Integrated Genomics Reveals Convergent Transcriptomic Networks Underlying Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis

Abstract

Rationale: Despite shared environmental exposures, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease are usually studied in isolation, and the presence of shared molecular mechanisms is unknown.

Objectives: We applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF.

Methods: We defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87).

Measurements and main results: Genes increased in both emphysema and IPF relative to control were enriched for the p53/hypoxia pathway, a finding confirmed in an independent cohort using both gene expression arrays and the nCounter Analysis System (n = 193). Immunohistochemistry confirmed overexpression of HIF1A, MDM2, and NFKBIB members of this pathway in tissues from patients with emphysema or IPF. Using reads aligned across splice junctions, we determined that alternative splicing of p53/hypoxia pathway-associated molecules NUMB and PDGFA occurred more frequently in IPF or emphysema compared with control and validated these findings by quantitative polymerase chain reaction and the nCounter Analysis System on an independent sample set (n = 193). Finally, by integrating parallel microRNA and mRNA-Seq data on the same samples, we identified MIR96 as a key novel regulatory hub in the p53/hypoxia gene-expression network and confirmed that modulation of MIR96 in vitro recapitulates the disease-associated gene-expression network.

Conclusions: Our results suggest convergent transcriptional regulatory hubs in diseases as varied phenotypically as chronic obstructive pulmonary disease and IPF and suggest that these hubs may represent shared key responses of the lung to environmental stresses.

Keywords: COPD; ILD; IPF; network; transcriptome.

Figure 1.

Overview of analysis. IHC = immunohistochemistry; LTRC = Lung Tissue Research Consortium; miRNA = microRNA; PCR = polymerase chain reaction.

Figure 2.

Overview of the transcriptomic landscape of Lung Genomic Research Consortium lung tissue samples. (A) Box plot of the number of genes characterized as real signal, missing, or noise in all sequenced samples (Table 2). (B) Distribution of genes present in all 87 samples (core lung transcriptome), those never expressed, and those that are variably expressed. (C) Venn diagram of genes always present within the unique lung condition signatures (Table 1). (D) Circos plot highlighting the overlap of functional enrichment of genes always expressed in the core lung transcriptome and across the unique lung conditions. COPD = chronic obstructive pulmonary disease; EMP = emphysema; IPF = idiopathic pulmonary fibrosis; KEGG = Kyoto Encyclopedia for Genes and Genomes.

Figure 3.

Convergent gene expression patterns in idiopathic pulmonary fibrosis (IPF) and emphysema. (A) Scatter plot of fold ratios of emphysema to control (y-axis) versus fold ratio of IPF to control (x-axis). Fold ratios for all genes are depicted in log base 2. Spots are colored in increasing shades of purple based on concurrent directions and yellow if going to the opposite direction. (B) Heatmap of significant shared changes in gene expression in emphysema and IPF. Red indicates relative higher expression, blue means relative lower expression. (C) Immunohistochemistry for NFκBiB, HIF1α, and MDM2 (n = 5 per group). For all three targets, greater staining intensity was observed in IPF and emphysema samples compared with controls. For NFκBiB and HIF1α, there is no clear localization to specific lung cell lineages in contrast to MDM2, which seems to be localized to epithelial cells and macrophages. Inset images show staining with nonimmune control IgG (magnification ×100; scale bar = 100 μm). Black arrows indicate epithelial cells, yellow arrows indicate macrophages, and pink arrows indicate cells in lymphoid aggregates. CTRL = control; EMP = emphysema; HIF-1α = hypoxia-inducible factor 1-α; IPF = idiopathic pulmonary fibrosis; MDM2 = E3 ubiquitin-protein ligase; PDGF-A = platelet-derived growth factor α.

Figure 4.

PDGFA and NUMB are differentially spliced in chronic lung disease. The structure of the two isoforms for both PDGFA and NUMB are shown at the top of A and D, respectively. (A) Box plots of PDGFA isoform 001 and 001 splice junctions. (B) Coverage plots showing the interquartile range (lighter shading) of normalized reads and the mean (darker line) for PDGFA. (C) Barplot of nanostring validation showing the mean and the SE of the normalized number of times a transcript was counted by the nanostring method. (D) Box plots of the splice junctions in NUMB. (E) Coverage plots of NUMB. (F) Barplot of NUMB alternative splicing nanostring validation. IPF = idiopathic pulmonary fibrosis; RPM = reads per million.

Figure 5.

Shared emphysema (EMP) and idiopathic pulmonary fibrosis (IPF) microRNA (miRNA) regulatory network. Regulatory miRNA–mRNA network showing regulation in both diseases. Red lines indicate direction of repression. Bold red lines indicate interactions that were selected and validated by polymerase chain reaction (PCR).