Circulating microRNAs associated with bronchodilator response in childhood asthma

Abstract

Background: Bronchodilator response (BDR) is a measure of improvement in airway smooth muscle tone, inhibition of liquid accumulation and mucus section into the lumen in response to short-acting beta-2 agonists that varies among asthmatic patients. MicroRNAs (miRNAs) are well-known post-translational regulators. Identifying miRNAs associated with BDR could lead to a better understanding of the underlying complex pathophysiology.

Objective: The purpose of this study is to identify circulating miRNAs associated with bronchodilator response in asthma and decipher possible mechanism of bronchodilator response variation.

Methods: We used available small RNA sequencing on blood serum from 1,134 asthmatic children aged 6 to 14 years who participated in the Genetics of Asthma in Costa Rica Study (GACRS). We filtered the participants into the highest and lowest bronchodilator response (BDR) quartiles and used DeSeq2 to identify miRNAs with differential expression (DE) in high (N = 277) vs. low (N = 278) BDR group. Replication was carried out in the Leukotriene modifier Or Corticosteroids or Corticosteroid-Salmeterol trial (LOCCS), an adult asthma cohort. The putative target genes of DE miRNAs were identified, and pathway enrichment analysis was performed.

Results: We identified 10 down-regulated miRNAs having odds ratios (OR) between 0.37 and 0.76 for a doubling of miRNA counts and one up-regulated miRNA (OR = 2.26) between high and low BDR group. These were assessed for replication in the LOCCS cohort, where two miRNAs (miR-200b-3p and miR-1246) were associated. Further, functional annotation of 11 DE miRNAs were performed as well as of two replicated miRs. Target genes of these miRs were enriched in regulation of cholesterol biosynthesis by SREBPs, ESR-mediated signaling, G1/S transition, RHO GTPase cycle, and signaling by TGFB family pathways.

Conclusion: MiRNAs miR-1246 and miR-200b-3p are associated with both childhood and adult asthma BDR. Our findings add to the growing body of evidence that miRNAs play a significant role in the difference of asthma treatment response among patients as it points to genomic regulatory machinery underlying difference in bronchodilator response among patients.

Trial registration: LOCCS cohort [ClinicalTrials.gov number NCT00156819, Registration date 20050912], GACRS cohort [ClinicalTrials.gov number NCT00021840].

Keywords: Asthma; Bronchodilator response; microRNA.

Fig. 1

Volcano plot for differential expression of miRNA between high and low BDR in the GACRS. Y-axis: represents the multiple testing corrected (FDR) p-value. Log fold change represents the log of the ratio of miRNA expression in the high-BDR quartile to the low-BDR quartile

Fig. 2

Clustered heatmap of all 11 differentially expressed miRs in the GACRS across conditions. DESeq2 normalized expression counts with shifted logarithm transformation was used. The heat map was created using unsupervised hierarchical clustering, and the distance metric was Pearson correlation. *Marked miRNAs were replicated in the LOCCS cohort

Fig. 3

Reactome pathways enriched for 11 DE miRNAs in the GACRS at 5% FDR cut-off. The target genes were identified using Micro T-CDS, TarBase, and Target Scan databases. The pairwise similarities of the enriched terms calculated by the pairwise_termsim function using Jaccard’s similarity index (JC) and the agglomeration method ward.Din is used for clustering in R. If a pathway was found to be enriched with a specific DE miRNA’s target gene, the DE miRNA name is written next to it

Fig. 4

miRNA-target gene network between two replicated miRNAs. Nodes with different colors represent the genes in selected Reactome pathways

Fig. 5

Bronchodilator response resistance mechanism: β2-Agonists cause relaxation by activating the β2-adrenoceptor, which then activates adenylyl cyclase (AC) to increase cAMP and cause bronchodilation. The increased resistance to β2-agonist-induced bronchodilation in asthmatics may be mediated by the effects of transforming growth factor (TGF)-β1. TGF-β1 activates the TGF-β receptor, causing phosphorylation of the transcription factors Smad2 and Smad3, which then translocate to the nucleus and form a complex with Smad4. This complex increases the expression of the PDE isomer PDE4DS, which leads to greater cAMP breakdown and, as a result, less bronchodilation. P = phosphorylation (Wortley et al., 2019). We found that BDR responsive DE miRs: miR-26b-5p, miR-378a-3p, miR-378i, miR-200b-3p, and miR-885-5p putatively target (TGFβ1, TGFβ2, TGFBR1, TGFBR2, and TGFBR3) genes encoding TGF-β and TGF-β receptor encoding, and miR-26-5p & miR-200b-3p putative targets were enriched in pathway associated with downregulation of SMAD2/3: SMAD4 transcriptional activity (FDR = 1.09 × 10^− 5)