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

Abstract

Purpose: Subcutaneous immunotherapy (SCIT) is an effective treatment for pediatric allergic rhinitis (AR), but its efficacy fluctuates among individuals. This study aims to identify the profile of serum exosomes derived microRNAs (miRNAs) and evaluate their capacities to early predict SCIT efficacy in pediatric AR.

Patients and methods: High-throughput sequencing was applied to identify the miRNA of serum exosomes in AR children. GO enrichment and KEGG pathway analysis were performed to enrich the biological annotations of target mRNAs of miRNAs. Then we validated differentially expressed miRNAs in two independent cohorts by RT-qPCR. Logistic regression and receiver operating characteristic curve (ROC) were applied to evaluate the abilities of identified miRNAs in predicting the efficacy of SCIT in AR children.

Results: A total of 812 miRNAs were detected in the serum exosomes, including 16 upregulated and 14 downregulated. Differentially expressed genes are enriched in the biological process of developmental process and regulation of cellular process, and gathered in pathways such as the signaling pathways regulating pluripotency of stem cells and the Wnt signaling pathway. In the first validation cohort, hsa-miR-4669 (P=0.009) and hsa-miR-4686 (P=0.032) were significantly downregulated in the effective group than the ineffective group, while hsa-miR-3196 (P=0.015) was upregulated. In the second cohort, hsa-miR-4669 level (P<0.0001) was downregulated in the effective group than the ineffective group. In addition, logistic regression revealed that hsa-miR-4669 level was correlated with the visual analogue scale (r=0.323, P=0.001) and total nasal symptoms score (r=0.269, P =0.007). ROC curve highlighted that hsa-miR-4669 level exhibited a reliable accuracy in predicting SCIT efficacy in pediatric AR (AUC=0.785).

Conclusion: Serum exosomes derived miRNA were associated with the efficacy of SCIT. Serum exosomes derived hsa-miR-4669 might serve as a novel biomarker for early predicting the response of SCIT in AR children.

Keywords: allergic rhinitis; children; exosome; miRNA; subcutaneous immunotherapy.

Figure 1

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

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.

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.

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.

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.