Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin

BACKGROUND: Abnormal methylation of N(6)-methyladenosine (m(6)A) is reportedly associated with central nervous system disorders. However, the role of m(6)A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity requires further research. METHODS: Rat pheochromocytoma PC12 cells treated with UCB were used as in vitro models. After the PC12 cells were treated with UCB (0, 12, 18, and 24 µM) for 24 h, the total RNA m(6)A levels were measured using an m(6)A RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the m(6)A mRNA methylation profile in PC12 cells exposed to UCB (0 and 18 µM) for 24 h using methylated RNA immunoprecipitation sequencing (MeRIP-seq). RESULTS: Compared with the control group, UCB (18 and 24 µM) treatment decreased the expression of the m(6)A demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total m(6)A levels in PC12 cells. Furthermore, 1533 m(6)A peaks were significantly elevated and 1331 peaks were reduced in the UCB (18 µM)-treated groups compared with those in the control group. Genes with differential m(6)A peaks were mainly enriched in protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, cell cycle, and endocytosis. Through combined analysis of the MeRIP-seq and RNA sequencing data, 129 genes with differentially methylated m(6)A peaks and differentially expressed mRNA levels were identified. CONCLUSION: Our study suggests that the modulation of m(6)A methylation modifications plays a significant role in UCB neurotoxicity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11033-023-08576-1.