IGF2BP3/CTCF Axis–Dependent NT5DC2 Promotes M2 Macrophage Polarization to Enhance the Malignant Progression of Lung Squamous Cell Carcinomas

IF 1.9 4区医学 Q3 RESPIRATORY SYSTEM Clinical Respiratory Journal Pub Date : 2024-11-06 DOI:10.1111/crj.70031

Jifeng Sun, Hao Wang, Ran Zhang, Xiaoxuan Sun, Zhanbo Wu, Jun Wang, Yuwen Wang

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Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

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.

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IGF2BP3/CTCF轴依赖性NT5DC2促进M2巨噬细胞极化，从而加强肺鳞癌的恶性进展

背景：肺鳞状细胞癌（LUSC）是一种发生在鳞状细胞中的肺癌。众所周知，肺鳞状细胞癌是由各种信号通路的激活和关键调控分子的失调引起的。5'-nucleotidase domain containing 2 (NT5DC2)就是这样一种分子，它已被确定为包括肺癌在内的各种癌症的关键调节因子。然而，目前还没有关于它在肺癌中作用的数据：方法：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，并使用 Western 印迹分析评估它们的蛋白表达。细胞增殖用细胞计数试剂盒-8（CCK-8）测定。使用流式细胞术分析细胞凋亡、CD11b 表达和 CD206 表达。通过试管形成试验评估试管形成情况。使用比色法测量葡萄糖消耗、乳酸生成和 ATP 水平。利用异种移植小鼠模型试验分析了 NT5DC2 对 LUSC 细胞恶性进展的影响。使用酶联免疫吸附试验检测了转化生长因子-β1（TGF-β1）和白细胞介素-10（IL-10）的水平。利用双荧光素酶报告实验、RNA免疫沉淀实验和 m6A RNA 免疫沉淀实验确定了 IGF2BP3、CTCF 和 NT5DC2 之间的关联：结果：与正常肺组织和正常人支气管上皮细胞相比，NT5DC2在LUSC组织和细胞中表达上调。沉默 NT5DC2 可抑制 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，研究还发现 CTCF 能转录激活 LUSC 细胞中的 NT5DC2。IGF2BP3 通过 m6A 甲基化稳定了 CTCF 的 mRNA 表达。此外，在 NCI-520 和 SK-MES-1 细胞中，过表达 CTCF 或 NT5DC2 可减弱 IGF2BP3 沉默的效果：结论：依赖于IGF2BP3/CTCF轴的NT5DC2促进了M2巨噬细胞的极化，从而增强了LUSC的恶性进展。该研究首次揭示了NT5DC2在LUSC中的作用及其内在机制。结果表明，靶向IGF2BP3/CTCF/NT5DC2轴可能对治疗LUSC具有临床意义。

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来源期刊

Clinical Respiratory Journal 医学-呼吸系统

CiteScore

3.70

自引率

0.00%

发文量

104

审稿时长

>12 weeks

期刊介绍： Overview Effective with the 2016 volume, this journal will be published in an online-only format. Aims and Scope The Clinical Respiratory Journal (CRJ) provides a forum for clinical research in all areas of respiratory medicine from clinical lung disease to basic research relevant to the clinic. We publish original research, review articles, case studies, editorials and book reviews in all areas of clinical lung disease including: Asthma Allergy COPD Non-invasive ventilation Sleep related breathing disorders Interstitial lung diseases Lung cancer Clinical genetics Rhinitis Airway and lung infection Epidemiology Pediatrics CRJ provides a fast-track service for selected Phase II and Phase III trial studies. Keywords Clinical Respiratory Journal, respiratory, pulmonary, medicine, clinical, lung disease, Abstracting and Indexing Information Academic Search (EBSCO Publishing) Academic Search Alumni Edition (EBSCO Publishing) Embase (Elsevier) Health & Medical Collection (ProQuest) Health Research Premium Collection (ProQuest) HEED: Health Economic Evaluations Database (Wiley-Blackwell) Hospital Premium Collection (ProQuest) Journal Citation Reports/Science Edition (Clarivate Analytics) MEDLINE/PubMed (NLM) ProQuest Central (ProQuest) Science Citation Index Expanded (Clarivate Analytics) SCOPUS (Elsevier)