MR Promotes Ferroptosis in Gastric Cancer by Regulating FANCD2 Expression Mediated by m6A Modification.

Abstract

Our previous work points out that methionine restriction (MR) treatment inhibits gastric cancer progression. Ferroptosis is a new form of cell death, and induction of ferroptosis has an inhibitory effect on tumors. Silencing of the ferroptosis inhibitory molecule FA complementation group D2 protein (FANCD2) has been reported to inhibit tumor growth. This investigation aims to explore whether MR treatment affects ferroptosis of gastric cancer cells by regulating FANCD2 expression, and thus affects the advancement of gastric cancer. Gastric cancer cells (AGS and HGC27) were cultured in MR condition. For ferroptosis detection, lipid ROS was examined by fluorescent staining; ACSL4 levels were estimated by western blot; malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) levels were measured via enzyme-linked immunosorbent assay. Transfection of FANCD2/METTL3 (methyltransferase-like 3) overexpression plasmids was to conduct in gain of function tests. SRAMP analysis was to predict the m6A methylation site of FANCD2, with methylated RNA immunoprecipitation detection of m6A levels of FANCD2 mRNA, and Actinomycin D experiments to evaluate its stability. Gastric cancer cells were administered through tail vein injection into BALB/c mice to conduct transplanted tumor models, and mice were given an MR diet or combined with an injection of oeFANCD2/oeMETTL3 lentivirus. The effect of FANCD2/METTL3 overexpression on tumor volume and ferroptosis was measured. The gastric cancer patient-derived organoids were also cultured and treated with MR, and the diameter was analyzed. MR treatment increased ferroptosis and reduced the volume of tumor tissue. FANCD2 levels were found to change dramatically following MR treatment, and overexpressing FANCD2 inhibited ferroptosis and promoted tumor formation. In addition, MR treatment decreased FANCD2 m6A abundance as well as FANCD2 mRNA stability. Database predictions suggested that METTL3 may be an m6A regulatory molecule influenced by MR, and our results showed that METTL3 was down-regulated under MR conditions, and METTL3 overexpression increased the m6A abundance and stability of FANCD2 mRNA. Further results showed that overexpressing METTL3 reduced ferroptosis-related indexes and increased the tumor volume, inhibiting METTL3 reversed the results. Furthermore, MR reduced the diameter of gastric cancer organoids. MR inhibits FANCD2 m6A levels and FANCD2 stability by inhibiting METTL3 expression, and then promotes ferroptosis in gastric cancer cells.