Multi-omics and multi-stages integration identified a novel variant associated with silicosis risk.

Assessing the association between candidate single-nucleotide polymorphisms (SNPs) identified by multi-omics approaches and susceptibility to silicosis. RNA-seq analysis was performed to screen the differentially expressed mRNAs in the fibrotic lung tissues of mice exposed to silica particles. Following this, we integrated the SNPs located in the above human homologenes with the silicosis-related genome-wide association study (GWAS) data to select the candidate SNPs. Then, expression quantitative trait locus (eQTL)-SNPs were identified by the GTEx database. Next, we validated the associations between the functional eQTL-SNPs and silicosis susceptibility by additional case-control study. And the contribution of the identified SNP and its host gene in the fibrosis process was further validated by functional experiments. A total of 12 eQTL-SNPs were identified in the screening stage. The results of the validation stage suggested that the variant T allele of rs419540 located in IL12RB1 significantly increased the risk of developing silicosis [additive model: odds ratio (OR) = 1.78, 95% confidence interval (CI) 1.11-2.85, P = 0.017]. Furthermore, the combination of GWAS and the results of validation stage also indicated that the variant T allele of rs419540 in IL12RB1 was associated with increased silicosis risk (additive model: OR = 2.07, 95% CI 1.38-3.12, P < 0.001). Additionally, after knockdown or overexpression of IL12RB1, the levels of pro-inflammatory factors, such as IL-12, IFN-γ, and other pro-inflammatory factors, were correspondingly decreased or increased. The novel eQTL-SNP, rs419540, might increase the risk of silicosis by modulating the expression levels of IL12RB1.

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.