FIGURE LEGENDS:
Figure 1. miRNA expression signatures in PBS (control) and house dust mite (HDM) exposed mouse lung. (A) Heatmap shows unsupervised hierarchical clustering of nine differentially expressed (DE) miRNAs in HDM vs. PBS exposed mouse lung at ZT0 analyzed by Morpheus tool. (B) Pairwise comparison analysis performed by nSolver 4.0 software showing DE miRNAs in HDM vs. PBS at ZT0.(C) Heatmap shows unsupervised hierarchical clustering of thirty-eight DE miRNAs in HDM vs. PBS exposed mouse lung at ZT12 analyzed by Morpheus tool. (D) Pairwise comparison analysis performed by nSolver 4.0 software showing DE miRNAs in HDM vs. PBS at ZT12. Data are shown as fold change/ratio, P -value (P< 0.05) and FDR adjusted P -value < 1 (n=6/group/time point).
Figure 2. Representative data from differentially expressed miRNAs and selected mRNA targets were analyzed by NanoString nCounter analysis. (A) DE 9 miRNAs were significantly downregulated at ZT0 and ZT12 pairwise comparison. We observed a time-of-day effect in the expression of miRNAs in HDM vs. PBS exposed mouse lung. (B)Selected miRNA-mRNA targets predicted by Ingenuity Pathway Analysis were further validated using NanoString mouse Myeloid Innate Immunity Panel and qPCR analysis (il13 mRNA). Normalized counts data are presented as analyzed by nSolver 4.0 software. (C)Bioinformatic analysis of DE miRNAs downregulated or upregulated in HDM exposed group at ZT12 using DIANA-miRPath v3.0. KEGG pathways marked in red color were pathways identified by miRPath analysis of DE miRNAs in HDM vs. PBS at ZT12. Data are shown as mean ± SEM (n=6/group; * P< 0.05, ** P < 0.01, *** P < 0.001, significant compare to respective PBS control;# P < 0.05, # #P < 0.01, # # # P< 0.001, significant compared to PBS or HDM from ZT0; 2way ANOVA with Tukey’s multiple comparison test).