m6A Methylation‐Modified hsa_circ_0044226 Attenuates Pulmonary Fibrosis by Suppressing Ferroptosis

Idiopathic Pulmonary fibrosis (IPF) is a debilitating lung condition marked by chronic progression and reduced lung function. This study aimed to explore the functional role and regulatory mechanism of circular RNA circ_0044226 in the pathogenesis of pulmonary fibrosis. The expression of circ_0044226 was analyzed in peripheral blood and TGF-β1-stimulated RLE-6TN cells using qRT-PCR. The cellular location of circ_0044226 was analyzed using FISH. The expression levels of circ_0044226 and METTL3 were evaluated in cells and clinical samples. Protein levels of GPX4 and FTH1 were examined using Western blot, while the levels of MDA, Fe²⁺ and ROS were determined using commercial kits. The association of circ_0044226 with m6A modifications was validated by Methylated-RNA immunoprecipitation assay (MeRIP), RNA pulldown, RIP and dot blot assay. Finally, a Bleomycin (BLM) treated rat model was also utilized to verify the function of circ_0044226 in pulmonary fibrosis. The primary localization of circ_0044226 was within the cytoplasm and was found to be upregulated in blood samples from IPF patients and TGF-β1-treated RLE-6TN cells. Knockdown of circ_0044226 reduced the accumulation of free iron and MDA, and suppressed α-SMA and FN1 in TGF-β1-induced cells. Furthermore, METTL3 mediated the m6A modification of circ_0044226. Downregulation of METTL3 mitigated ferroptosis by inhibiting hsa_circ_0044226. The rat pulmonary fibrosis model further demonstrated that METTL3 knockdown alleviated pulmonary fibrosis by blocking ferroptosis through circ_0044226. Our findings demonstrated that targeting the METTL3/circ_0044226 axis attenuated pulmonary fibrosis by inhibiting ferroptosis in the studied models.