FTO/m6A 介导 miR-138-5p 的成熟，并通过 miR-138-5p/LCN2 轴调控肺腺癌细胞对吉非替尼的耐药性。

BACKGROUND: Lung cancer (LC) occupies an important position in the lethality of cancer patients. Acquired resistance to gefitinib in lung adenocarcinoma (LUAD) seriously affects the therapeutic efficacy of LC. Thus, it is of major scientific and clinical significance to probe the mechanism of gefitinib resistance in LUAD for ameliorating the prognosis of patients. METHODS: The expression of miRNAs in gefitinib-resistant LUAD cells was validated using qRT-PCR. Cell viability was assessed through CCK-8, whereas cell death was examined through PI staining. Changes in the ferroptosis process were evaluated by detecting the intracellular Glutathione (GSH), Malondialdehyde (MDA), and Reactive Oxygen Species (ROS) levels. Downstream targets of miR-138-5p were verified via luciferase reporter and RNA pull-down assays. RIP and qRT-PCR were employed to evaluate pri-miR-138-5p binding to DiGeorge critical region 8 (DGCR8) and the pri-miR-138-5p m6A modification level. Additionally, the impact of fat mass and obesity-associated protein (FTO) on LUAD gefitinib sensitivity was assessed in vivo by constructing a xenograft model. RESULTS: We observed that miR-138-5p was notably diminished in gefitinib-resistant cells. Overexpression of miR-138-5p suppressed viability while facilitated cell death and intracellular ferroptosis in gefitinib-resistant cells. Moreover, lipocalin 2 (LCN2) was the downstream target of miR-138-5p. The biological functions of miR-138-5p on gefitinib-resistant cells was reversed by introduction of LCN2. FTO suppressed the binding of DGCR8 to pri-miR-138-5p through m6A modification, thereby restraining the processing of miR-138-5p. Meanwhile, silencing of FTO enhanced the sensitivity of LUAD to gefitinib treatment. CONCLUSION: FTO suppressed the processing of miR-138-5p and then modulated the proliferation, death, and ferroptosis of gefitinib-resistant cells through the miR-138-5p/LCN2 pathway, which may put forward novel insights for clinically ameliorating the therapeutic effect of gefitinib in LUAD.

背景：肺癌（LC）在癌症患者的致死率中占有重要地位。肺腺癌（LUAD）对吉非替尼的获得性耐药严重影响 LC 的治疗效果。因此，探索 LUAD 中吉非替尼耐药的机制对于改善患者预后具有重要的科学和临床意义。

方法：采用 qRT-PCR 验证吉非替尼耐药 LUAD 细胞中 miRNAs 的表达。通过 CCK-8 检测细胞活力，通过 PI 染色检测细胞死亡。通过检测细胞内谷胱甘肽（GSH）、丙二醛（MDA）和活性氧（ROS）水平来评估铁死亡过程的变化。通过荧光素酶报告和 RNA 下拉测定验证 miR-138-5p 的下游靶标。RIP 和 qRT-PCR 用于评估 pri-miR-138-5p 与 DiGeorge 关键区域 8（DGCR8）的结合和 pri-miR-138-5p 的 m6A 修饰水平。此外，通过构建异种移植模型评估肥胖相关蛋白（FTO）对 LUAD 吉非替尼敏感性的影响。

结果：我们观察到 miR-138-5p 在吉非替尼耐药细胞中明显减少。过表达 miR-138-5p 抑制了吉非替尼耐药细胞的活力，同时促进了细胞死亡和细胞内铁死亡。此外，脂钙蛋白 2（LCN2）是 miR-138-5p 的下游靶标。引入 LCN2 可逆转 miR-138-5p 对吉非替尼耐药细胞的生物学功能。FTO 通过 m6A 修饰抑制 DGCR8 与 pri-miR-138-5p 的结合，从而抑制 miR-138-5p 的加工。同时，沉默 FTO 增强了 LUAD 对吉非替尼治疗的敏感性。

结论：FTO 抑制 miR-138-5p 的加工，然后通过 miR-138-5p/LCN2 通路调节吉非替尼耐药细胞的增殖、死亡和铁死亡，这可能为临床上改善 LUAD 中吉非替尼的治疗效果提供新的思路。