The Role and Mechanism of Ferroptosis Mediated by METTL3-m6A Modification in Regulating Radioresistance of Esophageal Cancer

Abstract

Radioresistance remains the major cause of recurrence within two years after radical radiotherapy in approximately 50% of patients with esophageal cancer (ESCC). Recently, it has been reported that enhancing ferroptosis can reverse tumor radioresistance. However, the underlying mechanisms remain elusive. The aim of this study was to elucidate the role and mechanism of ferroptosis mediated by METTL3 N6-methyladenosine (m6A) modification in regulating radioresistance of Esophageal Cancer. We hypothesize that inhibition of ferroptosis confers radioresistance in Esophageal Cancer by METTL3-induced m6A modification which increase the expression of m6A in the 3'UTR region of SOCS6 and inhibits its expression. To explore the relationship between radioresistance and ferroptosis. Firstly, ferroptosis was analyzed by 4-HNE staining in ESCC specimens; Furthermore, cell death was detected by propidium iodide (PI) or SYTOX Green staining combined with microscopy or flow cytometry in ESCC radioresistance cells and parental cells, and Iron Assay Kit,C11-BODIPY were used to examine whether ferroptosis were inhibited in radioresistance cells. Subsequently, MeRIP-seq and MeRIP-PCR were performed for discussing the relationship between ferroptosis and m6A. Finally, Mettl3 depleted cells were generated with CRISPR/Cas9-mediated knockout system. Then, m6A methylation level, sensitivity to radiation and ferroptosis of Mettl3 depleted cells by EpiQuik m6A RNA methylation quantification kit, colony formation, C11-BODIPY and Western Blot. Firstly, we found that the level of lipid peroxide 4-HNE in ESCC samples that recurred after radical radiotherapy was significantly lower than that in radiotherapy (40Gy), and the ferroptosis of radioresistance cells was inhibited. MeRIP-seq found that the level of m6A in radioresistance cells increased significantly, and then the KEGG pathway analysis of MeRIP-seq results showed that hypermethylation of m6A was closely related to ferroptosis signal pathway. and we also found that the level of m6A was significantly decreased by adding agonist (Erastin), whereas the modification level was significantly increased by adding inhibitor Fer-1. Meanwhile, we found that the level of m6A was significantly decreased in radioresistance cells silencing METTL3, while the ferroptosis was activated, which caused the radiosensitivity of ESCC. Lastly, MeRIP-PCR data showed that METTLT3 induced a significant increase in m6A level in 3' UTR region of SOCS6 mRNA and inhibited its expression, which eventually led to radioresistance of ESCC. m6A-regulated ferroptosis inhibition confers radioresistance of ESCC. METTL3 up-regulated m6A in SOCS6 3' UTR and inhibited the expression of SOCS6, which caused the inhibition of ferroptosis.