关键词: 棘球蚴病, 外泌体, 微小RNA, 差异表达, 诊断标志物, qPCR

Abstract:

Objective To compare the differential expression of exosomal microRNAs (miRNAs) among alveolar echinococcosis (AE) patients pre- and post- surgery, and between AE and cystic echinococcosis (CE) patients, so as to investigate the value of exosomal miRNAs for diagnosis of echinococcosis. Methods Whole blood samples were collected from pre-surgery and 3 to 4 weeks post-surgery of 3 patients with clinically definitively diagnosed CE and 4 patients with clinically definitively diagnosed AE, 3 healthy individuals, and from 48 patients with typical AE lesions, 10 patients with typical CE lesions (all lesions detected by B-mode ultrasound), and 39 individuals negative for B-mode ultrasound. Whole blood samples were centrifuged and serum samples were collected. Exosomes were isolation and enriched using size exclusion chromatography and ultrafiltration, and RNA was extracted from exosomes for sequencing. MiRNAs were identified using miRDeep2, and miRNAs expression was quantified. The differential expression of exosomal miRNAs from patients was compared pre- and post-surgery using the edgeR software. Molecular probes were designed, and Echinococcus-derived exosomal miRNAs with high abundance were screened with qPCR assay and evaluated for their values in diagnosis of AE. In addition, the value of humans-derived differentially expressed miRNAs in differential diagnosis of echinococcosis was assessed. The comparison of relative expression levels of miRNA in human derived extracellular vesicles was performed using t-test. Results Exosomes appeared circular or circular-like vesicular structures of varying sizes and uneven distribution under a transmission electron microscope, with diameters ranging from 30 to 150 nm and distinct membranous structures. A total of 83 Echinococcus-derived miRNAs were identified in the exosomes from AE patients’ pre-surgical serum samples, including 64 known miRNAs and 19 novel miRNAs, and most miRNAs had low expression in serum samples, with emu-let-7-5p showing relatively high expression. Two miRNAs with high abundance (emu-miR-71-5p and emu-miR-10-5p) screened by qPCR assay and emu-let-7-5p that had been reported to exhibit a diagnostic value were included for further verification. The detection of emu-let-7-5p was 20.83% (10/48)；emu-miR-71-5p was detected in both positive and negative samples, with detection rates of 87.50% (42/48) and 30.76% (12/39), respectively; while emu-miR-10-5p was only detected in 52.08% (25/48) of positive samples. A total of 8 differentially expressed humans-derived exosomal miRNAs were identified, and 5 were selected for evaluation based on their expression abundance. Among them, 3 miRNAs (has-miR-183-5p, has-miR-222-3p and has-miR-196a-5p) had average expression levels of 355, 299, and 213 in CE positive serum samples, which were 39.5, 10.0, and 23.6 times higher than negative controls, respectively, and the difference was statistically significant (t = 7.15, 5.45, 6.26, all P < 0.01). Conclusion Due to trace levels in serum, Echinococcus-derived exosomal miRNAs still have limitations as diagnostic markers for echinococcosis. Three humans-derived exosomal miRNAs have potential in CE diagnosis.

Key words: Echinococcosis, Exosome, miRNA, Differential expression, Diagnostic marker, qPCR