Comprehensive transcriptome analysis of peripheral blood unravels key lncRNAs implicated in ABPA and asthma

Abstract

Allergic bronchopulmonary aspergillosis (ABPA) is a complex hypersensitivity lung disease caused by a fungus known as Aspergillus fumigatus. It complicates and aggravates asthma. Despite their potential associations, the underlying mechanisms of asthma developing into ABPA remain obscure. Here we performed an integrative transcriptome analysis based on three types of human peripheral blood, which derived from ABPA patients, asthmatic patients and health controls, aiming to identify crucial lncRNAs implicated in ABPA and asthma. Initially, a high-confidence dataset of lncRNAs was identified using a stringent filtering pipeline. A comparative mutational analysis revealed no significant difference among these samples. Differential expression analysis disclosed several immune-related mRNAs and lncRNAs differentially expressed in ABPA and asthma. For each disease, three sub-networks were established using differential network analysis. Many key lncRNAs implicated in ABPA and asthma were identified, respectively, i.e., AL139423.1-201, AC106028.4-201, HNRNPUL1-210, PUF60-218 and SREBF1-208. Our analysis indicated that these lncRNAs exhibits in the loss-of-function networks, and the expression of which were repressed in the occurrences of both diseases, implying their important roles in the immune-related processes in response to the occurrence of both diseases. Above all, our analysis proposed a new point of view to explore the relationship between ABPA and asthma, which might provide new clues to unveil the pathogenic mechanisms for both diseases.

Keywords: ABPA; Asthma; Long noncoding RNAs; Network analysis; RNA sequencing; Transcriptome analysis.

Figure 1. Bioinformatic analysis pipeline for the transcriptome analysis of RNA-seq data.

Figure 2. Transcriptome-level mutational landscape of ABPA and asthma compared to health controls.

(A) Each row in the figure corresponds to one gene/lncRNA, whereas each column corresponds to one sample (n = 28). (blue bar represents ABPA patients, dark red bar represents asthma patients and light green represents health controls). (Top) Bar plots describing the percentage of different type ofalternations identified in each sample across all the identified genes/lncRNAs. (Right) Bar plots of the percentage of different type of alternations of each gene/lncRNAs across all the sample. (Bottom) bar plot represents the type of samples, blue bar represents ABPA patients, dark red bar represents asthma patients and light green represents health controls. (B) The statistical analysis pipeline to detect whether the identified SSR event significantly existed between ABPA patients and health controls, as well as asthma patients and health controls. (C) The density distribution of the SSRs among the three types of samples. The graphs (D~I) plot the SSRs difference between ABPA patients, asthma patients as well as health controls.

Figure 3. Differential expression analysis of ABPA, asthma patients and health controls.

(A) Heatmap of all the differentially expressed transcripts identified by the comparison of ABPA with health controls and asthma with health controls. (B) Functional enrichment analysis based on the differentially expressed transcripts identified by the comparison of asthma with health controls. (C) Functional enrichment analysis based on the differentially expressed transcripts identified by the comparison of ABPA with health controls. (D) Overlap of the differentially expressed transcripts between two comparisons. (E) Functional enrichment analysis of the differentially expressed transcripts for both diseases.

Figure 4. Cluster analysis of differentially expressed mRNAs and lncRNAs in all human blood samples.

(A) Cluster 1 indicates the mRNAs and lncRNAs that were down-regulation expressed in health controls but were up-regulation in ABPA and asthma patients. (B) Bar plot shows the Gene ontology (GO) functional enrichment analysis based on the mRNAs of cluster 1. (C) Cluster 2 indicates the mRNAs and lncRNAs that specifically up-regulation expressed in ABPA patients. (D) Bar plot shows the Gene ontology (GO) functional enrichment analysis based on the mRNAs of cluster 2. (E) Cluster 3 indicates the mRNAs and lncRNAs that were up-regulation expressed in health controls but were down-regulation in ABPA and asthma patients. (F) Bar plot shows the Gene ontology (GO) functional enrichment analysis based on the mRNAs of cluster 3.

Figure 5. Differential network analysis between ABPA, asthma patients and health controls.

(A) Visualization of the immune-related loss-of-function network of ABPA patients (|PCC_healthy| ≥ 0.95, |PCC_ABPA| ≤ 0.30, |PCC_{ABPA − healthy}| ≥ 1.00). (B) Visualization of the immune-related loss-of-function network of asthma patients (|PCC_healthy| ≥ 0.93, |PCC_AS| ≤ 0.30, |PCC_{AS − healthy}| ≥ 1.00). (C) Visualization of the immune-related gain-of-function network of ABPA patients (|PCC_healthy| ≤ 0.30, |PCC_ABPA| ≥ 0.80, |PCC_{ABPA − healthy}| ≥ 1.00). (D) Visualization of the immune-related gain-of-function network of asthma patients (|PCC_healthy| ≤ 0.30, |PCC_AS| ≥ 0.90, |PCC_{AS − healthy}| ≥ 1.00). (E) Visualization of the immune-related anti-function network of ABPA patients (|PCC_healthy| ≥ 0.70, |PCC_ABPA| ≥ 0.70, |PCC_{ABPA − healthy}| ≥ 1.00). (F) Visualization of the immune-related anti-function network of asthma patients ((|PCC_healthy| ≥ 0.70, |PCC_AS| ≥ 0.70, |PCC_{AS − healthy}| ≥ 1.00)).

Figure 6. Functional analysis and GEO validation of key lncRNAs (hub nodes) derived from the loss-of-function networks of two diseases.

(A) hub node: SREBF1-208. (B) hub node: PUF60-218. (C) hub node: HNRNPUL1-210. (D) hub node: AL139423.1-201. (E) hub node: AC106028.4-201. (Left) sub-networks display all the target mRNAs for the key lncRNAs. (Right) bar plots show Gene ontology (GO) functional enrichment analysis based on the corresponding target mRNAs. (F–I). Validation of the expression level of four selected hub nodes (ENST00000327423, ENST00000369443, ENST00000587128 and ENST00000595806) between asthmatic groups and health controls. (F) Validation based on microarray data of GSE35571. (G) Validation based on microarray data of GSE473. (H) Validation based on microarray data of GSE31773. (I) Validation based on microarray data of GSE2125.