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MiRNA sequencing of extracellular vesicles derived from podocytes under high glucose stimulation.

ABSTRACT: Extracellular vesicles were isolated from the cell culture supernatants of podocytes under high glucose(HG), normal glucose(NG) and iso-osmolality stimulation (three replicates/group). miRNA sequencing was performed to identify differentially expressed miRNAs in podocyte-derived extracellular vesicles. After sequencing, A total of 1915 miRNAs were annotated from all samples . A comparison of the HG and NG groups showed that 11 miRNAs were differentially expressed (4 for up-regulated and 7 for down-regulated,|log2(fold change)| > 1, p-value < 0.05), while a comparison of the HG and iso-osmolality groups showed that 18 miRNAs were differentially expressed (1 for up-regulated and 17 for down-regulated, |log2(fold change)| > 1, p-value < 0.05). This study provides the results of miRNA alteration in podocytes extracellular vesicles under HG stimulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE154284 | GEO | 2020/10/20

REPOSITORIES: GEO

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Project description:Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterise extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems.Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterisation was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analysed by high-resolution mass spectrometry.Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR- 323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response.Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

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MiRNA sequencing of extracellular vesicles derived from podocytes under high glucose stimulation.

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