Transcriptomic changes involved in the dedifferentiation of myofibroblasts derived from the lung of a patient with idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiology. Under pathological conditions in lungs with IPF, myofibroblasts serve a key role in fibrogenesis via the accumulation of an excessive amount of extracellular matrix. To develop effective therapeutic interventions against IPF, studies have recently focused on how to dedifferentiate established myofibroblasts. The present study revealed that JQ1, an inhibitor of bromodomain and extra‑terminal proteins, markedly suppressed the expression levels of α‑smooth muscle actin and ED‑A‑fibronectin in myofibroblasts prepared from the lung of a patient with end‑stage IPF. Furthermore, these findings were supported by transcriptome analysis using RNA sequencing, in which differentially expressed genes (DEGs) downregulated by JQ1 treatment were significantly enriched in the fibrosis‑related signaling pathway. On the other hand, the upregulated DEGs in response to JQ1 treatment were significantly enriched in glutathione metabolism, which may affect the cell status of fibroblast/myofibroblast. To the best of our knowledge, this was the first study to comprehensively analyze transcriptome profiles associated with dedifferentiation of IPF myofibroblasts.

Keywords: idiopathic pulmonary fibrosis; myofibroblast; dedifferentiation; transcriptome analysis; microrna array.

Figure 1.

JQ1-induced downregulation of myofibroblast markers. (A) Changes in expression of α-SMA and ED-A-FN in NHLF and 46G-F cells observed by western blotting. The signal of β-actin for each lane was determined as an internal control. (B) The signal of each sample was determined using a densitometer and normalized to each internal control. Signal values are presented as fold changes from the control value of NHLF and as means ± SEM (n=3). *P<0.05 (ANOVA followed by Tukey's test). (C) Quantitative PCR analysis of ACTA2 and FN1 expression in 46G-F cells treated with PBS (NC) or JQ1. Quantitative data are presented as fold-changes from the control value and as means ± SEM (n=3). *P<0.05 (unpaired Student's t-test). ACTA2, actin α 2, smooth muscle; FN1, fibronectin 1; NC, normal control; NHLF, normal human lung fibroblasts; n.s., not significant.

Figure 2.

Identification of DEGs between NC and JQ-1 treated cells. (A) PC analysis. PC1 (x-axis, 55.3%) and PC2 (y-axis, 14.8%) of the total variation in expressed genes of all cells. (B) Hierarchical clustering of the expression profiles of DEGs between NC and JQ-1-treated groups (adjusted FDR P<0.05). Red represents high relative expression, and blue represents low relative expression. Upregulated DEGs (1,330 genes; JQ-1 vs. NC) and downregulated DEGs (2,825 genes; JQ-1 vs. NC). (C) GO and pathway analysis of downregulated DEGs. Top 10 enriched GO terms associated with molecular functions (top) and biological processes (middle), and KEGG pathway analysis (bottom). (D) Gene set enrichment analysis plots show enrichment of fibrosis-related gene sets (extracellular matrix organization, collagen formation and elastic fiber formation) in two groups. The NES, normal P-value and FDR q value are also indicated. DEGs, differentially expressed genes; FDR, false discovery rate; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, normal control; NES, normalized enrichment score; PC, principal component.

Figure 3.

Identification and enrichment analysis of upregulated DEGs in JQ-1-treated cells. (A) Scatter plot and (B) volcano plot of expression profiles between two groups. Points colored in red were significantly different (adjusted false discovery rate P<0.05). (C) Functional enrichment analysis of 1,330 upregulated DEGs. Top 10 enriched Gene Ontology terms associated with molecular functions and biological processes, and KEGG pathways, and the negative log₁₀ of the P-value. (D) Hierarchical cluster analysis of DEGs in the KEGG pathway for glutathione metabolism between two groups. Red represents high relative expression and blue represents low relative expression. DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, normal control.