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 mechanisms 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 1,048; 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. Differences in the abundance levels of the identified EV miRNAs could discriminate between the three EV populations. These data contribute substantially to knowledge within the EV-identified miRNA database, especially with regard to keratinocyte-derived EV miRNA content.
Highlights
Extracellular membrane vesicles (EVs) are secreted by various cell types and can be isolated from body fluids such as breast milk, urine, amniotic fluids, plasma and saliva, as well as from cell-conditioned culture media [1,2,3,4,5,6,7]
As previously described [10, 37], we found that vesicles from the apoptotic bodies (APs) fraction were typically larger than 1 μm; the MV fraction were irregular and 300 nm to 700 nm in size; and the EX fraction were relatively homogenous at 50 nm to 120 nm with a cupshaped morphology (Figure 1A)
Low level Extracellular vesicles (EVs) contamination of AP preparations may have resulted from a sub-population of EVs with low buoyant density that pelleted at low g forces
Summary
Extracellular membrane vesicles (EVs) are secreted by various cell types and can be isolated from body fluids such as breast milk, urine, amniotic fluids, plasma and saliva, as well as from cell-conditioned culture media [1,2,3,4,5,6,7]. EVs have been categorized based upon physical parameters in (i) apoptotic bodies (Aps; ∼1–5 μm); (ii) microvesicles (MVs; ∼100–1,000 nm); MicroRNA Cargoes Discriminate Extracellular Vesicles and (iii) exosomes (EXs; ∼40–100 nm) [8]. During the formation and release process, EVs are assembled as a lipid bilayer membrane encapsulating cellderived components such as proteins, lipids, organelles and genetic materials including nucleic acids [9,10,11] In this regard, statistical data in Vesiclepedia report that the EVs database contains 92,897 protein, 32,576 RNA and 584 lipid molecules from 538 studies in 33 species (Vesiclepedia version 3.1 released 9/1/2015, http://www.microvesicles.org/). Recent studies have demonstrated that miRNAs can be detected in blood plasma within EXs, MVs and APs derived from cancer cells [19,20,21,22,23]. This study is the first to isolate APs, MVs and EXs released from keratinocytes in order to analyse their miRNA content and identify the discriminant molecular features of EXs, Aps, and MVs
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