Department of Radiotherapy, Tianjin Cancer Hospital Airport Hospital, Tianjin, China.
Department of Breast Cancer, Tianjin Cancer Hospital Airport Hospital, Tianjin, China.
Clin Respir J. 2024 Nov;18(11):e70031. doi: 10.1111/crj.70031.
Lung squamous cell carcinoma (LUSC) is a type of lung cancer that develops in the squamous cells. It is known to be promoted by the activation of various signaling pathways and the dysregulation of key regulatory molecules. One such molecule, 5'-nucleotidase domain containing 2 (NT5DC2), has been identified as a critical regulator in various cancers including lung cancer. However, there are no data regarding its role in LUSC.
The mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), CCCTC-binding factor (CTCF), and NT5DC2 was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR), whereas their protein expression was assessed using a western blotting assay. Cell proliferation was determined using a cell counting kit-8 (CCK-8) assay. Cell apoptosis, CD11b expression, and CD206 expression were analyzed using flow cytometry. Tube formation was assessed through a tube formation assay. Glucose consumption, lactate production, and ATP levels were measured using colorimetric methods. The effect of NT5DC2 on the malignant progression of LUSC cells was analyzed using a xenograft mouse model assay. The levels of transforming growth factor-beta 1 (TGF-β1) and interleukin-10 (IL-10) were detected using enzyme-linked immunosorbent assays. The associations among IGF2BP3, CTCF and NT5DC2 were identified using dual-luciferase reporter assay, RNA immunoprecipitation assay and m6A RNA immunoprecipitation assay.
The expression of NT5DC2 was found to be upregulated in LUSC tissues and cells when compared with normal lung tissues and normal human bronchial epithelial cells. Silencing of NT5DC2 inhibited LUSC cell proliferation, tube formation, glycolysis, M2 macrophage polarization, and tumor formation while inducing cell apoptosis. In addition, CTCF was found to transcriptionally activate NT5DC2 in LUSC cells. IGF2BP3 stabilized the mRNA expression of CTCF through m6A methylation. Further, overexpression of CTCF or NT5DC2 attenuated the effects of IGF2BP3 silencing in both NCI-520 and SK-MES-1 cells.
The IGF2BP3/CTCF axis-dependent NT5DC2 promotes M2 macrophage polarization, thereby enhancing the malignant progression of LUSC. This study was the first to reveal the role of NT5DC2 in LUSC and the underlying mechanism. The result suggests that targeting the IGF2BP3/CTCF/NT5DC2 axis may have clinical significance in the treatment of LUSC.
肺鳞状细胞癌（LUSC）是一种在鳞状细胞中发展的肺癌。已知它由各种信号通路的激活和关键调节分子的失调所促进。一种这样的分子，5'-核苷酸酶结构域包含 2（NT5DC2），已被确定为包括肺癌在内的各种癌症中的关键调节剂。然而，关于它在 LUSC 中的作用尚无数据。
使用实时定量聚合酶链反应（qRT-PCR）分析胰岛素样生长因子 2 mRNA 结合蛋白 3（IGF2BP3）、CCCTC 结合因子（CTCF）和 NT5DC2 的 mRNA 表达，并用 Western blot 测定法评估其蛋白表达。使用细胞计数试剂盒-8（CCK-8）测定细胞增殖。通过流式细胞术分析细胞凋亡、CD11b 表达和 CD206 表达。通过管形成测定评估管形成。使用比色法测量葡萄糖消耗、乳酸生成和 ATP 水平。使用异种移植小鼠模型测定分析 NT5DC2 对 LUSC 细胞恶性进展的影响。使用酶联免疫吸附测定法检测转化生长因子-β1（TGF-β1）和白细胞介素 10（IL-10）的水平。使用双荧光素酶报告基因测定、RNA 免疫沉淀测定和 m6A RNA 免疫沉淀测定鉴定 IGF2BP3、CTCF 和 NT5DC2 之间的关联。
与正常肺组织和正常人支气管上皮细胞相比，LUSC 组织和细胞中发现 NT5DC2 的表达上调。沉默 NT5DC2 抑制 LUSC 细胞增殖、管形成、糖酵解、M2 巨噬细胞极化和肿瘤形成，同时诱导细胞凋亡。此外，在 LUSC 细胞中发现 CTCF 转录激活 NT5DC2。IGF2BP3 通过 m6A 甲基化稳定 CTCF 的 mRNA 表达。此外，在 NCI-520 和 SK-MES-1 细胞中，CTCF 或 NT5DC2 的过表达减弱了 IGF2BP3 沉默的影响。
IGF2BP3/CTCF 轴依赖性 NT5DC2 促进 M2 巨噬细胞极化，从而增强 LUSC 的恶性进展。这项研究首次揭示了 NT5DC2 在 LUSC 中的作用及其潜在机制。结果表明，靶向 IGF2BP3/CTCF/NT5DC2 轴可能在 LUSC 的治疗中具有临床意义。
