癌相关成纤维细胞（CAFs）通过维持细胞外间充质（ECM）的产生和促进肿瘤的发展，对肿瘤进展起着重要作用。环氧合酶-2（COX-2）已被证实可促进ECM的形成和肿瘤的进展。然而，COX-2介导CAFs激活的机制尚未阐明。因此，我们进行了这项研究，以鉴定肿瘤衍生外泌体miRNA诱导的COX-2对CAFs激活的影响和机制。作为CAFs激活的指标，我们通过Western blotting和免疫组织化学检测成纤维细胞激活蛋白-1（FAP-1）和α-平滑肌肌动蛋白（α-SMA），即主要的CAF标志物的表达情况。我们使用纤维连接蛋白（FN1）的表达情况分析CAFs的ECM产生。我们通过超速离心法提取外泌体，并通过qRT-PCR检测exomiRNA。在此基础上，我们使用在线预测软件、荧光素酶报告基因实验、共免疫共沉淀和实验动物模型来进一步阐明涉及的机制。在体内，我们观察到LUAD病灶中COX-2表达水平与间充质中α-SMA / FN1表达水平之间存在正相关。然而，COX-2的主要产物之一PGE2并未直接影响CAF的激活。COX-2过表达增加了exo-miR-1290的表达，进而促进了CAF的激活。此外，发现miR-1290的潜在靶点Cullin3（CUL3）能抑制COX-2 / exomiR-1290介导的CAF激活和ECM产生，从而阻碍肿瘤的进展。我们发现CUL3能诱导核因子红细胞2相关因子2（NFE2L2，Nrf2）的泛素化和降解，而exo-miR-1290通过靶向CUL3防止Nrf2的泛素化并增加其蛋白稳定性。此外，我们还发现Nrf2直接与FAP-1和FN1的启动子结合，通过促进FAP-1和FN1的转录来增强CAF的激活。我们的数据揭示了一种由高表达COX-2的肿瘤细胞衍生的外泌体引起的CAF激活新机制。具体而言，COX-2 / exomiR-1290 / CUL3被认为是LUAD中介导CAF激活和肿瘤进展的新信号通路。因此，这一发现为癌症治疗提供了一种可能以后能够抑制肿瘤进展的新策略。

Cancer-associated fibroblasts (CAFs) are critically involved in tumor progression by maintaining extracellular mesenchyma (ECM) production and improving tumor development. Cyclooxygenase-2 (COX-2) has been proved to promote ECM formation and tumor progression. However, the mechanisms of COX-2 mediated CAFs activation have not yet been elucidated. Therefore, we conducted this study to identify the effects and mechanisms of COX-2 underlying CAFs activation by tumor-derived exosomal miRNAs in lung adenocarcinoma (LUAD) progression.As measures of CAFs activation, the expressions of fibroblasts activated protein-1 (FAP-1) and α-smooth muscle actin (α-SMA), the main CAFs markers, were detected by Western blotting and Immunohistochemistry. And the expression of Fibronectin (FN1) was used to analyze ECM production by CAFs. The exosomes were extracted by ultracentrifugation and exo-miRNAs were detected by qRT-PCR. Herein, we further elucidated the implicated mechanisms using online prediction software, luciferase reporter assays, co-immunoprecipitation, and experimental animal models.In vivo, a positive correlation was observed between the COX-2 expression levels in parenchyma and α-SMA/FN1 expression levels in mesenchyma in LUAD. However, PGE2, one of major product of COX-2, did not affect CAFs activation directly. COX-2 overexpression increased exo-miR-1290 expression, which promoted CAFs activation. Furthermore, Cullin3 (CUL3), a potential target of miR-1290, was found to suppress COX-2/exo-miR-1290-mediated CAFs activation and ECM production, consequently impeding tumor progression. CUL3 is identified to induce the Nuclear Factor Erythroid 2-Related Factor 2 (NFE2L2, Nrf2) ubiquitination and degradation, while exo-miR-1290 can prevent Nrf2 ubiquitination and increase its protein stability by targeting CUL3. Additionally, we identified that Nrf2 is direcctly bound with promoters of FAP-1 and FN1, which enhanced CAFs activation by promoting FAP-1 and FN1 transcription.Our data identify a new CAFs activation mechanism by exosomes derived from cancer cells that overexpress COX-2. Specifically, COX-2/exo-miR-1290/CUL3 is suggested as a novel signaling pathway for mediating CAFs activation and tumor progression in LUAD. Consequently, this finding suggests a novel strategy for cancer treatment that may tackle tumor progression in the future. Video Abstract.© 2023. BioMed Central Ltd., part of Springer Nature.