Exosomes Derived hsa-miR-4669 as a Novel Biomarker for Early Predicting the Response of Subcutaneous Immunotherapy in Pediatric Allergic Rhinitis

Figures & data

Figure 1 Graphic workflow of this study, including miRNA sequencing, validation and clinical significance.

Table 1 The Demographic Character of the First Validation Group

Variables	Effective Group (n=26)	Ineffective Group (n=26)	P-value
Gender			0.760
Male	18	19
Female	8	7
Age (years)	9.6±2.3	9.3±2.2	0.627
BMI (kg/m^2)	17.2±3.2	16.1±3.5	0.275
Concomitant diseases
Asthma	4/22	4/22	1.000
Allergic conjunctivitis	3/23	4/22	0.685
Baseline VAS	6.4 ± 0.3	7.1 ± 0.4	0.124
Baseline TNSS	8.5 ± 0.3	9.3 ± 0.3	0.087
Multiple allergens			0.638
Yes	2	3
No	24	23

Abbreviations: VAS, Visual analogue scale; TNSS, Total nasal symptoms score.

Table 2 The Demographic Character of the Second Validation Group

Variables	Effective Group (n=24)	Ineffective Group (n=24)	P-value
Gender			0.540
Male	17	15
Female	7	9
Age (years)	9.5±2.5	10.8±2.4	0.061
BMI (kg/m^2)	16.6±2.1	17.4±2.9	0.845
Concomitant diseases
Asthma	5/19	5/19	1.000
Allergic conjunctivitis	2/22	2/22	1.000
Baseline VAS	6.6 ± 0.3	5.9 ± 0.3	0.072
Baseline TNSS	8.6 ± 0.3	8.9 ± 0.4	0.621
Multiple allergens			0.551
Yes	1	2
No	23	22

Abbreviations: VAS, Visual analogue scale; TNSS, Total nasal symptoms score.

Figure 2 Identification of serum exosomes. (A) The morphology of serum-derived exosomes was observed by TEM. Scale bar= 100 nm. (B) Particle diameter detection of exosomes.

Table 3 Differentially Expressed miRNAs Between the Effective and Ineffective Groups

MiRNA ID	Pre Locus	FC-value	P-value
hsa-miR-449c-5p	chr5:55172262–55172353(-)	0.009765985	0.022
hsa-miR-12135	chr15:73299910–73299979(+)	0.010086941	0.026
hsa-miR-3196	chr20:63238779–63238842(+)	0.014304379	0.049
hsa-miR-4669	chr9:134379411–134379472(+)	104.4577892	0.028
hsa-miR-4482-5p	chr10:104268336–104268405(-)	93.86569420	0.029
hsa-miR-4686	chr11:2173063–2173138(+)	87.58597604	0.033

Figure 3 MiRNA sequencing of serum exosomes. (A) The volcano plots displayed identified miRNAs in the effective and ineffective groups. (B) The heat map showed the cluster analysis of differentially expressed miRNAs (P<0.05).

Figure 4 GO analysis of the parental genes of the target mRNAs of the differentially expressed miRNAs, includes the following categories: biological process (BP), cellular component (CC) and molecular function (MF). GO analysis of the target mRNAs of (A–C) upregulated and (D–F) downregulated miRNAs in the effective group.

Figure 5 KEGG pathway analysis of the target mRNAs of the differentially expressed miRNAs. (A) Upregulated miRNAs and (B) downregulated miRNAs.

Figure 6 Relative expression level of differentially expressed miRNAs in serum exosomes in the first validation. The identified top three (A) downregulated miRNAs and (B) upregulated miRNAs in the effective group. *P <0.05, **P <0.01.

Abbreviation: ns, no significance.

Figure 7 Exosomes derived hsa-miR-4669 was significantly downregulated in the effective group. (A) The relative expression level in nasal mucous of the effective group versus the ineffective group in the second validation. (B) Prediction of potential target genes of hsa-miR-4669. ****P <0.0001.

Abbreviation: ns, no significance.

Figure 8 Altered hsa-miR-4669 was correlated with disease severity. Correlation between exosomes derived hsa-miR-4669 level and (A) VAS and (B) TNSS.