The Specific microRNA Profile and Functional Networks for Children with Allergic Asthma

Figures & data

Figure 1 Study flowchart.

Notes: The peripheral blood mononuclear cells (PBMCs) of healthy children and children suffer from allergic asthma were deep sequenced for small RNA expression profiles, and systematically analyzed using bioinformatics databases for deduction of novel molecular signatures in allergic asthma.

Table 1 Summary Characteristics of Allergic Asthma Patients and Controls

Groups	Small RNA Sequencing Sample			Validation Sample
	Control (n=3)	Allergic Asthma (n=3)	pValue	Control (n=15)	Allergic Asthma (n=15)	pValue
Age (yrs)	4.7±2.05	5±2.16	p=0.882	5.6±2.24	6.13±2.50	p=0.557
Sex (n %)
Women	1 (33.33)	1 (33.33)		4 (26.67)	6 (40)
Men	2 (66.67)	2 (66.67)		11 (73.33)	9 (60)
House dust mite	–	1/3 (moderate) 2/3 (Moderately severe/ severe)		–	1/3 (Moderate) 2/3 (Moderately severe/ severe)

Figure 2 Length distribution and abundance of the small RNA sequences.

Notes: As shown by their frequencies, 21-nt length reads were the most abundant. (A)–(C) Healthy children and (D)–(F) Allergic asthma. Healthy children: Control1, Control2, and Control3; allergic asthma: Case1, Case2, and Case3. Healthy children (n=3): Control1, Control2, and Control3; Allergic asthma (n=3): case1, case2, and case3.

Figure 3 The category annotation of the six libraries: (A)–(C) Healthy children and (D)–(F) Allergic asthma.

Notes: Healthy children: Control1, Control2, and Control3; Allergic asthma: case1, case2, and case3.

Figure 4 Base bias and length confirmation of known miRNAs.

Notes: (A): The length distribution of conserved miRNAs. As shown by their frequencies, 22nt length reads were the most abundant. (B): The bias of the bases of conserved miRNAs. (A)–(C) Healthy children and (D)–(F) Allergic asthma. Healthy children: Control1, Control2, and Control3; Allergic asthma: Case1, Case2, and Case3.

Table 2 Top 50 of Differentially Expressed miRNAs Between Allergic Asthma and Healthy Controls

Number	miRNA_id	FoldChange	log2FoldChange	pValue	qValue	Regulation
1	hsa-miR-143-5p	0.051618845	−4.275958321	4.70E-12	5.40E-09	Down
2	hsa-miR-369-3p	0.037134702	−4.751088171	2.90E-10	1.67E-07	Down
3	hsa-miR-487b-3p	0.060847938	−4.038647814	7.12E-10	2.73E-07	Down
4	hsa-miR-539-3p	0.014334259	−6.124388832	1.42E-09	4.07E-07	Down
5	hsa-miR-136-3p	0.052162853	−4.260833407	1.08E-08	1.94E-06	Down
6	hsa-miR-655-3p	0.032835197	−4.928613073	1.15E-08	1.94E-06	Down
7	hsa-miR-329-3p	0.072833871	−3.779246669	2.54E-08	3.33E-06	Down
8	hsa-miR-409-5p	0.083049618	−3.589882659	2.61E-08	3.33E-06	Down
9	hsa-miR-493-5p	0.053192381	−4.232636579	3.44E-08	3.95E-06	Down
10	hsa-miR-758-3p	0.074723148	−3.742300954	5.75E-08	5.91E-06	Down
11	hsa-miR-410-3p	0.065796976	−3.92583491	6.62E-08	5.91E-06	Down
12	hsa-miR-656-3p	0.061715206	−4.018230203	6.69E-08	5.91E-06	Down
13	hsa-miR-127-3p	0.136511851	−2.872901893	9.53E-08	7.82E-06	Down
14	hsa-miR-889-3p	0.056425905	−4.147498527	1.04E-07	7.96E-06	Down
15	hsa-miR-134-5p	0.122506393	−3.029071052	1.41E-07	1.01E-05	Down
16	hsa-miR-382-3p	0.068550874	−3.866681133	1.61E-07	1.09E-05	Down
17	hsa-miR-1197	0.037991693	−4.71817218	1.89E-07	1.20E-05	Down
18	hsa-miR-493-3p	0.06919629	−3.853161496	2.39E-07	1.45E-05	Down
19	hsa-miR-431-5p	0.077975386	−3.680837396	3.04E-07	1.69E-05	Down
20	hsa-miR-654-3p	0.082943049	−3.59173511	3.12E-07	1.69E-05	Down
21	hsa-miR-370-3p	0.115519549	−3.113791079	3.24E-07	1.69E-05	Down
22	hsa-miR-485-3p	0.113831648	−3.135026381	3.66E-07	1.83E-05	Down
23	hsa-miR-409-3p	0.094932135	−3.396959657	4.80E-07	2.30E-05	Down
24	hsa-miR-381-3p	0.091252102	−3.453998401	7.94E-07	3.65E-05	Down
25	hsa-miR-323a-3p	0.098471937	−3.344143559	8.78E-07	3.88E-05	Down
1	hsa-miR-708-5p	13.79602275	3.786180508	1.18E-08	1.94E-06	Up
2	novel771_mature	5.423108724	2.439120094	4.35E-06	0.000138831	Up
3	hsa-miR-4482-3p	42.07237771	5.394801449	1.40E-05	0.000413678	Up
4	hsa-miR-6868-3p	87.51513746	6.451460676	1.70E-05	0.000488882	Up
5	hsa-miR-1275	3.117091995	1.640200734	0.000166147	0.004150073	Up
6	hsa-miR-4482-5p	31.99442738	4.999748741	0.000208203	0.004945436	Up
7	hsa-miR-615-3p	5.694680501	2.509614902	0.000478065	0.01056339	Up
8	hsa-miR-150-5p	2.763207533	1.466343921	0.000517914	0.01102005	Up
9	hsa-miR-1248	9.279624324	3.214066401	0.00064147	0.013161593	Up
10	hsa-miR-642a-5p	2.869757112	1.520928636	0.00091724	0.017277196	Up
11	hsa-miR-4685-3p	5.696746252	2.510138146	0.001259055	0.022256223	Up
12	hsa-miR-708-3p	3.144856733	1.652994295	0.001441826	0.025100874	Up
13	hsa-miR-10395-3p	2.740029784	1.454191575	0.001848889	0.029920759	Up
14	hsa-miR-451a	2.484543166	1.312980607	0.001962729	0.031321884	Up
15	hsa-miR-342-5p	2.035752251	1.025561998	0.003791667	0.054457811	Up
16	hsa-miR-150-3p	2.152335969	1.105903293	0.004314203	0.059723126	Up
17	hsa-miR-342-3p	2.852794847	1.512376002	0.005455184	0.072046052	Up
18	hsa-miR-2110	2.089113952	1.062891187	0.00572415	0.074664202	Up
19	hsa-miR-132-3p	2.26858702	1.181794001	0.005783389	0.074664202	Up
20	novel23_mature	21.08374851	4.398059483	0.008906226	0.105953496	Up
21	hsa-miR-125a-5p	2.033383427	1.023882284	0.01068444	0.124004255	Up
22	hsa-miR-486-5p	2.214627728	1.147064206	0.011191045	0.128270373	Up
23	hsa-miR-129-1-3p	10.96145359	3.454367221	0.01155965	0.130216059	Up
24	hsa-miR-95-3p	2.116066601	1.081385036	0.014080888	0.155379628	Up
25	hsa-miR-181a-2-3p	2.582808272	1.368940553	0.014199183	0.155379628	Up

Figure 5 Differential expression and characteristics of miRNAs between allergic asthma and healthy controls.

Notes: (A) PCA analysis indicated significant differences; (B) Boxplot showing expression of miRNAs; (C) Number of up-/down-expressed miRNAs between allergic asthma and healthy controls. “Up” means that these miRNAs were higher expressed in children with allergic asthma compared to the healthy, and “down” means that these miRNAs were lower expressed in children with allergic asthma compared to the healthy; (D) A volcano plot showing differentially expressed miRNAs with various fold changes and p-values. Vertical line, fold change = 2 (log₂ transformed); horizontal line, p-value = 0.05 (-log₁₀ transformed). Red dots, significantly up-regulated miRNAs; green dots, significantly down-regulated miRNAs; gray dots, insignificantly changed miRNAs; (E) Hierarchical clustering of differentially expressed miRNAs.

Figure 6 Secondary structures of the top 10 novel miRNAs.

Notes: (A) novel23_mature, (B) novel79_mature, (C) novel95_mature, (D) novel156_mature, (E) novel191_mature, (F) novel206_mature, (G) novel234_mature, (H) novel305_mature, (I) novel498_mature, and (J) novel771_mature.

Figure 7 KEGG pathway enrichment analysis and Gene Ontology (GO) classification for targeted mRNAs of differentially expressed miRNAs.

Notes: (A): GO classification of targeted mRNAs was drawn based on the top30 GO terms listed in ascending order by P-value. The x-axis shows the diverse biological functions of targeted mRNAs according to the three GO categories (biological process, cellular component and molecular function). The y-axis shows the percentage and number of these targeted mRNAs. (B): Pathway analysis was performed based on KEGG databases, and sorted by the P-value of hypergeometric distribution. The enrichment score is calculated according to the formula “Enrichment score = (m/n)/(M/N)”. (N, the number of mRNAs with annotation in KEGG; n, the number of potential targeted mRNAs with annotation in KEGG; M, the number of mRNAs annotated as a designated KEGG Term; m, the number of potential targeted mRNAs annotated as a designated KEGG Term).

Table 3 Statistics of KEGG Enrichment Top 20

KEGG_id	Term	pValue	qValue	Enrichment_score
path:hsa04022	cGMP-PKG signaling pathway	3.20E-29	1.04E-26	1.42
path:hsa04725	Cholinergic synapse	3.93E-23	6.38E-21	1.43
path:hsa04970	Salivary secretion	2.14E-22	2.31E-20	1.51
path:hsa04972	Pancreatic secretion	4.59E-21	3.73E-19	1.51
path:hsa04974	Protein digestion and absorption	1.40E-20	9.12E-19	1.53
path:hsa04924	Renin secretion	4.39E-20	2.38E-18	1.51
path:hsa04921	Oxytocin signaling pathway	1.43E-19	6.63E-18	1.33
path:hsa04713	Circadian entrainment	5.85E-19	2.38E-17	1.39
path:hsa05414	Dilated cardiomyopathy (DCM)	4.41E-18	1.59E-16	1.41
path:hsa04270	Vascular smooth muscle contraction	5.24E-18	1.70E-16	1.36
path:hsa05410	Hypertrophic cardiomyopathy (HCM)	1.63E-17	4.81E-16	1.42
path:hsa04512	ECM-receptor interaction	6.48E-16	1.75E-14	1.45
path:hsa04724	Glutamatergic synapse	2.82E-14	6.65E-13	1.34
path:hsa04927	Cortisol synthesis and secretion	2.87E-14	6.65E-13	1.42
path:hsa04810	Regulation of actin cytoskeleton	1.16E-13	2.51E-12	1.23
path:hsa04020	Calcium signaling pathway	1.73E-13	3.52E-12	1.25
path:hsa04918	Thyroid hormone synthesis	2.85E-13	5.45E-12	1.44
path:hsa04371	Apelin signaling pathway	3.64E-13	6.57E-12	1.30
path:hsa04925	Aldosterone synthesis and secretion	4.16E-12	7.11E-11	1.31
path:hsa04911	Insulin secretion	5.77E-12	9.37E-11	1.31

Figure 8 The network of differentially expressed miRNAs-mRNAs in the cGMP-PKG signalling pathway.

Notes: (A): The network of differentially expressed miRNAs-mRNAs in cGMP-PKG signaling pathway using Cytoscape_v3.5.1 software and the result of KEGG classifications. Orange is differentially expressed miRNAs, green is mRNA with the interactions between 4 or more miRNA, yellow is mRNA with the interactions between only one miRNA, and blue is mRNA with the interactions between 2–3 miRNA. (B): Intracellular mechanism pathway of differentially expressed miRNAs-mRNAs in the cGMP-PKG signalling pathway.

Figure 9 Expression analysis of selected candidate miRNAs assessed by qRT-PCR between allergic asthma (n = 15) and healthy controls (n = 15).

Notes: (A): has-miR-708-5P; (B): novel771_mature; (C): hsa-miR-370-3p; (D): hsa-miR-1275; (E) hsa-miR-134-5p. The relative expression of miRNAs was calculated by applying the comparative cycle threshold method (2^−∆∆Ct), where 5S was an internal control. All data in Figure 5 were statistically analyzed by Student’s t-test or non-parametric Mann–Whitney test and P < 0.05 was considered statistically significant (P*<0.05, P**<0.01 between allergic asthma and healthy controls).

Abbreviation: NS, no significant changes.