Department of Thoracic Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Department of Thoracic Surgery, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China.
J Gene Med. 2024 Jan;26(1):e3573. doi: 10.1002/jgm.3573. Epub 2023 Aug 7.
BACKGROUND: Lung adenocarcinoma (LUAD), as the most common type of lung cancer, poses a significant threat to public health. Tumor heterogeneity plays a crucial role in carcinogenesis, which could be largely deciphered by next-generation sequencing (NGS). METHODS: We obtained and screened single-cell RNA sequencing (scRNA-seq) data from 16 LUAD samples, and endothelial cells (ECs) were grouped into three clusters. The origin of EC differentiation was explored by pseudo-time analysis. CellChat analysis was used to detect potential communication between ECs and malignant cells, and gene regulatory network analysis was used to identify changes in transcription factor activity. We explored the prognosis of specific ECs clusters and their effects on the tumor microenvironment (TME) at the bulk transcriptome level. 5-Ethynyl-2'- deoxyuridine (EdU) and Ki-67 staining were conducted to study the proliferative phenotype of LUAD cell lines. Western blotting targeting the phosphorylation of PI3K-AKT proteins was utilized for determination of the downstream pathway of NCL. RESULTS: COL3A1-positive ECs showed the highest crosstalk interaction with malignant cells, indicating that they have important effects on driving LUAD carcinogenesis. Vascular endothelial growth factor (VEGF) signaling pathway was identified as the main signaling pathway, mediating signal transduction from malignant cells. The TME-related genes of COL3A1-positive ECs were significantly more highly expressed. COL3A1-positive ECs showed unique metabolic and immune characteristics, as well as highly activated metabolic signaling pathways and inflammatory responses. Importantly, LUAD patients with low COL3A1-positive ECs scores displayed an inferior prognosis outcome and a higher risk of metastasis. The key target gene NCL, which is involved in the interaction between epithelial cells and cancer cells, has been identified through screening. Flow cytometry showed that knockdown of NCL prompted the apoptosis of A549 and NCI-H1299. Western blotting showed that knockdown of NCL decreased the phosphorylation of AKT and PI3K, which identified the downstream pathway of NCL. CONCLUSIONS: COL3A1-positive ECs have important effects on the development of LUAD and the formation of an immune microenvironment. Furthermore, we identified a key target gene, NCL, which is involved in the interaction between endothelial cells and cancer cells. NCL also affected the apoptosis and proliferation in LUAD through the PI3K-AKT pathway.
背景：肺腺癌（LUAD）作为最常见的肺癌类型，对公众健康构成重大威胁。肿瘤异质性在致癌过程中起着关键作用，这可以通过下一代测序（NGS）在很大程度上揭示。
方法：我们从 16 个 LUAD 样本中获取并筛选了单细胞 RNA 测序（scRNA-seq）数据，并将内皮细胞（ECs）分为三个簇。通过拟时间分析探索 EC 分化的起源。使用 CellChat 分析检测 ECs 与恶性细胞之间的潜在通信，并使用基因调控网络分析识别转录因子活性的变化。我们在 bulk 转录组水平上探索了特定 ECs 簇的预后及其对肿瘤微环境（TME）的影响。用 5-乙炔基-2'-脱氧尿苷（EdU）和 Ki-67 染色研究 LUAD 细胞系的增殖表型。用靶向 PI3K-AKT 蛋白磷酸化的 Western 印迹法测定 NCL 的下游途径。
结果：COL3A1 阳性 ECs 与恶性细胞的相互作用最强，表明它们对驱动 LUAD 癌变具有重要作用。血管内皮生长因子（VEGF）信号通路被确定为主要信号通路，介导恶性细胞的信号转导。COL3A1 阳性 ECs 的 TME 相关基因表达显著更高。COL3A1 阳性 ECs 表现出独特的代谢和免疫特征，以及高度激活的代谢信号通路和炎症反应。重要的是，LUAD 患者中 COL3A1 阳性 ECs 评分较低的患者预后较差，转移风险较高。通过筛选发现，参与上皮细胞和癌细胞相互作用的关键靶基因 NCL。流式细胞术显示，敲低 NCL 可促进 A549 和 NCI-H1299 的凋亡。Western 印迹显示，敲低 NCL 降低了 AKT 和 PI3K 的磷酸化，从而确定了 NCL 的下游途径。
结论：COL3A1 阳性 ECs 对 LUAD 的发展和免疫微环境的形成有重要影响。此外，我们确定了一个关键靶基因 NCL，它参与了内皮细胞和癌细胞之间的相互作用。NCL 通过 PI3K-AKT 通路影响 LUAD 的凋亡和增殖。
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