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

Background: Abnormal methylation of N-methyladenosine (mA) is reportedly associated with central nervous system disorders. However, the role of mA 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 mA levels were measured using an mA RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the mA 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 mA demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total mA levels in PC12 cells. Furthermore, 1533 mA 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 mA 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 mA peaks and differentially expressed mRNA levels were identified.

Conclusion: Our study suggests that the modulation of mA methylation modifications plays a significant role in UCB neurotoxicity.