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MiRNA sequencing of extracellular vesicles derived from rat peripheral nerve fibroblasts

ABSTRACT: Peripheral nerve fibroblasts were isolated from the sciatic nerves of Sprague-Dawley (SD) rats. Extracellular vesicles were extracted from the culture supernatant of fibroblasts by differential ultracentrifugation and subsequently identified. Small RNA libraries were constructed using total RNA isolated from extracellular vesicles and sequenced using the Illumina miRNA-seq platform. Bioinformatics analyses were performed, including quality control of raw sequencing data, alignment against the miRBase database, quantification of miRNA expression, and screening of differentially expressed miRNAs, to explore the potential roles of extracellular vesicles miRNAs derived from nerve fibroblasts in peripheral nerve regeneration and related physiological and pathological processes.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE328541 | GEO | 2026/07/15

REPOSITORIES: GEO

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Project description:Objective: To study the uptake by human embryos of extracellular vesicles secreted by the maternal endometrium, and to investigate their miRNA cargo, in order to describe their role in implantation and early embryo development. Design: Prospective descriptive study. Subjects: Healthy women oocyte donors with confirmed fertility and day 5 human blastocysts. Intervention: Endometrial biopsies were collected from healthy oocyte donors undergoing transvaginal ultrasound-guided cyst aspiration for oocyte retrieval. Main Outcome Measures: Extracellular vesicle were isolated from culture media of primary human endometrial epithelial cells by ultracentrifugation. Concentration and size were analyzed by nanoparticle tracking analysis, their morphology visualized by transmission electron microscopy and extracellular vesicle protein markers expression was determined by western blotting. Vesicles were fluorescently labelled with Bodipy-TR ceramide, and their uptake by human blastocysts was analyzed using confocal microscopy. Analysis of the miRNA cargo of extracellular vesicles was performed using miRNAseq, target genes of the most expressed miRNAs were annotated and functional enrichment analysis was performed. Results: Extracellular vesicle characterization revealed a size within 100-300 nm, and expression of extracellular vesicle protein markers HSP70, TSG101, CD9 and CD81. Fluorescent microscopy showed an efficient extracellular vesicle internalization by human blastocysts within 1-2h, being the fluorescent signal stronger in the hatched area of the embryo. miRNAseq described 149 annotated miRNAs and top 37 most expressed miRNAs targeted 6,592 genes. Functional enrichment analysis of these targeted genes indicated that they participate in several processes related to embryo development, oxygen metabolism, cell cycle, cell differentiation, apoptosis, metabolism, cellular organization or gene expression. Among miRNAs contained in these EVs, hsa-miR-92a-3p, hsa-let-7b-5p, hsa-miR-30a-5p, hsa-miR-24-3p, hsa-miR-21-5p and hsa-let-7a-5p were highly implicated in all these biological processes. Conclusion: Data suggest that extracellular vesicles secreted by human endometrial epithelial cells are internalized by human blastocysts, and transport miRNAs to modulate biological processes related to implantation events and early embryo development. Knowledge of the communication system between human endometrium and embryo via miRNA cargo of these vesicles could describe new biomarkers of implantation success and embryo competence.

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MiRNA sequencing of extracellular vesicles derived from rat peripheral nerve fibroblasts

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