Zhang Daquan, Liu Xiaoyu, Luo Binyu, Zhang Xiao, Teng Qing, Xia Xingmei, Liao Bin
Department of Gastrointestinal, Anorectal, and Hernia Surgery, Beijing Anzhen Nanchong Hospital Affiliated to Capital Medical University & Nanchong Central Hospital, The Second Clinical Medical College of North Sichuan Medical College, Nanchong, 637000, Sichuan, China.
Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital & Chongqing Cancer Institute, Chongqing University Cancer Hospital, 181 Hanyu Road, Shapingba District, Chongqing, 400030, China.
J Mol Histol. 2025 Jul 21;56(4):233. doi: 10.1007/s10735-025-10496-2.
Ferroptosis, a form of regulated cell death caused by iron-dependent accumulation of lipid peroxides, is recently demonstrated as a vital player in cancer development. Tribbles homolog 3 (TRIB3) is a contributing factor to the malignant progression of several human cancers, including colorectal cancer (CRC). However, its regulatory effect and mechanism in CRC are obscure. qRT-PCR and western blot assays determined the mRNA and protein expression of TRIB3, methyltransferase-like 14 (METTL14), and YT521-B homology domain family 2 (YTHDF2). Cell ferroptosis was evaluated by measuring the levels of intracellular reactive oxygen species (ROS), lipid ROS, malondialdehyde (MDA), and glutathione (GSH). Cell malignant progression were assessed by CCK-8, EdU, transwell, and xenograft assays. The m6A sites of TRIB3 were confirmed using m6A RNA immunoprecipitation (Me-RIP) assay. The binding between TRIB3 and METTL14 or YTHDF2 was validated using RIP or luciferase reporter experiments. We observed higher expression of TRIB3 and lower expression of METTL14 in CRC tissues and cells. Knockdown of TRIB3 increased ferroptosis by promoting the generation of intracellular ROS, lipid ROS, and MDA and inhibiting the production of GSH. Suppressing TRIB3 also decreased tumor growth by increasing ferroptosis in mice. Mechanistically, knockdown of METTL14 reduced the m6A modification of TRIB3 and elevated TRIB3 mRNA expression. Moreover, METTL14-methylated TRIB3 was was recognized by YTHDF2, which resulted in the degradation of TRIB3 mRNA. TRIB3 overexpression reversed METTL14-mediated ferroptosis in CRC cells. Silencing YTHDF2 also abrogated the promotive effect of METTL14 on ferroptosis in CRC cells. Additionally, knockdown of TRIB3 induced ferroptosis by inactivating the SLC7A11/GPX4 signaling. METTL14 suppressed the SLC7A11/GPX4 signaling by targeting TRIB3. METTL14 suppressed CRC cell proliferation, migration, and invasion by downregulating TRIB3. Our findings suggest that METTL14 suppressed TRIB3 expression via an m6A-YTHDF2-dependent manner, thus inducing ferroptosis to inhibit the malignant progression of CRC. TRIB3 is potentially exploited as a molecular target for CRC treatment based on ferroptosis.
铁死亡是一种由铁依赖性脂质过氧化物积累引起的程序性细胞死亡形式，最近被证明是癌症发展中的一个重要因素。 Tribbles 同源物 3（TRIB3）是包括结直肠癌（CRC）在内的几种人类癌症恶性进展的一个促成因素。然而，其在 CRC 中的调节作用和机制尚不清楚。qRT-PCR 和蛋白质免疫印迹分析确定了 TRIB3、甲基转移酶样 14（METTL14）和 YT521-B 同源结构域家族 2（YTHDF2）的 mRNA 和蛋白质表达。通过测量细胞内活性氧（ROS）、脂质 ROS、丙二醛（MDA）和谷胱甘肽（GSH）的水平来评估细胞铁死亡。通过 CCK-8、EdU、Transwell 和异种移植试验评估细胞恶性进展。使用 m6A RNA 免疫沉淀（Me-RIP）试验确认 TRIB3 的 m6A 位点。使用 RIP 或荧光素酶报告实验验证 TRIB3 与 METTL14 或 YTHDF2 之间的结合。我们观察到 CRC 组织和细胞中 TRIB3 表达较高而 METTL14 表达较低。敲低 TRIB3 通过促进细胞内 ROS、脂质 ROS 和 MDA 的产生并抑制 GSH 的产生来增加铁死亡。抑制 TRIB3 还通过增加小鼠体内的铁死亡来减少肿瘤生长。机制上，敲低 METTL14 降低了 TRIB3 的 m6A 修饰并提高了 TRIB3 mRNA 的表达。此外，METTL14 甲基化的 TRIB3 被 YTHDF2 识别，导致 TRIB3 mRNA 的降解。TRIB3 过表达逆转了 METTL14 介导的 CRC 细胞铁死亡。沉默 YTHDF2 也消除了 METTL14 对 CRC 细胞铁死亡的促进作用。此外，敲低 TRIB3 通过使 SLC7A11/GPX4 信号失活诱导铁死亡。METTL14 通过靶向 TRIB3 抑制 SLC7A11/GPX4 信号。METTL14 通过下调 TRIB3 抑制 CRC 细胞的增殖、迁移和侵袭。我们的研究结果表明，METTL14 通过 m6A-YTHDF2 依赖性方式抑制 TRIB3 表达，从而诱导铁死亡以抑制 CRC 的恶性进展。基于铁死亡，TRIB3 有可能被用作 CRC 治疗的分子靶点。
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