Identification of miRNAs Potentially Involved in Bronchiolitis Obliterans Syndrome: A Computational Study

Abstract

The pathogenesis of Bronchiolitis Obliterans Syndrome (BOS), the main clinical phenotype of chronic lung allograft dysfunction, is poorly understood. Recent studies suggest that epigenetic regulation of microRNAs might play a role in its development. In this paper we present the application of a complex computational pipeline to perform enrichment analysis of miRNAs in pathways applied to the study of BOS. The analysis considered the full set of miRNAs annotated in miRBase (version 21), and applied a sequence of filtering approaches and statistical analyses to reduce this set and to score the candidate miRNAs according to their potential involvement in BOS development. Dysregulation of two of the selected candidate miRNAs-miR-34a and miR-21 -was clearly shown in in-situ hybridization (ISH) on five explanted human BOS lungs and on a rat model of acute and chronic lung rejection, thus definitely identifying miR-34a and miR-21 as pathogenic factors in BOS and confirming the effectiveness of the computational pipeline.

Fig 1. Computational workflow followed to identify relevant miRNAs (green and orange blocks).

Two selected miRNAs (i.e., miR-34a and miR-21) obtained from the computational analysis were then validated in wet lab experiments (blue boxes).

Fig 2. Pathway selection process.

The NCBI E-Utilities Application Programming Interface (API) and KEGG API were used to mine the available literature and to identify a preliminary set of relevant pathways connected to BOS pathogenesis.

Fig 3. miRNA vs. pathway contingency table.

Np+: number of targeted genes in the pathway, Np-: number of non-targeted genes in the pathway, Nm+: number of total targets of the miRNA, Nm-: number of total genes not targeted by the miRNA.

Fig 4. Example of step by step processing of a single miRNA using VMTs.

(1) miR-34a, used as an example in the figure, is queried on TargetHUB to obtain its list of targets (VMTs); (2) The list of targets is intersected with the list of genes for each considered pathway to obtain a matrix showing how many targets belong to each pathway; (3) The matrix built during step 2, together with the total gene count for each pathway, is used to construct a set of contingency tables (one for each miRNA/pathway pair) as shown in Fig 3; (4) For each miRNA/pathway pair, the related contingency table is used to perform a one-tailed Fisher’s exact test to compute the enrichment of the miRNA targets in the pathway, obtaining a p-value that measures the significance of the test; and (5) p-values obtained for each miRNA/pathway pair are combined into a single significance score by the application of the modified Lancaster's method proposed in [39].

Fig 5. Ranked list of miRNAs most significantly enriched in BOS-related pathways (p<0.01) considering VMTs.

Results are plotted using a -log(p-value).

Fig 6. Ranked list of the top 30 miRNAs most significantly enriched in BOS-related pathways (p<0.01) considering CMTs.

Results are plotted using a -log(p-value).

Fig 7. Ranked list of miRNAs highly significantly enriched in a set of 39 randomly selected pathways (p<0.01) considering VMTs.

Results are plotted using a -log(p-value).

Fig 8. Ranked list of miRNAs highly significantly enriched in a set of 39 randomly selected pathways (p<0.01) considering CMTs.

Results are plotted using a -log(p-value).

Fig 9. miR-34a and miR-21 expression in human and rat transplanted lungs.

Upper panel: miR-34a expression in bronchial epithelial cells in normal human lung (A, blue staining), in myofibroblasts in lung explants from a BOS patient (B), in proliferating fibroblasts in rat lungs with chronic rejection (C) and in epithelial cells in remodeled areas in the animal model of acute cellular rejection (D). Lower panel: miR-21 ISH (E) and Movat pentachrome stains (F) highlighting miR-21 expression in BO myofibroblasts in lung explants from BOS patients. The airway lumen, which can be recognized by the peripheral elastic fibers, is totally occluded by collagen deposition and myofibroblasts (F), which were strongly positive for miR-21 (E, blue staining). In rat lungs with chronic rejection, miR-21 expression was similarly observed in BO myofibroblasts (G), while in acute cellular rejection, miR-21 expression was localized in epithelia and in interstitial fibroblasts associated with inflammatory infiltrates (H).

Fig 10. P-values obtained from the miRNA targets enrichment analysis for miR-34a and miR-21 paired with all considered pathways.

The color scale indicates the significance of the enrichment (green = significant enrichment). A: analysis with VMTs; B: analysis with CMTs.

Fig 11. Gene regulatory network showing interaction between miRNAs identified in the VMTs list and relevant genes implicated in BOS as identified from the literature (see the S1 Table).

The network is hierarchically organized into four levels. Brown squares: miRNA host genes for intragenic miRNAs; red and purple rhombi: intragenic and intergenic miRNAs respectively; cyan squares TFs; green hexagons: target genes.