Wang Jingting, Cai Jiaying, Wang Zengping, Yang Shuran, Wang Jing, Jia Yanfei, Sun Haiji, Ma Xiaoli
Research Center of Basic Medicine, Central Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250013, China.
Department of Medical Laboratory, Shandong Second Medical University, Weifang, Shandong, 261053, China.
Cancer Cell Int. 2025 Feb 25;25(1):67. doi: 10.1186/s12935-025-03701-5.
α5-nicotinic acetylcholine receptor (α5-nAChR) participates in chronic stress-promoted lung adenocarcinoma (LUAD) progression. Neuropilin and tolloid-like 2 (NETO2) contributes to fear expression and extinction, which is related to tumorigenesis. CHRNA5 (encoding α5-nAChR) gene profiling revealed a reduction in NETO2 expression following CHRNA5 knockdown. Nevertheless, the connection between α5-nAChR and NETO2 in LUAD progression induced by chronic stress remains unclear.
RNA-Seq and bioinformatics database were used for analyzing the expression as well as correlation of α5-nAChR, together with NETO2 in LUAD. α5-nAChR and NETO2 expression were detected using immunohistochemistry in LUAD tissue microarrays, chronic restraint stress (CRS) and chronic unpredictable stress (CUMS) mice tissues. In lung adenocarcinoma A549 and H1299 cells, the expression of α5-nAChR, NETO2, p-CAMKII, p-STAT3 and vimentin induced by acetylcholine/nicotine was examined by western blot. The interaction of α5-nAChR with NETO2 in lung adenocarcinoma cells was detected by Co-immunoprecipitation assay and modeled using molecular docking. EdU assay and colony formation assay were conducted to evaluate cell proliferation, while wound healing assay as well as transwell assay assessed the migration and invasion of lung adenocarcinoma cells.
α5-nAChR expression was related to NETO2 expression, low survival rate, staging as well as smoking status in LUAD dataset as well as tissue microarrays. The correlation between α5-nAChR and NETO2 was validated in nude mice xenograft tissues. α5-nAChR as well as NETO2 expression correlated in CRS and CUMS mice tissues. In vitro, acetylcholine/nicotine mediated NETO2, p-CAMKII, p-STAT3 and vimentin expression via α5-nAChR. α5-nAChR interacted with NETO2 as well as CAMKII in LUAD cells. α5-nAChR/NETO2 signaling contributed to LUAD cell proliferation, migration and invasion.
The above results uncover a new chronic stress-promoted LUAD signaling pathway: α5-nAChR/NETO2 axis contributes to chronic stress-promoted LUAD cell proliferation, migration and invasion.
α5-烟碱型乙酰胆碱受体（α5-nAChR）参与慢性应激促进的肺腺癌（LUAD）进展。神经纤毛蛋白和类 tolloid 样蛋白 2（NETO2）参与恐惧表达和消退，这与肿瘤发生有关。CHRNA5（编码α5-nAChR）基因分析显示，CHRNA5 基因敲低后 NETO2 表达降低。然而，慢性应激诱导的 LUAD 进展中α5-nAChR 与 NETO2 之间的联系仍不清楚。
利用 RNA 测序和生物信息学数据库分析 LUAD 中α5-nAChR 以及 NETO2 的表达及其相关性。在 LUAD 组织芯片、慢性束缚应激（CRS）和慢性不可预测应激（CUMS）小鼠组织中，采用免疫组织化学法检测α5-nAChR 和 NETO2 的表达。在肺腺癌 A549 和 H1299 细胞中，通过蛋白质免疫印迹法检测乙酰胆碱/尼古丁诱导的α5-nAChR、NETO2、磷酸化钙/钙调蛋白依赖性蛋白激酶 II（p-CAMKII）、磷酸化信号转导和转录激活因子 3（p-STAT3）和波形蛋白的表达。通过免疫共沉淀试验检测肺腺癌细胞中α5-nAChR 与 NETO2 的相互作用，并利用分子对接进行建模。采用 EdU 试验和集落形成试验评估细胞增殖，同时采用伤口愈合试验和 Transwell 试验评估肺腺癌细胞的迁移和侵袭能力。
在 LUAD 数据集以及组织芯片中，α5-nAChR 表达与 NETO2 表达、低生存率、分期以及吸烟状态相关。在裸鼠异种移植组织中验证了α5-nAChR 与 NETO2 之间的相关性。在 CRS 和 CUMS 小鼠组织中，α5-nAChR 以及 NETO2 表达相关。在体外，乙酰胆碱/尼古丁通过α5-nAChR 介导 NETO2、p-CAMKII、p-STAT3 和波形蛋白的表达。在 LUAD 细胞中，α5-nAChR 与 NETO2 以及钙/钙调蛋白依赖性蛋白激酶 II（CAMKII）相互作用。α5-nAChR/NETO2 信号通路促进 LUAD 细胞的增殖、迁移和侵袭。
上述结果揭示了一条新的慢性应激促进的 LUAD 信号通路：α5-nAChR/NETO2 轴促进慢性应激诱导的 LUAD 细胞增殖、迁移和侵袭。
