Renal Unit, Department of Medicine, University of Verona, Piazzale Stefani 1, 37126 Verona, Italy.
Department of Emergency and Organ Transplantation, University of Bari "Aldo Moro", Piazza Giulio Cesare 11, 70124 Bari, Italy.
Int J Mol Sci. 2018 Apr 20;19(4):1250. doi: 10.3390/ijms19041250.
The administration of Everolimus (EVE), a mTOR inhibitor used in transplantation and cancer, is often associated with adverse effects including pulmonary fibrosis. Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. To improve our knowledge, primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 h. EMT markers (α-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. Our results revealed a complex biological network implicated in EVE-related pulmonary fibrosis and underlined new potential disease biomarkers and therapeutic targets.
雷帕霉素（EVE）是一种用于移植和癌症的 mTOR 抑制剂，其给药通常与包括肺纤维化在内的不良反应相关。尽管其潜在机制尚未完全阐明，但这种情况可能部分是由气道细胞的上皮间质转化（EMT）引起的。为了增加我们的知识，用 EVE（5 和 100 nM）处理原代支气管上皮细胞（BE63/3）24 小时。通过 RT-PCR 测量 EMT 标志物（α-SMA、波形蛋白、纤连蛋白）。通过 Millicell-ERS 欧姆计测量跨上皮电阻。通过 Illumina 和 Agilent 试剂盒分别进行 mRNA 和 microRNA 分析。只有高剂量 EVE 增加 EMT 标志物并降低 BE63/3 的跨上皮电阻。生物信息学显示 125 个失调基因，根据富集分析，这些基因与胶原蛋白合成/代谢有关。结缔组织生长因子（CTGF）是上调 mRNA 之一。5 nM EVE 对促纤维化机制无效。此外，100 nM EVE 后有 5 个 miRNA 表达上调，并能调节转录组分析中选择的 31 个基因（包括 CTGF）。MMP12 和 CTGF 的 RT-PCR 和 Western blot 验证了高通量结果。我们的结果揭示了与 EVE 相关的肺纤维化中涉及的复杂生物学网络，并强调了新的潜在疾病生物标志物和治疗靶点。
