87MG cells relative to the control. FA caused a downregulation of mRNA expression for METTL3 (0.2605-fold; p = 0.0007); METTL14 (0.4137-fold; p = 0.0068); WTAP (0.2740-fold; p = 0.0004); YTHDF1 (0.7170-fold; p = 0.0793); YTHDF2 (0.6269-fold; p = 0.0224); YTHDC1 (0.9867-fold; p = 0.0008); YTHDC2 (0.0570-fold; p = 0.0003); FTO (0.4534-fold; p = 0.0039); ALKBH5 (0.0066-fold; p = 0.0004); BDNF (0.0106-fold; p = 0.0006) and CREB (0.9172- fold; p = 0.0003). However, YTHDF3 (1.335-fold; p = 0.0647) was upregulated. FA increased protein expression of BDNF (1,205-fold; p = 0.0173) and P-CREB (1.5537-fold; p = 0.0002) and decreased protein expression of PI3K (0.8411-fold; p = 0.0346) and P-Akt (0.8274-fold; p = 0.0614). The observed increase in global m6A, despite downregulation of ‘writers’ and ‘erasers’ underscores a complex interplay of compensatory mechanisms resulting from FA exposure. The differential expression of m6A ‘readers’, particularly the upregulation of YTHDF3 and downregulation of YTHDF1/2 and YTHDC1/2, suggests selective stabilization of survival-related transcripts to counteract FA-induced neurotoxicity. FA exposure resulted in the upregulation of BDNF and P-CREB protein levels which indicates a compensatory mechanism aimed at preserving neuroprotective signalling despite transcriptional repression. PI3K and P-AKT were downregulated indicating a suppression of growth and survival pathways which are potentially linked to oxidative stress and energy conservation under toxic stress.