突发性感音神经性听力损失患者血清来源外泌体中差异表达的miRNA谱

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

作者信息

Zhang Juhong, Ma Haizhu, Yang Guijun, Ke Jing, Sun Wenfang, Yang Li, Kuang Shaojing, Li Hai, Yuan Wei

机构信息

Department of Otorhinolaryngology Head and Neck Surgery, Chongqing General Hospital, Chongqing, China.

School of Basic Medicine, Chongqing Medical University, Chongqing, China.

出版信息

Front Neurol. 2023 Jun 2;14:1177988. doi: 10.3389/fneur.2023.1177988. eCollection 2023.

DOI:10.3389/fneur.2023.1177988

PMID:37332997

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10273844/

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 < 0.05 and |logfold 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.

摘要

目的

本研究旨在比较突发性感音神经性听力损失（SSNHL）患者和听力正常对照者血清来源外泌体中表达的微小RNA（miRNA）谱，以鉴定可能与SSNHL相关或作为SSNHL生物标志物的外泌体miRNA。

方法

收集SSNHL患者和健康对照者的外周静脉血以分离外泌体。采用纳米颗粒跟踪分析、透射电子显微镜和蛋白质印迹法鉴定分离出的外泌体，之后提取总RNA并用于miRNA转录组测序。基于<0.05和|log倍变化|>1的阈值鉴定差异表达的miRNA（DE-miRNA），并进行功能分析。最后，选择包括PC-5p-38556_39、PC-5p-29163_54、PC-5p-31742_49和hsa-miR-93-3p_R+1在内的4种外泌体DE-miRNA，使用定量实时聚合酶链反应（RT-qPCR）进行验证。

结果

从血清中分离出外泌体，并根据粒径、形态学检查和外泌体标志物蛋白的表达进行鉴定。在SSNHL病例中总共发现了18种外泌体DE-miRNA，包括3种上调的miRNA和15种下调的miRNA。基因本体（GO）功能注释分析显示，前20个术语中的靶基因主要与“蛋白质结合”、“金属离子结合”、“ATP结合”和“细胞内信号转导”有关。京都基因与基因组百科全书（KEGG）通路富集分析显示，这些靶基因在功能上富集于“Ras”、“Hippo”、“cGMP-PKG”和“AMPK信号通路”。在SSNHL中，PC-5p-38556_39和PC-5p-29163_54的表达水平显著下调，而miR-93-3p_R+1的表达水平高度上调。因此，测序与RT-qPCR之间的一致性率为75%，测序结果高度可靠。