Toll-like receptor 9 arginine methylation promotes ferroptosis in prostate cancer through the NRF2/GPX4 signaling pathway.

Background: A balance between positive and negative regulation of the Toll-like receptor (TLR) signaling pathway is required to prevent harmful or inappropriate inflammatory responses. Although TLR9 has been reported to be expressed in many mammalian tissues and cells, its function and exact mechanism are not fully understood. Some protein post-translational modifications (PTMs), such as phosphorylation and ubiquitination, are involved in the development of tumors. An important PTM, methylation, which controls the TLR9 signaling pathway, still remains unclear. Methods: Using human prostate cancer (PCa) cells PC3 and clinical PCa samples, this study employed LipidTOX staining, RNA interference, Western blot, immunoprecipitation, GST fusion protein sedimentation, and immunofluorescence techniques to investigate the molecular mechanism of TLR9 protein arginine methylation modification in PCa ferroptosis. Results: In this study, we found that the total level of methylation was reduced in PCa, the expression of TLR9 was decreased, and the level of reactive oxygen species (ROS) was increased in PCa cells, indicating that TLR9 may be involved in the regulation of ferroptosis in PCa. In addition, protein arginine methyltransferase 5 and 9 (PRMT5 and PRMT9) are recruited to mediate methylation modification of TLR9 after stimulation of unmethylated cytosine guanine [oligodeoxynucleotide (ODN)], but PRMT9 has the strongest effect, which prompted us to focus on PRMT9. Decreased expression of PRMT9 down-regulates arginine (R) methylation levels of TLR9. Methionine 260 (G260) mediates PRMT9 to catalyze arginine methylation of TLR9 on R216. In vitro transcriptomic results showed that the expression level of acyl synthetase medium chain family 1 (ACSM1) in cancer tissues was significantly higher than that in the para-cancer group. BODIPY-C11 immunofluorescence results indicated that TLR9 methylation could regulate the growth of PC3 cells mediated by ACSM1. In addition, arginine methylation enhancement of TLR9 induced by PRMT5 or 9 increased the transcription activity of nuclear factor erythroid 2-related factor 2 (NRF2), and arginine methylation of TLR9 at sites 216 and 305 mediated the interaction between TLR9 and NRF2 and enhanced the transcription activity of NRF2. PRMT9 mutation and (R216K) mutant TLR9 decreased NRF2 transcriptional activity, and R216 mutation decreased PRMT9-mediated arginine methylation GPX4 and other related iron-death protein levels. Conclusions: Our study reveals a key role of TLR9 protein arginine methylation in the regulation of ferroptosis, which may provide a therapeutic strategy for controlling the development and progression of PCa.