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Personne :
Gilbert, Caroline

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Gilbert

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Caroline

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Université Laval. Département de microbiologie-infectiologie et d'immunologie

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ncf11849137

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  • PublicationAccès libre
    A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk
    (Co-Action Publishing, 2017-11-21) Gilbert, Caroline; Shan, Si Ting; Benmoussa, Abderrahim; Provost, Patrick; Ly, Sophia; Laugier, Jonathan; Boilard, Éric
    MicroRNAs are small gene-regulatory RNAs that are found in various biological fluids, including milk, where they are often contained inside extracellular vesicles (EVs), like exosomes. In a previous study, we reported that commercial dairy cow’s milk microRNAs resisted simulated digestion and were not exclusively associated with canonical exosomes. Here, we report the characterization of a milk EV subset that sediments at lower ultracentrifugation speeds and that contains the bulk of microRNAs. Milk EVs were isolated by differential ultracentrifugation and Iodixanol density gradient (IDG), and analysed for (1) microRNA enrichment by reverse transcription and quantitative polymerase chain reaction (RT-qPCR), and (2) EV-associated proteins by Western blot. Milk EVs were characterized further by dynamic light scattering (DLS), density measurements, fluorescent DiR and RNA labelling, high-sensitivity flow cytometry (HS-FCM), transmission electron microscopy (TEM), proteinase K and RNase A assay, and liquid chromatography tandem-mass spectrometry (LC-MS/MS). We found that the bulk of milk microRNAs (e.g., bta-miR-125b, bta-miR-148a, etc.) sediment at 12,000 g and 35,000 g. Their distribution pattern was different from that of exosome-enriched proteins, but similar to that of several proteins commonly found in milk fat globule membranes (MFGM), including xanthine dehydrogenase (XDH). These low-speed ultracentrifugation pellets contained cytoplasm-enclosing phospholipid bilayered membrane vesicles of a density comprised between 1.11 and 1.14 g/mL in Iodixanol. This milk EV subset of ~100 nm in diameter/~200 nm hydrodynamic size resisted to proteinase K digestion and protected their microRNA content from RNase A digestion. Our results support the existence of a milk EV subset pelleting at low ultracentrifugations speeds, with a protein coating comparable with MFGM, which contains and protects the bulk of milk microRNAs from degradation. This milk EV subset may represent a new EV population of interest, whose content in microRNAs and proteins supports its potential bioactivity
  • PublicationRestreint
    Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions
    (Charles C. Thomas, Publisher, 2016-10-05) Gilbert, Caroline; Lee, Chan Ho C.; Savard, Patricia; Benmoussa, Abderrahim; Fliss, Ismaïl; Provost, Patrick; Laffont, Benoit; Laugier, Jonathan; Boilard, Éric
    Background: MicroRNAs are small, gene-regulatory noncoding RNA species present in large amounts in milk, where they seem to be protected against degradative conditions, presumably because of their association with exosomes. Objective: We monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined their associations with extracellular vesicles (EVs). Methods: We used a computer-controlled, in vitro, gastrointestinal model TNO intestinal model-1 (TIM-1) and analyzed, by quantitative polymerase chain reaction, the concentration of 2 microRNAs within gastrointestinal tract compartments at different points in time. EVs within TIM-1 digested and nondigested samples were studied by immunoblotting, dynamic light scattering, quantitative polymerase chain reaction, and density measurements. Results: A large quantity of dairy milk Bos taurus microRNA-223 (bta-miR-223) and bta-miR-125b (∼109–1010 copies/300 mL milk) withstood digestion under simulated gastrointestinal tract conditions, with the stomach causing the most important decrease in microRNA amounts. A large quantity of these 2 microRNAs (∼108–109 copies/300 mL milk) was detected in the upper small intestine compartments, which supports their potential bioaccessibility. A protocol optimized for the enrichment of dairy milk exosomes yielded a 100,000 × g pellet fraction that was positive for the exosomal markers tumor susceptibility gene-101 (TSG101), apoptosis-linked gene 2–interacting protein X (ALIX), and heat shock protein 70 (HSP70) and containing bta-miR-223 and bta-miR-125b. This approach, based on successive ultracentrifugation steps, also revealed the existence of ALIX−, HSP70−/low, and TSG101−/low EVs larger than exosomes and 2–6 times more enriched in bta-miR-223 and bta-miR-125b (P < 0.05). Conclusions: Our findings indicate that commercial dairy cow milk contains numerous microRNAs that can resist digestion and are associated mostly with ALIX−, HSP70−/low, and TSG101−/low EVs. Our results support the existence of interspecies transfer of microRNAs mediated by milk consumption and challenge our current view of exosomes as the sole carriers of milk-derived microRNAs.