Xin Lin, Fan Luo-Jun, Liu Chuan, Lu Hao, Zou Yong-Hui, Xu He-Song, Yue Zhen- Qi, Gan Jin-Heng, Liu Jiang, Zhou Qi
Department of General Surgery, The Second Affiliated Hospital of Nanchang University, No. 1 Minde Road, Donghu District, Nanchang, 330006, Jiangxi Province, China.
Intelligent Medical Imaging of Jiangxi Key Laboratory, Nanchang, 330006, China.
Appl Biochem Biotechnol. 2025 Aug 12. doi: 10.1007/s12010-025-05355-5.
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.
我们之前的研究指出，蛋氨酸限制（MR）治疗可抑制胃癌进展。铁死亡是一种新的细胞死亡形式，诱导铁死亡对肿瘤具有抑制作用。据报道，铁死亡抑制分子范可尼贫血互补组D2蛋白（FANCD2）的沉默可抑制肿瘤生长。本研究旨在探讨MR治疗是否通过调节FANCD2表达影响胃癌细胞的铁死亡，进而影响胃癌的进展。将胃癌细胞（AGS和HGC27）在MR条件下培养。用于铁死亡检测时，通过荧光染色检测脂质活性氧；通过蛋白质免疫印迹法评估酰基辅酶A合成酶长链家族成员4（ACSL4）水平；通过酶联免疫吸附测定法测量丙二醛（MDA）和4-羟基壬烯醛（4-HNE）水平。转染FANCD2/甲基转移酶样3（METTL3）过表达质粒以进行功能获得试验。利用SRAMP分析预测FANCD2的m6A甲基化位点，通过甲基化RNA免疫沉淀检测FANCD2 mRNA的m6A水平，并通过放线菌素D实验评估其稳定性。将胃癌细胞通过尾静脉注射到BALB/c小鼠体内构建移植瘤模型，给小鼠喂食MR饮食或联合注射oeFANCD2/oeMETTL3慢病毒。检测FANCD2/METTL3过表达对肿瘤体积和铁死亡的影响。还培养了源自胃癌患者的类器官并用MR处理，分析其直径。MR治疗增加了铁死亡并减小了肿瘤组织的体积。发现MR治疗后FANCD2水平发生显著变化，过表达FANCD2可抑制铁死亡并促进肿瘤形成。此外，MR治疗降低了FANCD2的m6A丰度以及FANCD2 mRNA的稳定性。数据库预测表明METTL3可能是受MR影响的m6A调节分子，我们的结果表明在MR条件下METTL3表达下调，过表达METTL3可增加FANCD2 mRNA的m6A丰度和稳定性。进一步的结果表明，过表达METTL3降低了铁死亡相关指标并增加了肿瘤体积，抑制METTL3可逆转这些结果。此外，MR减小了胃癌类器官的直径。MR通过抑制METTL3表达抑制FANCD2的m6A水平和FANCD2稳定性，进而促进胃癌细胞的铁死亡。
