Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
Department of Otolaryngology, Head and Neck Surgery, Freeman Hospital, Newcastle upon Tyne, United Kingdom.
Transplantation. 2019 Nov;103(11):2275-2286. doi: 10.1097/TP.0000000000002845.
Dysregulation of microRNAs (miRNAs) has been implicated in airway diseases where transforming growth factor-β (TGF-β)-induced epithelial-mesenchymal transition (EMT) may contribute to pathophysiology. Our study investigated the role of miRNA-200b in TGF-β1-induced EMT in human bronchial epithelial cells.
NanoString nCounter miRNA assay was used to profile miRNA in control versus TGF-β1 (1, 4, and 24 h) stimulated BEAS-2B cells. Immortalized primary bronchial epithelial cell line (BEAS-2B cells), human primary bronchial epithelial cells (PBECs), and PBECs derived post-lung transplant were transfected with miR-200b-3p mimics and EMT marker expression was examined at RNA and protein level. miRNA target studies were performed and validated using computational tools and luciferase assay. In situ hybridization was done on normal lung tissue to localize miR-200b-3p in airway epithelium.
miR-200b-3p was downregulated post-TGF-β1 treatment compared with control in BEAS-2B. miR-200b-3p mimic transfection before TGF-β1 stimulation maintained epithelial marker expression and downregulated mesenchymal cell markers at RNA and protein level in BEAS-2B cells and PBECs. Furthermore, miR-200b-3p mimics reversed established TGF-β1-induced EMT in BEAS-2B cells. miR-200b-3p targets, ZNF532, and ZEB2 were validated as direct targets using luciferase assay. miR-200b-3p mimics suppress TGF-β1-induced EMT via inhibition of ZNF532 and ZEB2. In situ hybridization showed that miR-200b-3p is expressed in the normal lung epithelium. Additionally, miR-200b-3p mimics inhibit EMT in the presence of TGF-β1 in PBECs derived from lung allograft.
We provide proof of concept that miR-200b-3p protects airway epithelial cells from EMT. Manipulating miR-200b-3p expression may represent a novel therapeutic modulator in airway pathophysiology.
微小 RNA（miRNA）的失调与气道疾病有关，其中转化生长因子-β（TGF-β）诱导的上皮-间充质转化（EMT）可能导致病理生理学变化。我们的研究调查了 miRNA-200b 在 TGF-β1 诱导的人支气管上皮细胞 EMT 中的作用。
使用 NanoString nCounter miRNA 分析方法对对照与 TGF-β1（1、4 和 24 h）刺激 BEAS-2B 细胞中的 miRNA 进行分析。转染 miRNA-200b-3p 模拟物，检测 EMT 标志物在 RNA 和蛋白质水平的表达，研究永生化原代支气管上皮细胞系（BEAS-2B 细胞）、人原代支气管上皮细胞（PBECs）和肺移植后 PBECs。使用计算工具和荧光素酶测定法进行 miRNA 靶标研究，并进行验证。对正常肺组织进行原位杂交，以定位气道上皮中的 miR-200b-3p。
与对照相比，BEAS-2B 细胞中 TGF-β1 处理后 miR-200b-3p 下调。在 TGF-β1 刺激前转染 miR-200b-3p 模拟物可维持 BEAS-2B 细胞和 PBECs 中的上皮标志物表达，并在 RNA 和蛋白质水平下调间充质细胞标志物。此外，miR-200b-3p 模拟物可逆转 BEAS-2B 细胞中 TGF-β1 诱导的 EMT。荧光素酶测定法验证了 miR-200b-3p 靶标 ZNF532 和 ZEB2 是直接靶标。miR-200b-3p 模拟物通过抑制 ZNF532 和 ZEB2 抑制 TGF-β1 诱导的 EMT。原位杂交显示 miR-200b-3p 在正常肺上皮中表达。此外，miR-200b-3p 模拟物在肺移植衍生的 PBEC 中 TGF-β1 的存在下抑制 EMT。
我们提供了概念验证，表明 miR-200b-3p 可保护气道上皮细胞免受 EMT。操纵 miR-200b-3p 的表达可能代表气道病理生理学的一种新的治疗调节剂。
