IRF4 Participates in Pulmonary Fibrosis Induced by Silica Particles through Regulating Macrophage Polarization and Fibroblast Activation.

Long-term exposure to silica dust can cause silicosis, which is characterized by chronic progressive inflammatory injury, fibroblast activation, and the deposition of extracellular matrix. IRF4 is involved in immune response. However, the potential regulation of IRF4 in silicosis and pulmonary fibrosis remains largely unexplored. In this study, RNA-seq analysis identified the upregulated expression of IRF4 in fibrotic lung tissues of mice exposed to silica particles. And we verified the increased expression of IRF4 in SiO2-treated macrophages and TGF-ß1-treated fibroblasts. We further found that the down-regulation of IRF4 impeded the macrophage polarization and the release of pro-fibrotic factors. Moreover, the down-regulation of IRF4 alleviated the migration, invasion, and the expression of fibrotic molecules in fibroblasts. Using ChIP-qPCR assay, we confirmed that IRF4 regulated the transcriptional activity of the IL-17A promoter, thus stimulated fibroblast activation, migration and invasion. In vivo experiment, the AAV-siIRF4 was designed to interfere with the expression of IRF4 in lung tissues of mice exposed to silica particles. Whole blood, bronchoalveolar lavage fluid and lung tissues were obtained from mice at 7, 14, 28 and 56 days after silica exposure. The results showed that the leukocyte content and inflammatory factors reached a peak at day 14 and remained peak for a long time after IRF4 knockdown. Furthermore, the fibrotic responses of mouse lung tissues were alleviated after IRF4 knockdown. Our study explored the important roles of IRF4 in inflammatory and fibrotic responses, which provided a new target for the treatment of silicosis and pulmonary fibrosis.

Identification of circRNA expression profiles and the potential role of hsa_circ_0006916 in silicosis and pulmonary fibrosis.

Circular RNAs (circRNAs) are emerging as novel regulators in the biological development of various diseases, but their expression profiles, functions and mechanisms in silicosis and pulmonary fibrosis remain largely unexplored. In this study, we constructed a mouse model of pulmonary fibrosis by intratracheal injection of silica particles and then performed transcriptome RNA sequencing of lung tissues. The results showed that 78 circRNAs, 39 miRNAs and 262 mRNAs were differentially expressed. Among them, five circRNAs, three miRNAs and four mRNAs were further selected, and their abnormal expression was verified in mouse fibrotic lung tissues by RT-qPCR assay. The circRNA-associated ceRNA network including 206 ceRNA triplets was constructed based on abnormally expressed circRNAs, miRNAs and mRNAs, and miR-199b-5p, miR-296-5p and miR-708-5p were identified as hub miRNAs connected to circRNAs and mRNAs. Subsequently, GO and KEGG pathway enrichment analyses were performed to detect the potential roles of differentially expressed mRNAs in pulmonary fibrosis, which were mainly involved in immune response, Th17 cell differentiation, NF-κB signaling pathway and PI3K-Akt signaling pathway. Furthermore, we identified that hsa_circ_0006916 was up-regulated in pulmonary fibrosis. To characterize the potential role of hsa_circ_0006916, we transfected siRNA targeting hsa_circ_0006916 into alveolar macrophages and found that knockdown of hsa_circ_0006916 significantly increased the expression levels of M1 molecules IL-1ß and TNF-α and reduced the expression level of M2 molecule TGF-ß1, indicating that hsa_circ_0006916 may play an important role in the activation of M1-M2 polarization effect in macrophages. Our results provided important evidence on the possible contribution of these abnormal circRNAs to the development of silicosis and pulmonary fibrosis.

Long non-coding RNA SNHG1 promotes fibroblast-to-myofibroblast transition during the development of pulmonary fibrosis induced by silica particles exposure.

Inhaling silica dust in the environment can cause progressive pulmonary fibrosis, then silicosis. Silicosis is the most harmful occupational disease in the world, so the study of the mechanism is of great significance for the prevention and treatment of silicosis. Long non-coding RNAs (lncRNAs) are important players in the pathological process of fibrotic diseases. However, the function of specific lncRNA in regulating pulmonary fibrosis remains elusive. In this study, a mouse model of pulmonary fibrosis via intratracheal instillation of silica particles was established, and the differential expression of lnc-SNHG1 and miR-326 in lung tissues and TGF-ß1-treated fibroblasts was detected by the qRT-PCR method. Short interfering RNA (siRNA) and plasmid were designed for knockdown or overexpression of lnc-SNHG1 in fibroblasts. MiRNA simulant was designed for overexpression of miR-326 in vivo and in vitro. Dual-luciferase reporter system, immunofluorescence, western blot, wound healing and transwell assay were performed to investigate the function and the underlying mechanisms of lnc-SNHG1. As a result, we found that lnc-SNHG1 was highly expressed in fibrotic lung tissues of mice and TGF-ß1-treated fibroblasts. Moreover, the high expression of lnc-SNHG1 facilitated the migration and invasion of fibroblasts and the secretion of fibrotic molecules, while the low expression of lnc-SNHG1 exerted the opposite effects. Further mechanism studies showed that miR-326 was the potential target of lnc-SNHG1, and there is a negative correlation between the expression levels of lnc-SNHG1 and miR-326. Combined with mitigating fibrotic effects of miR-326 in a mouse model of silica particles exposure, we revealed that lnc-SNHG1 significantly sponged miR-326 and facilitated the expression of SP1, thus accelerating fibroblast-to-myofibroblast transition and synergistically promoting the development of pulmonary fibrosis. Our study uncovered a key mechanism by which lnc-SNHG1 regulated pulmonary fibrosis through miR-326/SP1 axis, and lnc-SNHG1 is a potential target for the prevention and treatment of silicosis.

miR-138 inhibits epithelial-mesenchymal transition in silica-induced pulmonary fibrosis by regulating ZEB2.

Silica dust is a common pollutant in the occupational environment, such as coal mines. Inhalation of silica dust can cause progressive pulmonary fibrosis and then silicosis. Silicosis is still one of the most harmful occupational diseases in the world, so the study of its pathogenesis is necessary for the treatment of silicosis. In this study, we constructed a mouse model of pulmonary fibrosis via intratracheal instillation of silica particles and identified the decreased expression of miR-138 in fibrotic lung tissues of mice. Moreover, the overexpression of miR-138 retarded the process of epithelial-mesenchymal transition (EMT) in a mouse model of silica particles exposure and epithelial cells stimulated by silica particles. Further studies showed that ZEB2 was one of the potential targets of miR-138, and the up-regulation of miR-138 reduced ZEB2 levels in mouse lung tissues and in epithelial cells. We next found that the expression levels of ɑ-SMA and Vimentin were significantly increased and E-cadherin levels were decreased after transfection with miR-138 inhibitor in epithelial cells. However, these effects were abated by the knockdown of ZEB2. Consistently, the increased migration ability of epithelial cells by miR-138 inhibitor transfection was also reversed by the knockdown of ZEB2. Collectively, we revealed that miR-138 significantly targeted ZEB2, thus inhibited the EMT process and mitigated the development of pulmonary fibrosis. miR-138 may be a potential target for the treatment of pulmonary fibrosis.