Transcriptomic analysis and validation reveal the pathogenesis and a novel biomarker of acute exacerbation of chronic obstructive pulmonary disease

Abstract

Background: Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is the main factor that leads to the deterioration of the disease. Currently, the diagnosis of AECOPD mainly relies on clinical manifestations, good predictors or biomarkers are lacking. We aim to reveal specific biomarkers and potential pathogenesis of AECOPD and provide a research basis for the diagnosis and treatment.

Methods: Four patients with AECOPD, four patients with stable COPD, and five control subjects were enrolled for RNA sequencing and KEGG analysis. The mRNA level of target genes was verified by quantitative real-time PCR (qPCR) with an expanded sample size (30 patients with AECOPD, 27 patients with stable COPD, and 35 control subjects). ELISA and immunofluorescence were used to identify the target proteins. Furthermore, the expression and function of WNT/β-catenin signaling pathway were assessed in animal models of COPD.

Results: RNA sequencing showed that 54 genes were up-regulated and 111 genes were down-regulated in the AECOPD. Differentially expressed genes were mainly enriched in WNT signaling pathway, et al. QPCR revealed that multi-genes of the WNT/β-catenin signaling were significantly down-regulated in AECOPD (P < 0.05), and β-catenin protein was significantly decreased in plasma of AECOPD and stable COPD (P < 0.01), while phosphorylated β-catenin was significantly up-regulated in peripheral blood mononuclear cells of AECOPD (P < 0.05). Similarly, WNT ligands, WNT receptors, and downstream signaling molecules were down-regulated, with an increased phosphorylated β-catenin protein in animal models of COPD. Activation of WNT/β-catenin signaling pathway by lithium chloride reduced the expression of phosphorylated β-catenin and ameliorated the COPD-like airway inflammation in mice.

Conclusion: WNT/β-catenin signaling pathway is down-regulated in AECOPD patients and in animal models of COPD. Increased expression of phosphorylated β-catenin in the blood might be a potential biomarker of AECOPD. Activation of WNT/β-catenin pathway may also represent a therapeutic target for AECOPD.

Keywords: Acute exacerbation; Biomarkers; Chronic obstructive pulmonary disease; Pathogenesis; Transcriptomic.

Fig. 1

AECOPD-related DEGs identified by differential expression analysis. A The number of DEGs between the stable COPD group and the control group, the AECOPD group and the control group, the AECOPD group and the stable COPD group, respectively. B The Volcano Plot of DEGs. Each point in the Volcano Plot represents a gene, the horizontal axis represents the − log₂ (fold change) of a gene expression in two groups. − log₁₀ (p value) as the ordinate represents the statistical significance of the change in gene expression. The yellow dots, green dots and purple dots represent up-regulated, down-regulated and non-differentially expressed genes, respectively. C According to the disease progression to screen DEGs. D The clustering heat maps of DEGs. The abscissa represents the three groups, the ordinate represents the DEGs and the clustering results of the genes. The color represents the gene expression level, the deeper the red color, the higher the gene expression level

Fig. 2

AECOPD-related signaling pathways obtained by KEGG analysis. A KEGG pathway enrichment analysis of DEGs. The vertical axis represents signaling pathways. The horizontal axis represents the gene ratio, which is, the number of DEGs to the number of annotated genes in pathways. The size of the dot indicates the number of DEGs in the pathway, and the color of the dot corresponds to different adjusted p value ranges. B The pathways and DEGs associated with the pathogenesis of AECOPD. Selection criteria: 1. The number of DEGs enriched in pathways is not less than three. 2. Exclude specific pathogen infection, and disease pathways

Fig. 3

Validation of down-regulated WNT/β-catenin pathway in AECOPD. A WNT/β-catenin signaling pathway. The WNT ligands bind to the receptors, including the FZD family, the coreceptor LRP 5/6 and LGR4-6, downstream signaling molecules are activated, the destruction complex (Axin/ APC/ CK1/ GSK-3) is inhibited. The accumulation and translocation of dephosphorylated β-catenin to the nucleus drives the expression of T-cell factor/lymphoid enhancer-binding factor (TCF/LEF)-dependent genes. When there are no WNT ligands or fewer WNT ligands and receptors, the destruction complex is activated, causing phosphorylation of β-catenin to increase and eventually be degraded. Exogenous addition of lithium chloride can also inhibit the destruction complex, promotes β-catenin—mediated gene transcription. B The mRNA levels of WNT10b, WNT2, LRP6, LGR6, FZD4, CTNNB1, LEF1, FOSL1, and FRAT2 in the control group (n = 35), the stable COPD group (n = 27) and the AECOPD group (n = 30). Results are presented as relative mRNA level (mean ± SEM). Stable or Acute vs. Normal, *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. Acute vs. Stable, ^###P < 0.001 by one-way ANOVA

Fig. 4

Alterations of β-catenin and its phosphorylation in AECOPD. A The expression of β-catenin protein in plasma of control subjects (n = 23), patients with stable COPD (n = 17) and patients with AECOPD (n = 22) was analyzed by ELISA. Results are presented as protein concentration (mean ± SEM). B, C The expression of phosphorylated β-catenin protein in PBMC of control subjects (n = 5), patients with stable COPD (n = 5) and patients with AECOPD (n = 5) was analyzed by immunofluorescence. B Results are presented as relative number of positive p-β-catenin cells (mean ± SEM). C Immunofluorescent staining of PBMC. Green fluorescence represents positive phosphorylated β-catenin cells (red arrowheads). Blue fluorescence represents DAPI. *P < 0.05, **P < 0.01 by one-way ANOVA

Fig. 5

Altered WNT/β-catenin pathway in experimental COPD models. A–D CS exposure following elastin challenge in mouse lung tissues. A Experimental outline. Mice were exposed to CS (n = 5) or room air (n = 4) for 2 weeks and were hosted at room air for another 2 weeks. Mice were then challenged with elastin (Eln, 100 μg) or normal saline (NS) intratracheally (i.t.) for 3 times at day 29, 30, and 31, and were sacrificed 48 h after the last elastin challenge. B The mRNA levels of WNT10b, WNT2, LRP6, LGR6, FZD4, CTNNB1, LEF1 and FOSL1 in lung tissue of CE and air control mouse models, were assessed by qPCR. Results are presented as relative mRNA level (mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001 by the Student t-test. C Representative immunofluorescent staining of lung tissue. Green fluorescence represents positive phosphorylated β-catenin cells. Blue fluorescence represents DAPI. D Semi-quantification of positive phosphorylated β-catenin cells. E, F CS exposure in mouse lung tissues. E Experimental outline. Mice were exposed to cigarette smoke (n = 6) or filtered room air (n = 5), and lung tissue was obtained after 3 months. F The mRNA levels of WNT10b, WNT2, LRP6, LGR6, FZD4, CTNNB1, LEF1, and FOSL1 in lung tissue of CS and air control mouse models, were assessed by qPCR. Results are presented as relative mRNA level (mean ± SEM). *P < 0.05, ***P < 0.001 by the Student t-test

Fig. 6

Activation of the WNT pathway reduces phosphorylated-β-catenin and COPD-like airway inflammation. Mice were exposed to CS or room air for 2 weeks and were hosted at room air for another 2 weeks. Mice were then challenged with elastin (Eln, 100 μg) or normal saline (NS) intratracheally (i.t.) for 3 times at day 29, 30, and 31, and were injected with lithium chloride (LiCl) or NS intraperitoneally for 5 times at day 29, 30, 31, 32 and 33 (Air + NS, n = 5; CE + NS, n = 4; Air + LiCl, n = 5; CE + LiCl, n = 6). Then mice were sacrificed 5 h after the last LiCl injection. A Representative expression and localization of phosphorylated β-catenin in mice lung tissues by immunofluorescence. B Semi-quantitative analysis of the immunofluorescence results. Results are presented as relative number of positive phosphorylated β-catenin cells (mean ± SEM). *P < 0.05, ***P < 0.001 by one-way ANOVA. C The expression of phosphorylated β-catenin in mice lung tissue was analyzed by Western Blot. D Semi-quantitative analysis of the Western Blot results. Results are presented as relative protein expression concentration (mean ± SEM). **P < 0.01 by one-way ANOVA. E H&E staining for histologic assessment of lung tissue. D The mRNA levels of IL-1β, IL-6 and IL-17A. Results are presented as relative mRNA level (mean ± SEM). CE + NS vs. Air + NS, *P < 0.05, **P < 0.01; CE + LiCl vs. CE + NS, ^#P < 0.05, ^##P < 0.01 by one-way ANOVA