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Differential expression of keratinocyte-derived extracellular vesicle miRNAs discriminate exosomes from apoptotic bodies and microvesicles

ABSTRACT: Extracellular vesicles (EVs) are mammalian cell-derived nano-scale structures enclosed by a lipid bilayer that were previously considered to be cell debris with little biological value. However, EVs are now recognized to possess biological function, acting as a packaging, transport and delivery mechanism by which functional molecules (i.e. miRNAs) can be transferred to target cells over some distance. To examine the miRNA from keratinocyte-derived EVs, we isolated three distinct populations of EVs from both HaCaT and primary human keratinocytes (PKCs) and characterized their biophysical, biochemical and functional features by using microscopy, immunoblotting, nanoparticle tracking, and next generation sequencing. We identified 1048; 906; and, 704 miRNAs, respectively, in apoptotic bodies (APs), microvesicles (MVs) and exosomes (EXs) released from HaCaT, and 608; 506; and, 622 miRNAs in APs, MVs and EXs released from PKCs. In which, there were 623 and 437 identified miRNAs common to three HaCaT-derived EVs and PKC-derived EVs, respectively. In addition, we found hundreds of exosomal miRNAs that were previously un-reported and that differences in the abundance levels of the identified EV miRNAs could discriminate between the three EV populations. These data contribute to EV-identified miRNA database, especially keratinocyte-derived EV miRNA content.

ORGANISM(S): Homo sapiens

PROVIDER: GSE106453 | GEO | 2019/05/20

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.

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Differential expression of keratinocyte-derived extracellular vesicle miRNAs discriminate exosomes from apoptotic bodies and microvesicles

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