Wilkus-Adamczyk Kinga, Brodaczewska Klaudia, Majewska Aleksandra, Kieda Claudine
Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine- National Research Institute, Warsaw, Poland.
Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland.
Front Cell Dev Biol. 2023 May 16;11:1125077. doi: 10.3389/fcell.2023.1125077. eCollection 2023.
Hypoxia shapes the tumor microenvironment, modulates distinct cell population activities, and activates pathological angiogenesis in cancer, where endothelial cells (ECs) are the most important players. This study aimed to evidence the influences of the tumor microenvironment on the global gene expression pattern characteristic for ECs and the distinct responses displayed by tumor-derived ECs in comparison to the healthy endothelium during endothelial to mesenchymal transition (EndMT) and its regulation by miR-200-b-3p. Immortalized lines of ECs from the same patient with breast cancer, healthy breast tissue (HBH.MEC), and primary tumor (HBCa.MEC) were used. The experiments were performed in normoxia and hypoxia for 48 h. By using the wound healing test, we investigated the migration abilities of ECs. Global gene expression analysis with NGS was carried out to detect new pathways altered in pathological ECs and find the most changed miRNAs. The validation of NGS data from RNA and miRNA was estimated by qPCRs. Mimic miR-200b-3p was used in HBH.MEC, and the targets , , , and were checked. Hypoxia influences EC migration properties in wound healing assays. In hypoxia, healthy ECs migrate slower than they do in normoxia, as opposed to HBCa.MEC, where no decreased migration ability is induced by hypoxia due to EndMT features. NGS data identified this process to be altered in cancer ECs through extracellular matrix (ECM) organization. The deregulated genes, validated by qPCR, included , , , , , , , , , , and . NGS also identified collagens, laminins, fibronectins, and integrins, as being deregulated in tumor-derived ECs. Moreover, the analysis of the 10 most intensively modified miRNAs, when breast tumor-derived ECs were compared to healthy ECs, shed light on miR-200b-3p, which is strongly upregulated in HBCa.MECs when compared to HBH.MECs. The pathological ECs differed significantly, both phenotypically and functionally, from the normal corresponding tissue, thus influencing their microenvironment cross-talk. The gene expression profile confirms the EndMT phenotype of tumor-derived ECs and migratory properties acquisition. Moreover, it indicates the role of miR-200b-3p, that is, regulating EndMT in pathological ECs and silencing several angiogenic growth factors and their receptors by directly targeting their mRNA transcripts.
缺氧塑造肿瘤微环境，调节不同细胞群体的活性，并激活癌症中的病理性血管生成，其中内皮细胞（ECs）是最重要的参与者。本研究旨在证明肿瘤微环境对ECs的整体基因表达模式特征的影响，以及与健康内皮相比，肿瘤来源的ECs在内皮-间质转化（EndMT）过程中表现出的不同反应及其受miR-200-b-3p的调控。使用了来自同一乳腺癌患者的永生化ECs系、健康乳腺组织（HBH.MEC）和原发性肿瘤（HBCa.MEC）。实验在常氧和缺氧条件下进行48小时。通过伤口愈合试验，我们研究了ECs的迁移能力。利用NGS进行全基因表达分析，以检测病理性ECs中改变的新途径，并找出变化最大的miRNAs。通过qPCR对RNA和miRNA的NGS数据进行验证。在HBH.MEC中使用miR-200b-3p模拟物，并检测其靶标、、、和。缺氧在伤口愈合试验中影响ECs的迁移特性。在缺氧条件下，健康的ECs迁移速度比在常氧条件下慢，而对于HBCa.MEC，由于EndMT特征，缺氧不会诱导其迁移能力下降。NGS数据表明，该过程在癌症ECs中通过细胞外基质（ECM）组织发生改变。经qPCR验证的失调基因包括、、、、、、、、、、和。NGS还鉴定出胶原蛋白、层粘连蛋白、纤连蛋白和整合素在肿瘤来源的ECs中失调。此外，将乳腺肿瘤来源的ECs与健康ECs进行比较时，对10个变化最强烈的miRNAs的分析揭示了miR-200b-3p，与HBH.MEC相比，其在HBCa.MEC中强烈上调。病理性ECs在表型和功能上与正常相应组织有显著差异，从而影响它们与微环境的相互作用。基因表达谱证实了肿瘤来源ECs的EndMT表型和迁移特性的获得。此外，它表明了miR-200b-3p的作用，即在病理性ECs中调节EndMT，并通过直接靶向其mRNA转录本来沉默几种血管生成生长因子及其受体。
