Mettl3-mediated m⁶A RNA methylation regulates osteolysis induced by titanium particles

Peri-prosthetic osteolysis (PPO) induced by wear particles is considered the primary cause of titanium prosthesis failure and revision surgery. The specific molecular mechanisms involve titanium particles inducing multiple intracellular pathways, which impact disease prevention and the targeted therapy of PPO. Notably, N6-methyladenosine (m6A) serves critical roles in epigenetic regulation, particularly in bone metabolism and inflammatory responses. Thus, the present study aimed to determine the role of RNA methylation in titanium particle-induced osteolysis. Results of reverse transcription-quantitative PCR (RT-qPCR), western blotting, ELISA and RNA dot blot assays revealed that titanium particles induced osteogenic inhibition and proinflammatory responses, accompanied by the reduced expression of methyltransferase-like (Mettl) 3, a key component of m6A methyltransferase. Specific lentiviruses vectors were employed for Mettl3 knockdown and overexpression experiments. RT-qPCR, western blotting and ELISA revealed that the knockdown of Mettl3 induced osteogenic inhibition and proinflammatory responses comparable with that induced by titanium particle, while Mettl3 overexpression attenuated titanium particle-induced cellular reactions. Methylated RNA immunoprecipitation-qPCR results revealed that titanium particles mediated the methylation of two inhibitory molecules, namely Smad7 and SMAD specific E3 ubiquitin protein ligase 1, via Mettl3 in bone morphogenetic protein signaling, leading to osteogenic inhibition. Furthermore, titanium particles induced activation of the nucleotide binding oligomerization domain 1 signaling pathway through methylation regulation, and the subsequent activation of the MAPK and NF-kappa B pathways. Collectively, the results of the present study indicated that titanium particles utilized Mettl3 as an upstream regulatory molecule to induce osteogenic inhibition and inflammatory responses. Thus, the present study may provide novel insights into potential therapeutic targets for aseptic loosening in titanium prostheses.