Deducting MicroRNA-Mediated Changes Common in Bronchial Epithelial Cells of Asthma and Chronic Obstructive Pulmonary Disease-A Next-Generation Sequencing-Guided Bioinformatic Approach

Abstract

Asthma and chronic obstructive pulmonary disease (COPD) are chronic airway inflammatory diseases that share some common features, although these diseases are somewhat different in etiologies, clinical features, and treatment policies. The aim of this study is to investigate the common microRNA-mediated changes in bronchial epithelial cells of asthma and COPD. The microRNA profiles in primary bronchial epithelial cells from asthma (AHBE) and COPD (CHBE) patients and healthy subjects (NHBE) were analyzed with next-generation sequencing (NGS) and the significant microRNA changes common in AHBE and CHBE were extracted. The upregulation of hsa-miR-10a-5p and hsa-miR-146a-5p in both AHBE and CHBE was confirmed with quantitative polymerase chain reaction (qPCR). Using bioinformatic methods, we further identified putative targets of these microRNAs, which were downregulated in both AHBE and CHBE: miR-10a-5p might suppress BCL2, FGFR3, FOXO3, PDE4A, PDE4C, and PDE7A; miR-146a-5p might suppress BCL2, INSR, PDE4D, PDE7A, PDE7B, and PDE11A. We further validated significantly decreased expression levels of FOXO3 and PDE7A in AHBE and CHBE than in NHBE with qPCR. Increased serum miR-146a-5p level was also noted in patients with asthma and COPD as compared with normal control subjects. In summary, our study revealed possible mechanisms mediated by miR-10a-5p and miR-146a-5p in the pathogenesis of both asthma and COPD. The findings might provide a scientific basis for developing novel diagnostic and therapeutic strategies.

Keywords: COPD; asthma; bioinformatics; bronchial epithelial cells; epithelium; next-generation sequencing.

Figure 1

The expression levels of microRNAs in primary human bronchial epithelial cells (HBEs) from a normal subject (NHBE), an asthmatic patient (AHBE), and a patient with chronic obstructive pulmonary disease (CHBE). The expression levels of microRNAs were assessed with quantitative polymerase chain reaction (qPCR). Using the 2^−ΔΔCt method, the relative microRNA levels in various cells were calculated. All results were expressed as the mean ± standard error of mean of three independent experiments. The expression levels were compared with analysis of variances followed by Dunnett’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with NHBE.

Figure 2

The levels of miR-10a-5p (a,c) and miR-146a-5p (b,d) in human bronchial epithelial cells obtained from GSE25230 (a,b) and GSE38974 (c,d). The data form Gene Expression Omnibus (GEO) database were analyzed with GEO2R. The p values adjusted with method by Benjamini and Hochberg (false discovery rate) (adj. p) were shown.

Figure 3

Venn diagrams showing the intersections of asthma-associated molecules, COPD-associated molecules, and the putative targets of miR-10a-5p (a,c) and miR-146a-5p (b,d) suggested by at least two (a,b) or four (c,d) databases in miRWalk 2.0.

Figure 4

The expression levels of predicted microRNA targets in asthma/chronic obstructive pulmonary disease (COPD) bronchial epithelial cells. The data from the Gene Expression Omnibus (GEO) database were analyzed with GEO2R. The log₂-fold change (log₂FC) between two groups (asthma/COPD vs. control) were shown. * The p value adjusted with method by Benjamini and Hochberg (false discovery rate) (adj. p) < 0.25. The detailed information is presented in Table A2 of Appendix A.

Figure 5

Gene ontology (GO) analyses using the Database for Annotation, Visualization and Integrated Discovery (DAVID) about the putative targets of miR-10a-5p and miR-146a-5p which were significantly down-regulated in bronchial epithelial cells from both asthma and COPD patients. Arabic numerals represent numbers of genes in the (a) biological process and (b) molecular function.

Figure 6

The expression levels of selected genes in primary human bronchial epithelial cells (HBEs) from a normal subject (NHBE), an asthmatic patient (AHBE), and a patient with chronic obstructive pulmonary disease (CHBE). The expression levels of selected genes were assessed with qPCR. Using the 2^−ΔΔCt method, the relative mRNA levels in various cells were calculated. All results were expressed as the mean ± standard error of mean of three independent experiments. The expression levels were compared with analysis of variances followed by Dunnett’s test. * p < 0.05, ** p <0.01, *** p < 0.001, as compared with NHBE.

Figure 7

The serum levels of microRNAs in normal subjects, asthmatic patients, and COPD patients. The expression levels of microRNAs were assessed with qPCR. Using the 2^−ΔΔCt method, the relative microRNA levels were assessed. The data of subjects were plotted and the mean ± standard error of mean of the group were shown. The expression levels were compared between groups with analysis of variances. Using Dunnett’s post hoc analyses which took the levels of normal subjects as the reference, only the serum miR-146a-5p level was significantly higher in COPD group than that in the normal subjects (p = 0.0215), while the serum miR-146a-5p level was not significantly higher in asthma group than in the normal subjects (p = 0.1143). For other microRNAs, no significant difference was found with Dunnett’s post hoc analyses.