Identification of Potential Pathogenic Super-Enhancers-Driven Genes in Pulmonary Fibrosis

Abstract

Abnormal fibroblast differentiation into myofibroblast is a crucial pathological mechanism of pulmonary fibrosis (PF). Super-enhancers, a newly discovered cluster of regulatory elements, are regarded as the regulators of cell identity. We speculate that abnormal activation of super-enhancers must be involved in the pathological process of PF. This study aims to identify potential pathogenic super-enhancer-driven genes in PF. Differentially expressed genes (DEGs) in PF mouse lungs were identified from a GEO dataset (GDS1492). We collected super-enhancers and their associated genes in human lung fibroblasts and mouse embryonic fibroblasts from SEA version 3.0, a network database that provides comprehensive information on super-enhancers. We crosslinked upregulated DEGs and super-enhancer-associated genes in fibroblasts to predict potential super-enhancer-driven pathogenic genes in PF. A total of 25 genes formed an overlap, and the protein-protein interaction network of these genes was constructed by the STRING database. An interaction network of transcription factors (TFs), super-enhancers, and associated genes was constructed using the Cytoscape software. Gene enrichment analyses, including KEGG pathway and GO analysis, were performed for these genes. Latent transforming growth factor beta (TGF-β) binding protein 2 (LTBP2), one of the predicted super-enhancer-driven pathogenic genes, was used to verify the predicted network's accuracy. LTBP2 was upregulated in the lungs of the bleomycin-induced PF mouse model and TGF-β1-stimulated mouse and human fibroblasts. Myc is one of the TFs binding to the LTBP2 super-enhancer. Knockout of super-enhancer sequences with a CRISPR/Cas9 plasmid or inhibition of Myc all decreased TGF-β1-induced LTBP2 expression in NIH/3 T3 cells. Identifying and interfering super-enhancers might be a new way to explore possible therapeutic methods for PF.

Keywords: LTBP2; fibroblast; pulmonary fibrosis; super-enhancer; transcription factor.

Figure 1

Prediction of potential super-enhancer-driven pathogenic genes in PF. (A) Venn diagram of upregulated DEGs in lungs of PF mice from a GEO dataset (GDS1492) and super-enhancer-associated genes in human lung fibroblasts and mouse embryonic fibroblasts from the SEA database. (B) A PPI network of the 25 super-enhancer-driven pathogenic genes was generated using the STRING database. (C) The interaction network of TFs, super-enhancers, and associated genes in PF.

Figure 2

KEGG pathway analysis (A) and GO analysis (B) of super-enhancer-driven pathogenic genes in PF.

Figure 3

LTBP2 was highly expressed in lungs of mice with PF. (A) Representative images show H&E and Masson’s trichrome staining of lung sections from the indicated groups of mice. Scale bars: 100 μm. (B) Representative bands of western blots showing expression of COL1A1 and β-tubulin in lungs of indicated mice. (C) Quantitative analysis of hydroxyproline in lung homogenates from indicated mice. (D) Representative bands of western blots showing expression of LTBP2 and β-tubulin in lungs of indicated mice. (E) The mRNA expressions of a-SMA, COL1A1, fibronectin, and LTBP2 in lungs were detected by real-time PCR. (F) Co-immunofluorescence staining for collagen I (green), LTBP2 (red), and DAPI (blue) in bleomycin-treated mice. Original magnification, scale bars: 100 μm. Data presented as mean ± SD, n = 4–5. ^*p < 0.05; ^**p < 0.01.

Figure 4

LTBP2 was highly expressed in TGF-β1-induced myofibroblasts. The mRNA expressions of a-SMA, COL1A1, fibronectin, and LTBP2 in mouse embryonic fibroblasts (NIH/3 T3; A) and human fetal lung fibroblasts (HFL1; B) were detected by real-time PCR after 5 ng/ml TGF-β1 stimulation for 12 h. Data presented as mean ± SD, n = 3–4. ^*p < 0.05; ^**p < 0.01.

Figure 5

Knockout of super-enhancer sequences and inhibition of myc decreased LTBP2 expression. (A) Genomic DNA of NIH/3 T3 cells transfected with or without a CRISPR/Cas9 knockout plasmid was extracted. PCR was used to detect the expression of targeted genes. Products were electrophoresed in a 1% agarose gel, and images were captured under ultraviolet light. (B) The mRNA expressions of LTBP2 in NIH/3 T3 cells transfected with or without a CRISPR/Cas9 knockout plasmid were detected by real-time PCR after TGF-β1 stimulation for 12 h. (C) TFs binding the LTBP2 super-enhancer (SE:237549). Squares represent TFs. Arrows represent the super-enhancer. Circles represent targeted genes. (D) The mRNA expressions of LTBP2 in NIH/3 T3 cells were detected by real-time PCR after 5 ng/ml TGF-β1 and 20 μM myc inhibitor (10058-F4) treatment for 12 h. Data presented as mean ± SD, n = 3. ^**p < 0.01.