To the Editor
Asthma is a chronic inflammatory lung disease that shows a time-of-day
effect (airway inflammation and lung function) on the severity of the
disease 1. Recent studies support the functional role
of miRNAs in the molecular pathophysiology of asthma
phenotypes/endotypes 2,3 through post-transcriptional
gene regulation of key signaling pathways and cellular processes in
human airways and immune cells of asthmatics and mouse model of asthma4,5,6. However, whether rhythmic changes in the
expression of miRNAs are responsible for the time-of-day effects
observed during asthma and its exacerbations is not known.
In this study, we performed miRNA and mRNA expression profiling using
NanoString to determine the time-of-day effects using house dust mite
(HDM)-induced allergic asthma model. Differential expression (DE)
analysis revealed a strong time-of-day difference in the miRNA
expression at ZT0 and ZT12 in HDM vs. PBS (control), as shown by
hierarchical cluster analysis (Figure 1A-D ). We found 6 miRNAs
(downregulated: miR-125b-5p, miR-125a-5p, miR-150, miR-23a, miR-23b and
miR-15b) and 3 miRNAs (upregulated: miR-652, miR-200b and miR-200c)
significantly in HDM vs. PBS at ZT0 (Figure 1A-B ), and 21
miRNAs downregulated and 17 miRNAs upregulated significantly in HDM vs.
PBS at ZT12 (Figure 1C-D ).
When we compared the DE miRNAs in HDM vs. PBS group at ZT0 and ZT12, we
found the same 6 miRNAs (downregulated) and 3 miRNAs (upregulated) at
ZT0 pairwise comparisons were present (Figure S1A-B ).
Additionally, unsupervised clustering and PCA of normalized counts from
HDM vs. PBS at ZT0 and ZT12 showed clusters separated based on the
treatment groups (Figure S1C-D ). Hierarchical cluster analysis
of HDM vs. PBS groups at ZT0 and ZT12 together revealed close clustering
of all DE miRNAs in PBS and HDM groups at ZT12 except for few HDM ZT0
samples that were clustered with either the PBS ZT0 or HDM ZT12
(Figure S1E ).
Multiple comparison analyses revealed several miRNAs that were either
significantly downregulated or upregulated in HDM vs. PBS at ZT0 and
ZT12 (Figure 2A and Figure S2A-B ). We found about 11
miRNAs downregulated and 8 miRNAs upregulated significantly in HDM vs.
PBS at ZT12, and 3 miRNAs were upregulated in HDM vs. PBS at ZT0
(Figure S2A-B ). However, several miRNAs were either
downregulated (2 miRNAs: miR-720 and miR-1944) or upregulated (9 miRNAs:
miR-151-5p, miR-199a-3p, miR-30c, let-7d, miR-15a, miR-181a, miR-25,
let-7e, and miR-126-3p) in PBS ZT0 vs. ZT12 comparison (Figure
S2A-B ). The pairwise comparison between HDM vs. PBS at ZT0 and ZT12
revealed 9 miRNAs and 38 miRNAs DE at ZT0 and ZT12 respectively
(Figure S3A-C ). Additional pairwise comparison between PBS at
ZT0 vs. ZT12 and HDM ZT0 vs. ZT12 revealed several miRNAs DE were among
those identified in HDM vs. PBS at ZT12 (Figure S1F ).
NanoString Myeloid Innate Immunity panel was utilized to validate the
miRNA-mRNA targets predicted by Ingenuity Pathway Analysis (IPA). The
predicted mRNA target genes for DE miRNAs were validated based on
normalized counts and qPCR analysis (Figure 2B andFigure S4A ). The predicted mRNA target genes includeil13, tnfrsf1b, arg1, fgf2, fut4, ccr2, vamp2, adamts3, cxcl10,
cxcr5, yap1, ptgs2, ikbke, hla-a, prg2, lat2, tlr2, pdcd1lg2, andusp18 . These miRNA-mRNA predictions were based on IPA and miRPath
analyses and their gene annotation to specific signaling pathways
including the cell type associated with allergic asthma are summarized
(Table S1, Figure 2C, and Figure S4A-C ). We speculate that the
time-of-day difference in DE miRNAs in allergic asthma (ZT0 vs. ZT12)
may be due to disruption of circadian rhythms in the lung which were
among the KEGG pathway predicted showing >1 miRNAs
interacting with 15 circadian genes (Figure S4C ).
Overall, we found a significant time-of-day difference in the DE of
miRNAs in HDM vs. PBS exposed mouse lungs at ZT0 and ZT12. DE miRNA and
IPA-predicted mRNA target genes belong to cytokine/chemokine signaling,
growth factor signaling, Th2 activation, etc. that was further validated
by NanoString and qPCR analyses. miRPath analysis support our findings
from DE miRNA-mRNA, which were linked to KEGG pathways directly
associated with asthma and inflammation. This is the first report that
shows a time-of-day difference in miRNA expression in the lung following
HDM exposure that may involve the interaction of circadian clock genes
which need to be further investigated.
Isaac Kirubakaran Sundar, PhD
Ashokkumar Srinivasan, PhD