Dataset Information

Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages.

ABSTRACT: Exposure to crystalline silica is considered to increase the risk of lung fibrosis. The primary effector cell, the myofibroblast, plays an important role in the deposition of extracellular matrix (ECM). DNA methylation change is considered to have a potential effect on myofibroblast differentiation. Therefore, the present study was designed to investigate the genome-wide DNA methylation profiles of lung fibroblasts co-cultured with alveolar macrophages exposed to crystalline silica in vitro.AM/fibroblast co-culture system was established. CCK8 was used to assess the toxicity of AMs. mRNA and protein expression of collagen I, ?-SMA, MAPK9 and TGF-?1 of fibroblasts after AMs exposed to 100 ?g /ml SiO2 for 0-, 24-, or 48 h were determined by means of quantitative real-time PCR, immunoblotting and immunohistochemistry. Genomic DNA of fibroblasts was isolated using MeDIP-Seq to sequence. R software, GO, KEGG and Cytoscape were used to analyze the data.SiO2 exposure increased the expression of collagen I and ?-SMA in fibroblasts in co-culture system. Analysis of fibroblast methylome identified extensive methylation changes involved in several signaling pathways, such as the MAPK signaling pathway and metabolic pathways. Several candidates, including Tgfb1 and Mapk9, are hubs who can connect the gene clusters. MAPK9 mRNA expression was significantly higher in fibroblast exposed to SiO2 in co-culture system for 48 h. MAPK9 protein expression was increased at both 24-h and 48-h treatment groups. TGF-?1 mRNA expression of fibroblast has a time-dependent manner, but we didn't observe the TGF-?1 protein expression.Tgfb1 and Mapk9 are helpful to explore the mechanism of myofibroblast differentiation. The genome-wide DNA methylation profiles of fibroblasts in this experimental silicosis model will be useful for future studies on epigenetic gene regulation during myofibroblast differentiation.

SUBMITTER: Li J

PROVIDER: S-EPMC5429546 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages.

Li Juan J Yao Wu W Zhang Lin L Bao Lei L Chen Huiting H Wang Di D Yue Zhongzheng Z Li Yiping Y Zhang Miao M Hao Changfu C

Respiratory research 20170512 1

<h4>Background</h4>Exposure to crystalline silica is considered to increase the risk of lung fibrosis. The primary effector cell, the myofibroblast, plays an important role in the deposition of extracellular matrix (ECM). DNA methylation change is considered to have a potential effect on myofibroblast differentiation. Therefore, the present study was designed to investigate the genome-wide DNA methylation profiles of lung fibroblasts co-cultured with alveolar macrophages exposed to crystalline s ...[more]

PMID: 28499430

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Project description:Excessive fibroproliferation is a central hallmark of idiopathic pulmonary fibrosis (IPF), a chronic, progressive disorder that results in impaired gas exchange and respiratory failure. Fibroblasts are the key effector cells in IPF, and aberrant expression of multiple genes contributes to their excessive fibroproliferative phenotype. DNA methylation changes are critical to the development of many diseases, but the DNA methylome of IPF fibroblasts has never been characterized. Here, we utilized the HumanMethylation 27 array, which assays the DNA methylation level of 27,568 CpG sites across the genome, to compare the DNA methylation patterns of IPF fibroblasts (n?=?6) with those of nonfibrotic patient controls (n?=?3) and commercially available normal lung fibroblast cell lines (n?=?3). We found that multiple CpG sites across the genome are differentially methylated (as defined by P value less than 0.05 and fold change greater than 2) in IPF fibroblasts compared to fibroblasts from nonfibrotic controls. These methylation differences occurred both in genes recognized to be important in fibroproliferation and extracellular matrix generation, as well as in genes not previously recognized to participate in those processes (including organ morphogenesis and potassium ion channels). We used bisulfite sequencing to independently verify DNA methylation differences in 3 genes (CDKN2B, CARD10, and MGMT); these methylation changes corresponded with differences in gene expression at the mRNA and protein level. These differences in DNA methylation were stable throughout multiple cell passages. DNA methylation differences may thus help to explain a proportion of the differences in gene expression previously observed in studies of IPF fibroblasts. Moreover, significant variability in DNA methylation was observed among individual IPF cell lines, suggesting that differences in DNA methylation may contribute to fibroblast heterogeneity among patients with IPF. These results demonstrate that IPF fibroblasts exhibit global differences in DNA methylation that may contribute to the excessive fibroproliferation associated with this disease.

Dataset Information

Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages.

Publications

Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages.

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