School of Public Health, Southwest Medical University, Luzhou, 646000, Sichuan, People's Republic of China.
Department of Clinical Laboratory, 404 Hospital of Mianyang, Mianyang, 621000, Sichuan, People's Republic of China.
Toxicol Lett. 2021 Sep 1;347:58-66. doi: 10.1016/j.toxlet.2021.04.017. Epub 2021 May 4.
For smoking-induced pulmonary fibrosis (PF), a serious disease endangering human health, there is no effective clinical treatment. Aberrant epithelium-fibroblast cross-talk is involved in formation of the excessive extracellular matrix (ECM) that contributes to PF. Circular RNAs have been associated with various pulmonary diseases. However, the mechanisms of circRNAs in PF are not clear. Herein, our goals were to investigate the involvement of circRNA_0026344 in the aberrant epithelium-fibroblast cross-talk induced by cigarette smoke (CS) and to define its mechanism. Chronic exposure (16 weeks) of BALB/c mice to 500 mg/m CS induced lung injury and fibrosis in lung tissues. From HBE cells, circRNA_0026344 was selected by microarray analysis and verified as that with the most severe down-regulation caused by cigarette smoke extract (CSE). The regulatory relationship between circRNA_0026344 and miR-21 was assessed by use of bioinformatics, RNA pull-down assays, and qRT-PCR. We found that miR-21 binding sites were present in circRNA_0026344 and, in HBE cells, it could act as a sponge for miR-21. When pcDNA3.0-circRNA_0026344, a high expression plasmid of circRNA_0026344, was transfected into HBE cells, the CSE-induced up-regulation of miR-21 levels was reversed. In MRC-5 cells, HBE-secreted exosomal miR-21 decreased levels of Smad7 and activated the TGF-β1/Smad3 pathway. By using the Targetscan database, the presence of species-conserved miR-21 binding sites in the Smad7 3'UTR region were predicted. We verified, by use of a luciferase reporter gene, that miR-21 bound to the 3'UTR region of Smad7 mRNA to inhibit its transcription. In conclusion, the results reveal that, in CS-induced pulmonary fibrosis, circRNA_0026344, via exosomal miR-21 regulation of Smad7, is involved in aberrant cross-talk of epithelium-fibroblasts. These results will be useful for the discovery of early biomarkers and for providing therapeutic targets for smoking-induced pulmonary fibrosis.
对于吸烟引起的肺纤维化（PF）这种危害人类健康的严重疾病，目前临床上尚无有效的治疗方法。异常的上皮-成纤维细胞相互作用参与了细胞外基质（ECM）的过度形成，这是 PF 的一个重要特征。环状 RNA 与各种肺部疾病有关。然而，环状 RNA 在 PF 中的作用机制尚不清楚。在此，我们的目标是研究环状 RNA_0026344 是否参与香烟烟雾（CS）诱导的异常上皮-成纤维细胞相互作用，并确定其作用机制。将 BALB/c 小鼠慢性暴露（16 周）于 500mg/m3 CS 中可诱导肺组织损伤和纤维化。通过微阵列分析从 HBE 细胞中选择环状 RNA_0026344，并通过香烟烟雾提取物（CSE）验证其表达下调最显著。使用生物信息学、RNA 下拉测定和 qRT-PCR 评估环状 RNA_0026344 与 miR-21 之间的调控关系。我们发现 miR-21 的结合位点存在于环状 RNA_0026344 中，并且在 HBE 细胞中，它可以作为 miR-21 的海绵。当转染 pcDNA3.0-circRNA_0026344（环状 RNA_0026344 的高表达质粒）时，CSE 诱导的 miR-21 水平上调得到逆转。在 MRC-5 细胞中，HBE 细胞分泌的外泌体 miR-21 降低了 Smad7 水平并激活了 TGF-β1/Smad3 途径。通过使用 Targetscan 数据库，预测了 Smad7 3'UTR 区域存在物种保守的 miR-21 结合位点。通过使用荧光素酶报告基因，我们验证了 miR-21 结合到 Smad7 mRNA 的 3'UTR 区域以抑制其转录。总之，这些结果表明，在 CS 诱导的肺纤维化中，环状 RNA_0026344 通过外泌体 miR-21 调节 Smad7，参与上皮-成纤维细胞的异常相互作用。这些结果将有助于发现早期生物标志物，并为吸烟引起的肺纤维化提供治疗靶点。
