Chen Lingxiao, He Cheng, Ou Zhenyu, Zhao Cheng
Department of Urology, Xiangya Hospital, Central South University, Changsha City, Hunan Province, China.
Department of Urology, Xiangya Hospital, Central South University, Changsha City, Hunan Province, China.
Biochim Biophys Acta Mol Basis Dis. 2025 Jun;1871(5):167811. doi: 10.1016/j.bbadis.2025.167811. Epub 2025 Mar 19.
Bladder cancer (BCa) metastasis is a multi-step process triggered by cytoskeleton reorganization. However, the regulation mechanism of cytoskeleton reorganization in BCa remains ambiguous. This study elucidated the influence of tumor necrosis factor-alpha (TNF-α) in cytoskeleton remodeling during BCa metastasis and its possible mechanisms.
Colony formation, scratch, transwell, and the nude mouse model were adopted to evaluate the growth and metastasis. Molecular expression was assessed by immunohistochemical staining, quantitative real-time PCR (qRT-PCR), and Western blotting. The N6-methyladenosine (m6A) level was detected by methylated RNA immunoprecipitation (MeRIP). Protein interaction was validated by Co-immunoprecipitation (Co-IP). Immunofluorescence staining was used to identify rearrangement of actin filament fibers (F-actin) and protein colocalization.
TNF-α facilitated cytoplasmic linker associated protein 2 (CLASP2) and methyltransferase like 3 (METTL3) expression in a dose (10-50 ng/mL)-dependent manner in BCa. CLASP2 high expression suggested a shorter overall survival of BCa patients. CLASP2 deficiency suppressed BCa cell proliferation, migration, and invasion via disrupting F-actin cytoskeleton. Mechanistically, TNF-α promoted METTL3-mediated m6A modification of CLASP2 to enhance CLASP2 mRNA stability. Moreover, CLASP2 directly interplayed with IQ motif containing GTPase activating protein 1 (IQGAP1) to regulate F-actin cytoskeleton remodeling. In vivo data demonstrated that inhibition of METTL3/CLASP2 axis delayed lung metastasis in nude mice.
TNF-α favors BCa cell metastasis, which involves METTL3-mediated m6A modification of CLASP2 that interacts with IQGAP1, thus leading to F-actin cytoskeleton remodeling. Our findings suggest targeting TNF-α/METTL3/CLASP2/IQGAP1 axis as a potential avenue for promising treatment for BCa.
膀胱癌（BCa）转移是一个由细胞骨架重组引发的多步骤过程。然而，BCa中细胞骨架重组的调控机制仍不明确。本研究阐明了肿瘤坏死因子-α（TNF-α）在BCa转移过程中对细胞骨架重塑的影响及其可能机制。
采用集落形成、划痕、Transwell实验以及裸鼠模型评估生长和转移情况。通过免疫组织化学染色、定量实时聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法评估分子表达。通过甲基化RNA免疫沉淀（MeRIP）检测N6-甲基腺苷（m6A）水平。通过免疫共沉淀（Co-IP）验证蛋白质相互作用。利用免疫荧光染色鉴定肌动蛋白丝纤维（F-肌动蛋白）的重排和蛋白质共定位。
TNF-α以剂量（10 - 50 ng/mL）依赖的方式促进BCa中细胞质连接相关蛋白2（CLASP2）和甲基转移酶样3（METTL3）的表达。CLASP2高表达提示BCa患者总生存期较短。CLASP2缺陷通过破坏F-肌动蛋白细胞骨架抑制BCa细胞增殖、迁移和侵袭。机制上，TNF-α促进METTL3介导的CLASP2的m6A修饰以增强CLASP2 mRNA稳定性。此外，CLASP2直接与含IQ模体的GTP酶激活蛋白1（IQGAP1）相互作用以调节F-肌动蛋白细胞骨架重塑。体内数据表明抑制METTL3/CLASP2轴可延缓裸鼠肺转移。
TNF-α促进BCa细胞转移，这涉及METTL3介导的与IQGAP1相互作用的CLASP2的m6A修饰，从而导致F-肌动蛋白细胞骨架重塑。我们的研究结果表明，靶向TNF-α/METTL3/CLASP2/IQGAP1轴是BCa有前景的潜在治疗途径。
