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Silicosis is a progressive and chronic occupational lung disease characterized by lung inflammation, silicotic nodule formation, and diffuse pulmonary fibrosis. Emerging evidence indicates that endothelial-mesenchymal transition (EndoMT) plays a crucial role in the development of silicosis. Herein, we conducted a SiO 2 -induced EndoMT model and established a mouse model with pulmonary fibrosis by silica. We identified that SiO 2 effectively increased the expression of mesenchymal markers while decreasing the levels of endothelial markers in endothelial cells. It's further demonstrated that SiO 2 induced the PI3K/Akt signaling pathway activation via LGALS3 synthesis. Next, interfering LGALS3 blocked the process of EndoMT by inhibiting the activity of PI3K/AKT signaling. In vivo, the administration of a specific PI3K inhibitor LY294002 significantly alleviated silica-induced pulmonary fibrosis. Collectively, these results identified that the LGALS3/PI3K/AKT pathway provided a rationale target for the clinical treatment and intervention of silicosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As a fatal occupational disease with limited therapeutic options, molecular mechanisms underpinning silicosis are still undefined. Herein, single-cell RNA sequencing of the lung tissue of silicosis mice identified two monocyte subsets, which were characterized by Cxcl10 and Mmp14 and enriched in fibrotic mouse lungs. Both Cxcl10 + and Mmp14 + monocyte subsets exhibited activation of inflammatory marker genes and positive regulation of cytokine production. Another fibrosis-unique neutrophil population characterized by Ccl3 appeared to be related to the pro-fibrotic process, specifically the "inflammatory response". Meanwhile, the proportion of monocytes and neutrophils was significantly higher in the serum of silicosis patients and slices of lung tissue from patients with silicosis further validated the over-expression of Cxcl10 and Mmp14 in monocytes, also Ccl3 in neutrophils, respectively. Mechanically, receptor-ligand interaction analysis identified the crosstalk of Cxcl10 + /Mmp14 + monocytes with Ccl3 + neutrophils promoting fibrogenesis via coupling of HBEGF-CD44 and CSF1-CSF1R. In vivo, administration of clodronate liposomes, Cxcl10 or Mmp14 siRNA-loaded liposomes, Ccl3 receptor antagonist BX471, CD44 or CSF1R neutralizing antibodies significantly alleviated silica-induced lung fibrosis. Collectively, these results demonstrate that the newly defined Cxcl10 + /Mmp14 + monocytes and Ccl3 + neutrophils participate in the silicosis process and highlight anti-receptor-ligand pair treatment as a potentially effective therapeutic strategy in managing silicosis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Pulmonary fibrosis is a growing clinical problem that develops as a result of abnormal wound healing, leading to breathlessness, pulmonary dysfunction and ultimately death. However, therapeutic options for pulmonary fibrosis are limited because the underlying pathogenesis remains incompletely understood. Circular RNAs, as key regulators in various diseases, remain poorly understood in pulmonary fibrosis induced by silica., Methods: We performed studies with fibroblast cell lines and silica-induced mouse pulmonary fibrosis models. The expression of circZNF609, miR-145-5p, and KLF4 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RNA immunoprecipitation (RIP) assays and m6A RNA immunoprecipitation assays (MeRIP), Western blotting, immunofluorescence assays, and CCK8 were performed to investigate the role of the circZNF609/miR-145-5p/KLF4 axis and circZNF609-encoded peptides in fibroblast activation., Results: Our data showed that circZNF609 was downregulated in activated fibroblasts and silica-induced fibrotic mouse lung tissues. Overexpression of circZNF609 could inhibit fibroblast activation induced by transforming growth factor-β1 (TGF-β1). Mechanically, we revealed that circZNF609 regulates pulmonary fibrosis via miR-145-5p/KLF4 axis and circZNF609-encoded peptides. Furthermore, circZNF609 was highly methylated and its expression was controlled by N6-methyladenosine (m6A) modification. Lastly, in vivo studies revealed that overexpression of circZNF609 attenuates silica-induced lung fibrosis in mice., Conclusions: Our data indicate that circZNF609 is a critical regulator of fibroblast activation and silica-induced lung fibrosis. The circZNF609 and its derived peptides may represent novel promising targets for the treatment of pulmonary fibrosis., (© 2023. The Author(s).)

Silicosis is a global occupational pulmonary disease due to the accumulation of silica dust in the lung. Lacking effective clinical drugs makes the treatment of this disease quite challenging in clinics largely because the pathogenic mechanisms remain obscure. Interleukin 33 (IL33), a pleiotropic cytokine, could promote wound healing and tissue repair via the receptor ST2. However, the mechanisms governing the involvement of IL33 in silicosis progression remain to be further explored. Here, we demonstrated that the IL33 levels in the lung sections were significantly overexpressed after bleomycin and silica treatment. Chromatin immunoprecipitation assay, knockdown, and reverse experiments were performed in lung fibroblasts to prove gene interaction following exogenous IL33 treatment or cocultured with silica-treated lung epithelial cells. Mechanistically, we illustrated that silica-stimulated lung epithelial cells secreted IL33 and further promoted the activation, proliferation, and migration of pulmonary fibroblasts by activating the ERK/AP-1/NPM1 signaling pathway in vitro. And more, treatment with NPM1 siRNA-loaded liposomes markedly protected mice from silica-induced pulmonary fibrosis in vivo. In conclusion, the involvement of NPM1 in the progression of silicosis is regulated by the IL33/ERK/AP-1 signaling axis, which is the potential therapeutic target candidate in developing novel antifibrotic strategies for pulmonary fibrosis., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pulmonary fibrosis (PF), as an end-stage clinical phenotype of interstitial lung diseases (ILDs), is frequently initiated after alveolar injury, in which ferroptosis has been identified as a critical event aggravating the pathophysiological progression of this disease. Here in, a comprehensive analysis of two mouse models of pulmonary fibrosis developed in our lab demonstrated that lung damage-induced ferroptosis of alveolar epithelial Type2 cells (AEC2) significantly accumulates during the development of pulmonary fibrosis while ferroptosis suppressor genes GPX4 and FSP1 are dramatically inactivated. Mechanistically, upregulation of de novo methylation regulator Uhrf1 sensitively elevates CpG site methylation levels in promoters of both GPX4 and FSP1 genes and induces the epigenetic repression of both genes, subsequently leading to ferroptosis in chemically interfered AEC2 cells. Meanwhile, specific inhibition of UHRF1 highly arrests the ferroptosis formation and blocks the progression of pulmonary fibrosis in both of our research models. This study first, to our knowledge, identified the involvement of Uhrf1 in mediating the ferroptosis of chemically injured AEC2s via de novo promoter-specific methylation of both GPX4 and FSP1 genes, which consequently accelerates the process of pulmonary fibrosis. The above findings also strongly suggested Uhrf1 as a novel potential target in the treatment of pulmonary fibrosis., (© 2022. The Author(s).)

Pulmonary fibrosis is a chronic and progressive interstitial lung disease associated with the decay of pulmonary function, which leads to a fatal outcome. As an essential epigenetic regulator of DNA methylation, the involvement of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) in fibroblast activation remains largely undefined in pulmonary fibrosis. In the present study, we found that TGF-β1-mediated upregulation of UHRF1 repressed beclin 1 via methylated induction of its promoter, which finally resulted in fibroblast activation and lung fibrosis both in vitro and in vivo. Moreover, knockdown of UHRF1 significantly arrested fibroblast proliferation and reactivated beclin 1 in lung fibroblasts. Thus, intravenous administration of UHRF1 siRNA-loaded liposomes significantly protected mice against experimental pulmonary fibrosis. Accordingly, our data suggest that UHRF1 might be a novel potential therapeutic target in the pathogenesis of pulmonary fibrosis.

Background: Pulmonary fibrosis is a chronic progressive fibrotic interstitial lung disease characterized by excessive extracellular matrix (ECM) deposition caused by activated fibroblasts. Increasing evidence shows that matrix stiffness is essential in promoting fibroblast activation and profibrotic changes. Here, we investigated the expression and function of matrix stiffness-regulated ZNF416 in pulmonary fibrotic lung fibroblasts., Methods: 1 kappa (soft), 60 kappa (stiff) gel-coated coverslips, or transforming growth factor-beta 1 (TGF-β1)-cultured lung fibroblasts and the gain- or loss- of the ZNF416 function assays were performed in vitro. We also established two experimental pulmonary fibrosis mouse models by a single intratracheal instillation with 50 mg/kg silica or 6 mg/kg bleomycin (BLM). ZNF416 siRNA-loaded liposomes and TGF-β1 receptor inhibitor SB431542 were administrated in vivo., Results: Our study identified that ZNF416 could regulate fibroblast differentiation, proliferation, and contraction by promoting the nuclear accumulation of p-Smad2/3. Besides, ZNF416 siRNA-loaded liposome delivery by tail-vein could passively target the fibrotic area in the lung, and co-administration of ZNF416 siRNA-loaded liposomes and SB431542 significantly protects mice against silica or BLM-induced lung injury and fibrosis., Conclusion: In this study, our results indicate that mechanosensitive ZNF416 is a potential molecular target for the treatment of pulmonary fibrosis. Strategies aimed at silencing ZNF416 could be a promising approach to fight against pulmonary fibrosis., (© 2022. The Author(s).)

Exposure to silica is a cause of pulmonary fibrosis disease termed silicosis, which leads to respiratory failure and ultimately death. However, what drives fibrosis is not fully elucidated and therapeutic options remain limited. Our previous RNA-sequencing analysis showed that the expression of caveolin-1 (CAV1) was downregulated in silica-inhaled mouse lung tissues. Here, we not only verified that CAV1 was decreased in silica-induced fibrotic mouse lung tissues in both messenger RNA and protein levels, but also found that CSP7, a functional peptide of CAV1, could attenuate pulmonary fibrosis in vivo. Further in vitro experiments revealed that CAV1 reduced the expression of Yes-associated protein 1(YAP1) and affected its nuclear translocation in fibroblasts. In addition, Glutaminase 1 (GLS1), a key regulator of glutaminolysis, was identified to be a downstream effector of YAP1. CAV1 could suppress the activity of YAP1 to decrease the transcription of GLS1, thereby inhibiting fibroblast activation. Taken together, our results demonstrated that CAV1 and its functional peptide CSP7 may be potential molecules or drugs for the prevention and intervention of silicosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: N 6 -methyladenosine (m 6 A) is the most common and abundant internal modification of RNA. Its critical functions in multiple physiological and pathological processes have been reported. However, the role of m 6 A in silica-induced pulmonary fibrosis has not been fully elucidated. AlkB homolog 5 (ALKBH5), a well-known m 6 A demethylase, is upregulated in the silica-induced mouse pulmonary fibrosis model. Here, we sought to investigate the function of ALKBH5 in pulmonary fibrosis triggered by silica inhalation., Methods: We performed studies with fibroblast cell lines and silica-induced mouse pulmonary fibrosis models. The expression of ALKBH5, miR-320a-3p, and forkhead box protein M1 (FOXM1) was determined by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RNA immunoprecipitation (RIP) assays and m 6 A RNA immunoprecipitation assays (MeRIP), western bolt, immunofluorescence assays, and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining were performed to explore the roles of ALKBH5, miR-320a-3p, and FOXM1 in fibroblast activation., Results: ALKBH5 expression was increased in silica-inhaled mouse lung tissues and transforming growth factor (TGF)-β1-stimulated fibroblasts. Moreover, ALKBH5 knockdown exerted antifibrotic effects in vitro. Simultaneously, downregulation of ALKBH5 elevated miR-320a-3p but decreased pri-miR-320a-3p. Mechanically, ALKBH5 demethylated pri-miR-320a-3p, thus blocking the microprocessor protein DGCR8 from interacting with pri-miR-320a-3p and leading to mature process blockage of pri-miR-320a-3p. We further demonstrated that miR-320a-3p could regulate fibrosis by targeting FOXM1 messenger RNA (mRNA) 3'-untranslated region (UTR). Notably, our study also verified that ALKBH5 could also directly regulate FOXM1 in an m 6 A-dependent manner., Conclusions: Our findings suggest that ALKBH5 promotes silica-induced lung fibrosis via the miR-320a-3p/FOXM1 axis or targeting FOXM1 directly. Approaches aimed at ALKBH5 may be efficacious in treating lung fibrosis., (© 2022. The Author(s).)

Silicosis is a devastating interstitial lung disease arising from long-term exposure to inhalable silica. Regrettably, no therapy currently can effectively reverse the silica-induced fibrotic lesion. Emerging evidence has indicated that the dysregulation of microRNAs is involved in silica-induced pulmonary fibrosis. The aim of this study is to explore the expression pattern and underlying mechanisms of miR-770-5p in silica-induced pulmonary fibrosis. Consistent with our previous miRNA microarray analysis, the results of qRT-PCR showed that miR-770-5p expression was downregulated in silica-induced pulmonary fibrosis in humans and animal models. Administration of miR-770-5p agomir significantly reduced the fibrotic lesions in the lungs of mice exposed to silica dust. MiR-770-5p also exhibited a dramatic reduction in TGF-β1-activated human pulmonary fibroblasts (MRC-5). Transfection of miR-770-5p mimics significantly decreased the viability, migration ability, and S/G0 phase distribution, as well as the expression of fibronectin, collagen I, and α-SMA in TGF-β1-treated MRC-5 cells. Transforming growth factor-β receptor 1 (TGFBR1) was confirmed as a direct target of regulation by miR-770-5p. The expression of TGFBR1 was significantly increased in pulmonary fibrosis. Knockdown of TGFBR1 blocked the transduction of the TGF-β1 signaling pathway and attenuated the activation of MRC-5 cells, while overexpression of TGFBR1 effectively restored the activation of MRC-5 cells inhibited by miR-770-5p. Together, our results demonstrated that miR-770-5p exerted an anti-fibrotic effect in silica-induced pulmonary fibrosis by targeting TGFBR1. Targeting miR-770-5p might provide a new therapeutic strategy to prevent the abnormal activation of pulmonary fibroblasts in silicosis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Silicosis is one of the most common occupational pulmonary fibrosis caused by respirable silica-based particle exposure, with no ideal drugs at present. Metformin, a commonly used biguanide antidiabetic agent, could activate AMP-activated protein kinase (AMPK) to exert its pharmacological action. Therefore, we sought to investigate the role of metformin in silica-induced lung fibrosis., Methods: The anti-fibrotic role of metformin was assessed in 50 mg/kg silica-induced lung fibrosis model. Silicon dioxide (SiO 2 )-stimulated lung epithelial cells/macrophages and transforming growth factor-beta 1 (TGF-β1)-induced differentiated lung fibroblasts were used for in vitro models., Results: At the concentration of 300 mg/kg in the mouse model, metformin significantly reduced lung inflammation and fibrosis in SiO 2 -instilled mice at the early and late fibrotic stages. Besides, metformin (range 2-10 mM) reversed SiO 2 -induced cell toxicity, oxidative stress, and epithelial-mesenchymal transition process in epithelial cells (A549 and HBE), inhibited inflammation response in macrophages (THP-1), and alleviated TGF-β1-stimulated fibroblast activation in lung fibroblasts (MRC-5) via an AMPK-dependent pathway., Conclusions: In this study, we identified that metformin might be a potential drug for silicosis treatment., (© 2021. The Author(s).)

Dysregulation of non-coding RNAs (ncRNAs) has been proved to play pivotal roles in epithelial-mesenchymal transition (EMT) and fibrosis. We have previously demonstrated the crucial function of long non-coding RNA (lncRNA) ATB in silica-induced pulmonary fibrosis-related EMT progression. However, the underlying molecular mechanism has not been fully elucidated. Here, we verified miR-29b-2-5p and miR-34c-3p as two vital downstream targets of lncRNA-ATB. As opposed to lncRNA-ATB, a significant reduction of both miR-29b-2-5p and miR-34c-3p was observed in lung epithelial cells treated with TGF-β1 and a murine silicosis model. Overexpression miR-29b-2-5p or miR-34c-3p inhibited EMT process and abrogated the pro-fibrotic effects of lncRNA-ATB in vitro. Further, the ectopic expression of miR-29b-2-5p and miR-34c-3p with chemotherapy attenuated silica-induced pulmonary fibrosis in vivo. Mechanistically, TGF-β1-induced lncRNA-ATB accelerated EMT as a sponge of miR-29b-2-5p and miR-34c-3p and shared miRNA response elements with MEKK2 and NOTCH2, thus relieving these two molecules from miRNA-mediated translational repression. Interestingly, the co-transfection of miR-29b-2-5p and miR-34c-3p showed a synergistic suppression effect on EMT in vitro. Furthermore, the co-expression of these two miRNAs by using adeno-associated virus (AAV) better alleviated silica-induced fibrogenesis than single miRNA. Approaches aiming at lncRNA-ATB and its downstream effectors may represent new effective therapeutic strategies in pulmonary fibrosis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Pulmonary fibrosis develops when myofibroblasts and extracellular matrix excessively accumulate in the injured lung, but what drives fibrosis is not fully understood. Glycolysis has been linked to cell growth and proliferation, and several studies have shown enhanced glycolysis promotes pulmonary fibrosis. However, detailed studies describing this switch remain limited. Here, we identified that TGF-β1 effectively increased the expression of circHIPK3 in lung fibroblasts, and circHIPK3 inhibition attenuated the activation, proliferation, and glycolysis of fibroblasts in vitro . Dual-luciferase reporter gene assays, RNA immunoprecipitation (RIP), and RNA pull-down assays showed that circHIPK3 could function as a sponge of miR-30a-3p and inhibit its expression. Furthermore, FOXK2, a driver transcription factor of glycolysis, was identified to be a direct target of miR-30a-3p. Mechanistically, circHIPK3 could enhance the expression of FOXK2 via sponging miR-30a-3p, thereby facilitating fibroblast glycolysis and activation. Besides, miR-30a-3p overexpression or FOXK2 knockdown blocked fibroblast activation induced by TGF-β1 and abrogated the profibrotic effects of circHIPK3. Moreover, circHIPK3 and miR-30a-3p were also dysregulated in fibrotic murine lung tissues induced by silica. Adeno-associated virus (AAV)-mediated circHIPK3 silence or miR-30a-3p overexpression alleviated silica-induced pulmonary fibrosis in vivo . In conclusion, our results identified circHIPK3/miR-30a-3p/FOXK2 regulatory pathway as an important glycolysis cascade in pulmonary fibrosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Silicosis is a universal occupational disease, which is caused by long-term crystalline silica exposure. Recent studies have shown that noncoding RNAs participate in diverse pathological cellular pathways. However, the precise regulation mechanism remains limited in silicosis. Here, we established a silica-induced mouse fibrosis model (all mice received a one-time intratracheal instillation with 50 mg/kg of silica in 0.05 mL sterile saline). MiR-490-3p was significantly downregulated in silica-induced fibrotic mouse lung tissues and TGF-β1 treated fibroblasts. Moreover, overexpressed miR-490-3p could relieve silica-induced lung fibrosis in vivo, and prevent the process of fibroblast-to-myofibroblast transition(FMT)in vitro. Mechanistically, TGFBR1 was one of the major target genes of miR-490-3p, and tightly associated with the process of fibroblasts activation. SNHG20, as opposed to miR-490-3p expression, was elevated in TGF-β1-treated fibroblast cell lines and contributed to decreased levels of miR-490-3p. Taken together, these data indicated that miR-490-3p plays a key role in silica-induced pulmonary fibrosis. Our results suggested that SNHG20/miR-490-3p/TGFBR1 axis may provide a new treatment target of pulmonary fibrosis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Silicosis is a kind of irreversible pulmonary fibrosis induced by the long-term inhalation of silica particles. The therapeutic strategy based on the microRNAs might be an effective way for the treatment of silicosis. Our previous miRNA microarray data indicated that miR-326 was decreased in the mouse lung tissues of silica-induced pulmonary fibrosis. However, the specific functions of miR-326 on silica-induced pulmonary fibrosis remain unclear. The objective was to determine the expression and the biological effects of miR-326 in silica-induced pulmonary fibrosis. Methods included mouse models of silica-induced pulmonary fibrosis and miR-326 intervention that were established separately to explore the effect of miR-326 in vivo . The cell models of SiO 2 -treated lung epithelial cells (HBE and A549) and TGF-β1-stimulated lung fibroblast cells (MRC-5 and NIH/3T3) were used to investigate the mechanism of miR-326 in vitro . Hematoxylin and eosin staining was used to evaluate the severity and distribution of fibrosis of mouse lung tissues. Western blot and immunofluorescence assays were performed to measure the downstream molecules of miR-326. Transmission electron microscopy pictures showed the autophagy activity. The results showed miR-326 is down-regulated in the fibrotic lung tissues of silica-treated mice, while increased expression of miR-326 attenuates silica-induced pulmonary fibrosis in vivo . Tumor necrosis factor superfamily-14 (TNFSF14) and polypyrimidine tract-binding protein 1 (PTBP1) are identified as the targets of miR-326. MiR-326 dampens pulmonary inflammation through targeting TNFSF14 and promotes autophagy activity of fibroblasts through targeting PTBP1. LncRNA HOTAIR facilitates inflammation via sponging miR-326. In conclusion, we demonstrate that miR-326 inhibits inflammation and promotes autophagy activity by targeting TNFSF14 and PTBP1 separately to alleviate silica-induced pulmonary fibrosis. Our results might shed new light on the therapeutic strategies for silica-induced pulmonary fibrosis.

Silica-induced pulmonary fibrosis is a kind of worldwide occupational disease, and there is no effective treatment at present. Peptide therapy has attracted significant attention due to its simple structure, high selectiveness, strong bioactivity, relative safety, and high patient tolerance. In this study, we first confirmed that M10, a 10 amino acid peptide, has anti-fibrotic effects during the early and late stages of silica-induced fibrosis in mouse models and then partly explored the underlying mechanisms in vitro. M10 was detected in both the cell cytoplasm and nuclei. M10 showed no cytotoxicity to pulmonary epithelial cells and fibroblasts at the given concentrations. Functionally, M10 can reverse the silica-induced EMT process in epithelial cells and decrease TGF-β1-stimulated fibroblast activation. Further mechanism investigations supported that M10 can block TGF-β1 signalling by inhibiting phosphorylation of Smad2 protein in vitro and in vivo. All of the results indicate that M10 peptide may be a new method for the treatment of silica-induced pulmonary fibrosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Various miRNAs are dysregulated during initiation and progression of pulmonary fibrosis. However, their function remains limited in silicosis. Here, we observed that miR-125a-3p was downregulated in silica-induced fibrotic murine lung tissues. Ectopic miR-125a-3p expression with chemotherapy attenuated silica-induced pulmonary fibrosis. Further in vitro experiments revealed that TGF-β1 effectively decreased miR-125a-3p expression in fibroblast lines (NIH/3T3 and MRC-5). Overexpression of miR-125a-3p blocked fibroblast activation stimulated by TGF-β1. Mechanistically, miR-125a-3p could bind to the 3'-untranslated region of Fyn and inhibit its expression in both mRNA and protein levels, thus causing inactivation of Fyn downstream effector STAT3. Fyn and p-STAT3, as opposed to miR-125a-3p expression, were elevated in silica-induced fibrotic murine lung tissues and TGF-β1-treated fibroblast lines. Furthermore, Fyn knockdown or p-STAT3 suppression effectively attenuated fibroblast activation and ECM production. Taken together, miR-125a-3p is involved in fibrosis pathogenesis by fibroblast activation, suggesting that targeting miR-125a-3p/Fyn/STAT3 signaling pathway could be a potential therapeutic approach for pulmonary fibrosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Silicosis is one of the typical forms of pneumoconiosis characterized by abnormal proliferation of fibroblasts and deposition of extracellular matrix. Recent findings have shown that microRNAs and circular RNAs (circRNAs) are implicated in many diseases. However, the function of noncoding RNAs in pulmonary fibrosis remain to be elucidated. Here, miR-7 was found significantly decreased in silica-treated pulmonary epithelial cells as well as in fibrotic lung tissues of mice. Elevated expression of miR-7 via agomir injection relieved lung fibrosis in vivo. Further molecular study showed that miR-7 played its role against pulmonary fibrosis by blocking epithelial-mesenchymal transition (EMT) progression of human bronchial epithelial cells and A549 cells. Notably, transforming growth factor beta receptor 2 (TGFBR2) was identified as a target gene of miR-7 with bioinformatics tools, which was verified by dual luciferase receptor gene assay in human bronchial epithelial cells and A549 cells. Silica induced elevation of TGFBR2 could be abolished by exogenous expression of miR-7. Furthermore, bioinformatics software indicated that circRNA CDR1as had several binding sites for miR-7. The inhibitory effects of miR-7 on EMT and its target TGFBR2 were suppressed by circRNA CDR1as, which contributed to pulmonary fibrosis. Our studies also revealed overexpressed miR-7 could repress fibrogenesis of lung fibroblasts induced by TGF-β1. Collectively, circRNA CDR1as stimulated by silica could sponge miR-7 to release TGFBR2, plays an important role during pulmonary fibrosis by promoting EMT process. These results indicated that the interaction between miR-7 and circRNA CDR1as may exert important functions and provide potential therapeutic targets in lung fibrotic diseases.

Silicosis is a very serious occupational disease and it features pathological manifestations of inflammatory infiltration, excessive proliferation of fibroblasts and massive depositions of the extracellular matrix in the lungs. Recent studies described the roles of a variety of microRNAs (miRNAs) in fibrotic diseases. Here, we aimed to explore the potential mechanism of miR-542-5p in the activation of lung fibroblasts. To induce a pulmonary fibrosis mouse model, silica suspension and the miR-542-5p agomir were administered to mice by intratracheal instillation and tail vein injection. We found that miR-542-5p was significantly decreased in mouse fibrotic lung tissues and up-regulation of miR-542-5p visually attenuated a series of fibrotic lesions, including alveolar structural damage, alveolar interstitial thickening and silica-induced nodule formation. The down-regulation of miR-542-5p was also observed in mouse fibroblast (NIH-3T3) treated with transforming growth factor β1 (TGF-β1). The proliferation and migration ability of NIH-3T3 cells were also inhibited by the transfection of miR-542-5p mimic. Integrin α6 (Itga6), reported as a cell surface protein associated with fibroblast proliferation, was confirmed to be a direct target of miR-542-5p. The knockdown of Itga6 significantly inhibited the phosphorylation of FAK/PI3K/AKT. In conclusion, miR-542-5p has a potential function for reducing the proliferation of fibroblasts and inhibiting silica-induced pulmonary fibrosis, which might be partially realized by directly binding to Itga6. Our data suggested that miR-542-5p might be a new therapeutic target for silicosis or other pulmonary fibrosis., Competing Interests: The authors declare no conflict of interest.

Long non-coding RNAs (lncRNAs) are important signal transduction regulators that act by various patterns. However, little is known about the molecular mechanisms of lncRNA related pathways in occupational lung fibrosis. Our previous study found that epithelial-mesenchymal transition (EMT) was one of the key events in silica-induced pulmonary fibrosis. This study showed that the lncRNA-ATB promoted EMT by acting as a miR-200c sponge. miR-200c was identified by miRNA array as a potential target of lncRNA-ATB and verified by dual luciferase reporter gene together with RNA pull-down assays. Moreover, our findings demonstrated that lncRNA-ATB is abundantly expressed during EMT of lung epithelial cells, which contributes to decreased levels of miR-200c. miR-200c targeted ZEB1 to relief silicosis by blocking EMT in vivo and in vitro. The results also suggested M2 macrophages secreted transforming growth factor-β1 (TGF-β1) to induce EMT process by activating lncRNA-ATB in epithelial cells. Collectively, silica-stimulated macrophages secreted TGF-β1 to induce lncRNA-ATB in epithelia cells, promoting EMT by binding with miR-200c and releasing ZEB1. These observations provide further understanding of the regulatory network of silica-induced pulmonary fibrosis and identify new therapeutic targets hopefully., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Silicosis is associated with fibroblast proliferation and extracellular matrix deposition in lung tissues. The dysregulation of miR-1224-5p has been implicated in several human cancers; however, the expression and function of miR-1224-5p in silicosis is unknown. The mitochondrial dysfunctions play critical roles in some diseases, but how these processes are regulated in silicosis remains limited. Here, we explored the role of miR-1224-5p in a mouse model of silicosis. We showed that the expression of miR-1224-5p is increased both in lung tissues of silica-induced pulmonary fibrosis and fibroblasts exposed to TGF-β1. Repression of miR-1224-5p expression attenuated silica-induced fibrotic progression in vivo and TGF-β1-induced myofibroblast differentiation in vitro. Additionally, we demonstrated that miR-1224-5p facilitated silica-induced pulmonary fibrosis primarily by repressing one of target genes, BECN1, thereby blocking PARK2 translocation to mitochondria and inducing the accumulation of damaged mitochondria. Furthermore, the activation of PDGFR signal mediated by mitochondrial damage and insufficient mitophagy resulted in myofibroblast differentiation. Collectively, these data indicated that miR-1224-5p exerts key functions in silica-induced pulmonary fibrosis and may represent a potential therapeutic target for silicosis., Competing Interests: The authors declare no conflict of interest.

Silicosis is a kind of chronic, progressive and incurable lung fibrotic diseases with largely unknown and complex pathogenesis and molecular mechanisms. Mounting evidence suggests that microRNAs (miRNAs, miRs) are involved in the pathogenesis of silicosis. Our previous study based on miRNA microarray had shown that the expression levels of miR-503 were down-regulated in mouse lung tissues of silica-induced pulmonary fibrosis. Here, we validated the decreased expression of miR-503 in the fibrotic mouse lung tissues, human bronchial epithelial cells (HBE) and human lung adenocarcinoma A549 cells which were exposed to silica. In addition, overexpressed miR-503 inhibited silica-induced pulmonary fibrosis by attenuating the severity and the distribution of lesions in vivo and limiting the process of epithelial-mesenchymal transition (EMT) in vitro. Our molecular study further demonstrated that PI3K p85 is one of the target genes of miR-503 and the downstream molecules (Akt, mTOR and Snail) are tightly associated with EMT. Furthermore, the up-regulated lncRNA Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), acted as a competing endogenous RNA (ceRNA), can directly bound to miR-503, which indicated that lncRNA MALAT1 may modulate the expression of miR-503 thus triggering the activation of downstream fibrotic signaling pathways. Taken together, our data suggested that MALAT1-miR-503-PI3K/Akt/mTOR/Snail pathway plays critical roles in silica-induced pulmonary fibrosis.

Objective: To explore the potential therapeutic role of miR-489 in silica-induced pulmonary fibrosis mouse models. Methods: A total of 32 C57BL/6 male mice were randomly divided into four groups: saline, silica, silica plus miRNA control and silica plus miR-489 agomir ( n =8 in each group) . The mice were instilled with silica particles suspended in saline or sterile saline intratracheally. Subsequently, miR-489 agomir or miRNA control was injected via the tail vein into each mouse at days 28, 35, 42 and 49, the miR-489 levels, histological examination, collagen deposition, fibrotic biomarkers (E-cadherin, α-SMA, Vimentin, Fibronectin) and transforming growth factor-β(1) (TGF-β(1)) protein levels in mouse lung tissues were measured. Results: miR-489 levels in silica plus miR-489 group were significantly increased in lung tissues compared with silica plus miRNA control group ( P <0.05) . Histological examination showed attenuated inflammation, less severe fibrotic foci and less destruction of alveolar architecture in the silica plus miR-489 group. Additionally, both the severity and distribution of lung lesions were ameliorated in silica plus miR-489 group compared with the silica plus miRNA control group ( P <0.05) . The collagen deposition and hydroxyproline levels in silica plus miR-489 group were significantly decreased compared with the silica plus miRNA control group ( P <0.05) . These changes were supported by decreased protein levels of α-SMA, Vimentin, Fibronectin, TGF-β1 along with increased protein levels of E-cadherin in silica plus miR-489 group ( P <0.05) . Conclusion: Our data indicate that the upregulation of miR-489 has potential therapeutic role in silica-induced pulmonary fibrosis in vivo, which may be associated with the depression of TGF-β1 release.

Silicosis is an occupational pulmonary fibrosis caused by inhalation of silica (SiO 2 ) and there are no ideal drugs to treat this disease. Earthworm extract (EE), a natural nutrient, has been reported to have anti-inflammatory, antioxidant, and anti-apoptosis effects. The purpose of the current study was to test the protective effects of EE against SiO 2 -induced pulmonary fibrosis and to explore the underlying mechanisms using both in vivo and in vitro models. We found that treatment with EE significantly reduced lung inflammation and fibrosis and improved lung structure and function in SiO 2 -instilled mice. Further mechanistic investigations revealed that EE administration markedly inhibited SiO 2 -induced oxidative stress, mitochondrial apoptotic pathway, and epithelial-mesenchymal transition in HBE and A549 cells. Furthermore, we demonstrate that Nrf2 activation partly mediates the interventional effects of EE against SiO 2 -induced pulmonary fibrosis. Our study has identified EE to be a potential anti-oxidative, anti-inflammatory, and anti-fibrotic drug for silicosis.

Silicosis is a fatal pulmonary fibrotic disorder characterized by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. MiR-449a is a potential mediator of many cellular processes, including cell proliferation, differentiation, and apoptosis. We hypothesized that miR-449a may play a crucial role in the progression of pulmonary fibrogenesis. Here, we described miR-449a as a new autophagy-regulated miRNA. Importantly, miR-449a expression was significantly decreased in lung tissues of mice with silica treatment, and it was similarly expressed in NIH-3T3 and MRC-5 cells stimulated with TGF-β1. The activity of autophagy was inhibited in fibrotic lung tissues and TGF-β1-treated fibroblasts. To investigate the potential effect of miR-449a, we overexpressed miR-449a in mouse models and found that miR-449a significantly reduced both the distribution and severity of lung lesions induced by silica. In addition, miR-449a was observed to induce the activity of autophagy in vivo and in vitro. Notably, Bcl2 was identified as a target of miR-449a. Bcl2 levels were decreased in NIH-3T3 cells upon miR-449a overexpression. Indeed, the Bcl2 3' UTR contained functional miR-449a responsive sequences. Furthermore, TGF-β1 was observed to increase the expression of Bcl2 via the MAPK/ERK pathway. These results suggest that miR-449a is an important regulator of autophagy, as well as a novel endogenous suppressor of pulmonary fibrosis., Key Message: MiR-449a expression was decreased in fibrotic lungs and activated fibroblasts. Autophagy was inhibited in fibrotic lung tissues and TGF-β1-treated fibroblasts. MiR-449a had an antifibrotic effect in silica-induced lung fibrosis. MiR-449a upregulated autophagic activity in vitro. Bcl2 is the autophagy-related target of miR-449a.

Objective: To investigate the contributions and underlying molecular mechanisms of annexin A5 toward silica-induced pulmonary fibrosis., Methods: Male C57BL/6 mice were randomly divided into three groups and instilled intratracheally with silica, saline, or air. Mice were euthanized at 3, 7, 14, or 28 days following treatment. Annexin A5 levels in serum and lung tissues were detected by enzyme-linked immunosorbant assay (ELISA) assays or Western blots. The association of annexin A5 levels with silica-induced lung fibrosis was further investigated in the macrophage cell line, RAW264.7. Following exposure of these cells to silica at a concentration of 200 μg/ml for 6 or 12 h, the expression levels of transforming growth factor β1 (TGF-β1), interleukin 1α (IL-1α), Fas ligand (FasL), and their downstream targets were evaluated by Western blots. Furthermore, annexin A5 and FasL were knocked down by small interfering ribonucleic acid (siRNA) and TGF-β1 secretion into the cell culture medium was measured by ELISA assays or Western blots., Results: Mice treated with silica demonstrated lung fibrosis at 28 days following exposure, whereas, in controls, only mild and transient inflammation was evident at day 3 and day 7 postinstillation and was not present at day 14. Furthermore, silica-exposed mice exhibited significantly (p < 0.05) elevated levels of annexin A5 in serum and lung tissues, relative to control groups. Consistent with these findings, silica exposure of RAW264.7 cells for 6 or 12 h, led to an annexin A5-dependent increase in the expression levels of TGF-β1, IL-1α, FasL, and their downstream target molecules. These silica-induced changes were reversed by siRNA-mediated knockdown of annexin A5, but downregulation of FasL led to increased annexin A5 expression and reduced levels of TGF-β1, IL-1α, and FasL downstream target molecules., Conclusions: These findings define a role of annexin A5 in promoting macrophage activation via Fas/FasL pathways in silica-induced lung fibrosis., (© The Author(s) 2015.)

Silicosis is an incurable occupational disease associated with inflammation, fibroblast proliferation and the accumulation of extracellular matrix in lung tissues. The dysregulation of lncRNAs and miRNAs has been implicated in many complex diseases; however, the current understanding of their roles in fibrotic lung diseases, especially silicosis, remains limited. Our previous microRNA (miRNA, miR) microarray data have indicated decreased expression levels of miR-489 in lung tissues of silica-induced pulmonary fibrosis. Here, we further explored the role of miR-489 in a mouse model of silicosis. Interestingly, miR-489 levels were reduced in both macrophages that were exposed to silica and fibroblasts that were exposed to TGF-β1. Additionally, the overexpressed miR-489 carried out its anti-fibrotic role by attenuating inflammation and fibrotic progression in vivo. Our molecular study further demonstrated that miR-489 inhibited silica-induced pulmonary fibrosis primarily by repressing its target genes MyD88 and Smad3. Moreover, the up-regulated lncRNA cardiac hypertrophy-related factor (CHRF) reversed the inhibitory effect of miR-489 on MyD88 and Smad3 and then triggered the inflammation and fibrotic signaling pathways. Overall, our data indicate that the CHRF-miR-489-MyD88 Smad3 signaling axis exerts key functions in silica-induced pulmonary fibrosis and may represent a therapeutic target for silicosis.

To identify microRNAs (miRNAs, miRs) with potential roles in lung fibrogenesis, we performed genome-wide profiling of miRNA expression in lung tissues from a silica-induced mouse model of pulmonary fibrosis using microarrays. Seventeen miRNAs were selected for validation via qRT-PCR based on the fold changes between the silica and the control group. The dysregulation of five miRNAs, including miR-21, miR-455, miR-151-3p, miR-486-5p and miR-3107, were confirmed by qRT-PCRs in silica-induced mouse model of pulmonary fibrosis and were also confirmed in a bleomycin (BLM)-induced mouse lung fibrosis. Notably, miR-486-5p levels were decreased in the serum samples of patients with silicosis, as well as in the lung tissues of patients with silicosis and idiopathic pulmonary fibrosis (IPF). In addition, as determined by luciferase assays and Western blotting, SMAD2, a crucial mediator of pulmonary fibrosis, was identified to be one of target genes of miR-486-5p. To test the potential therapeutic significance of this miRNA, we overexpressed miR-486-5p in animal models. At day 28, miR-486-5p expression significantly decreased both the distribution and severity of lung lesions compared with the silica group (P < 0.01). In addition, miR-486-5p had a similar effect in the BLM group (P < 0.001). These results indicate that miR-486-5p may inhibit fibrosis.

Objective: To investigate the regulatory effect of miR-149 on interleukin-6 (IL-6) expression in silica-induced pulmonary fibrosis., Methods: A mouse model of pulmonary fibrosis was established using silica dust; the level of miR-149 in the lung tissues of mice with silica-induced pulmonary fibrosis was measured by quantitative real-time polymerase chain reaction (qRT-PCR), while the protein expression of IL-6 was measured by immunohistochemistry and Western blot. Type II alveolar epithelial cells (A549) and bronchial epithelial cells (HBE) were exposed to silica dust to establish a model; the level of miR-149 was measured by qRT-PCR, while the protein expression of IL-6 was measured by Western blot. A549 cells were transfected with miR-149 mimics and inhibitor in vitro, and the cellular expression of IL-6 was measured by Western blot. Serum samples from patients with coal workers' pneumoconiosis were examined by double-antibody sandwich ELISA to measure the protein expression of IL-6., Results: At three time points after silica treatment, the miR-149 expression in lung tissues was significantly down-regulated while an evident increase in IL-6 expression was observed in lung tissues (P < 0.01). Silica-stimulated epithelial cell (A549 and HBE) had up-regulated IL-6 expression and down-regulated miR-149 expression (P < 0.01). Increased levels of miR-149 attenuated IL-6 expression, whereas adverse results were found when miR-149 was inhibited. Compared with that in control group, serum level of IL-6 was significantly increased in patients with stage II and III coal workers' pneumoconiosis (P < 0.01)., Conclusion: Down-regulation of miR-149 and up-regulation of IL-6 might be involved in the progression of silica-induced pulmonary fibrosis; miR-149 could negatively regulate IL-6 expression.