A range of miRNAs including let d
A range of miRNAs, including let-7d, miR-21, miR-29, miR-200, miR-154, miR-17 cluster, miR-145, and miR-199-5p, have been shown to be differentially expressed in human IPF lung tissue, TGF-β1-stimulated fibroblasts, or both of them . However, the mechanisms of these miRNAs are still understood little. Meanwhile, the linkage between lncRNAs and miRNAs is commonly observed, one of the typical hypothesis is ceRNA. In the previous studies, lncRNA-ATB is considered to be one of the ceRNAs . In this work, our results also indicate lncRNA-ATB is enrichment in cytoplasm, a potential marker of ceRNA. To further prove this hypothesis, we performed miRNA array to seek for the targeted miRNAs. Our results pointed out the dramatic change of miR-200c after lncRNA-ATB knockdown in EMT model.
The participation of miRNA-200 in pulmonary fibrosis has been proved by different research groups. The down-regulated miR-200 BQ-788 sodium salt levels have been observed both in pulmonary fibrosis model and patient lungs , especially, miR-200 family members inhibit TGF-β1-induced EMT of AECs. It's well known that miR-200c involve in numerous diseases by regulating EMT process, such as cancer, fibrosis, and organ development . Also, our work suggested miR-200c was decreased in EMT process of silicosis, while elevated levels of miR-200c by mimic or agomir could revert TGF-β1-induced EMT and the degree of silicosis. In addition, we performed RNA-pull down and dual luciferase reporter gene assays to validate the binding between lncRNA-ATB and miR-200c. Furthermore, lncRNA-ATB blocked EMT could be strengthened by miR-200c mimic or relived by miR-200c inhibitor. All of these data indicate that miR-200c is negatively regulated by lncRNA-ATB and involved in EMT.
ZEB1 (also known as TCF8 and δEF1) is an important regulator in the complex network of transcriptional repressors, which is also been used as a mesenchymal marker. Former studies suggest ZEB1 regulates the expression of E-cadherin and EMT through repression of many master regulators of epithelial polarity . Also, miR-200 regulated EMT by targeting ZEB1 is classic . Therefore, we verified the regulatory role of miR-200c and lncRNA-ATB. These data indicate that ZEB1 is controlled by lncRNA-ATB-mediated inhibition of miR-200c.
At the beginning of silicosis, alveolar macrophages recognize and phagocyte the silica particles to remove them from alveoli or decompose them, then they secret cytokines including IL-1β, TNF-α, and TGF-β. Meanwhile, cells release chemokines that recruit new inflammatory cells and increase the inflammation . TGF-β, the most important cytokine for generating and maintaining a fibrotic milieu, is mainly secreted by M2 macrophages . In the microenvironment of lung alveoli, alveolar M2 macrophages influence surrounded AECs to promote pulmonary fibrosis. Here, our results showed macrophages could induce lung epithelial EMT by secreting TGF-β1, activating lncRNA-ATB/miR-200c/ZEB1 cascade. Particularly, different cells types influence and crosstalk with each other in the alveolar environment promoted the occurrence of pulmonary fibrosis jointly. Since M2 macrophage is one of the main sources of TGF-β1, we co-cultured M2 macrophages and lung epithelial cells, EMT was successfully observed along with activated lncRNA-ATB, which further proved our former data.
In conclusion, the present work established that lncRNA-ATB mediated EMT regulation through binding with miR-200c to relieve ZEB1, which is involved in silica-induced pulmonary fibrosis (Fig. 7). These results are helpful to complete the regulatory net and mechanisms of lncRNA-ATB in fibrosis. This new insight may provide strategy to intervene the process of occupational pulmonary fibrosis.
Funding This work was supported by the Natural Science Foundation of China (81573119), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Postgraduate Innovation Project of Jiangsu Province (JX22013368).