Differentially expressed miRNA profiles of serum-derived exosomes in patients with sudden sensorineural hearing loss

Abstract

Objectives: This study aimed to compare the expressed microRNA (miRNA) profiles of serum-derived exosomes of patients with sudden sensorineural hearing loss (SSNHL) and normal hearing controls to identify exosomal miRNAs that may be associated with SSNHL or serve as biomarkers for SSNHL.

Methods: Peripheral venous blood of patients with SSNHL and healthy controls was collected to isolate exosomes. Nanoparticle tracking analysis, transmission electron microscopy, and Western blotting were used to identify the isolated exosomes, after which total RNA was extracted and used for miRNA transcriptome sequencing. Differentially expressed miRNAs (DE-miRNAs) were identified based on the thresholds of P < 0.05 and |log₂fold change| > 1 and subjected to functional analyses. Finally, four exosomal DE-miRNAs, including PC-5p-38556_39, PC-5p-29163_54, PC-5p-31742_49, and hsa-miR-93-3p_R+1, were chosen for validation using quantitative real-time polymerase chain reaction (RT-qPCR).

Results: Exosomes were isolated from serum and identified based on particle size, morphological examination, and expression of exosome-marker proteins. A total of 18 exosomal DE-miRNAs, including three upregulated and 15 downregulated miRNAs, were found in SSNHL cases. Gene ontology (GO) functional annotation analysis revealed that target genes in the top 20 terms were mainly related to "protein binding," "metal ion binding," "ATP binding," and "intracellular signal transduction." Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these target genes were functionally enriched in the "Ras," "Hippo," "cGMP-PKG," and "AMPK signaling pathways." The expression levels of PC-5p-38556_39 and PC-5p-29163_54 were significantly downregulated and that of miR-93-3p_R+1 was highly upregulated in SSNHL. Consequently, the consistency rate between sequencing and RT-qPCR was 75% and sequencing results were highly reliable.

Conclusion: This study identified 18 exosomal DE-miRNAs, including PC-5p-38556_39, PC-5p-29163_54, and miR-93-3p, which may be closely related to SSNHL pathogenesis or serve as biomarkers for SSNHL.

Keywords: biomarkers; exosomes; miRNA; miRNA transcriptome sequencing; sudden sensorineural hearing loss.

Figure 1

Identification of exosomes isolated from serum of healthy individuals and sudden sensorineural hearing loss (SSNHL) patients. (A) Particle size distribution and number of exosomes measured by Nanosight. (B) Transmission electron microscopy revealed spherical or saucer-shaped morphology of exosomes with a double-membrane structure. (C) Western blotting showed that exosomal surface markers (HSP70, TSG101, and CD9) were all expressed. Healthy-exo, exosomes isolated from healthy individuals; SSNHL-exo, exosomes isolated from SSNHL patients; healthy-supernatant, supernatant from healthy individuals; SSNHL-supernatant, supernatant from SSNHL patients.

Figure 2

Database comparison and microRNA (miRNA) identification. (A) The ratio of rRNA, snoRNA, snRNA, tRNA, and other non-miRNA sequences in total reads to Rfam data. (B) The ratio of rRNA, snoRNA, snRNA, tRNA, and other non-miRNA sequences in unique reads to Rfam data. (C) The ratio of rRNA, snoRNA, snRNA, tRNA, and other non-miRNA sequences in total reads to Repbase data. (D) The ratio of rRNA, snoRNA, snRNA, tRNA, and other non-miRNA sequences in unique reads to Repbase data. (E) Venn diagram showing the number of miRNAs detected in the two groups of samples. (F) Length distribution of the detected miRNAs.

Figure 3

Screening of differentially expressed miRNAs (DE-miRNAs) in exosomes from SSNHL and healthy samples. Red: upregulated; blue: downregulated. (A) Statistical diagram of upregulated or downregulated miRNAs in the different groups. (B) Heat map of DE-miRNAs in exosomes from SSNHL and healthy samples. (C) Volcano map of DE-miRNAs from SSNHL and healthy samples. (D) Scatterplot of DE-miRNAs in SSNHL and healthy samples.

Figure 4

Gene Ontology (GO) enrichment scatterplot showing the top 20 terms with the lowest p-values. Rich factor: the proportion of the target gene number located in the GO to the total gene number located in the GO (rich factor = S gene number/B gene number).

Figure 5

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment scatterplot showing the top 20 terms with the lowest p-values.

Figure 6

Expression Analysis of exosomal PC-5p-38556_39, PC-5p-29163_54, PC-5p-31742_49, and miR-93-3p_R+1 in exosomes isolated from healthy individuals and SSNHL patients (n = 3). *P < 0.05, **P < 0.01 vs. healthy controls.