Dairy cow milk microRNAs associated with exosomes can resist digestion

rna-seq

Large amounts of nondigested commercial dairy milk microRNAs are associated with exosomes. Fresh commercial dairy milk (60 mL, n = 3) was subjected to the same exosome enrichment protocol described previously for the digested sample. The buoyant density of each IDG fraction was calculated (A), and the hydrodynamic size (diameter in nanometers) of the particles present in each fraction was measured (B); results are expressed as means ± SEMs (n = 3). Proteins from fractions F5–F8 containing exosomes and those from F4 and F9 used as controls were analyzed by immunoblotting to detect the exosome-enriched proteins TSG101 and ALIX (C) (52). Fraction F6–associated particles were studied by transmission electron microscopy (D). ALIX, apoptosis-linked gene 2–interacting protein X; F, fraction; HSP70, heat shock protein 70; IDG, iodixanol density gradient; TSG101, tumor susceptibility gene-101.

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.

Researchers from the University of Quebec monitored the relative stability of commercial dairy cow milk microRNAs during digestion and examined their associations with extracellular vesicles (EVs).

They 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.

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).

These 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. The 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.

Benmoussa A, Lee CH, Laffont B, Savard P, Laugier J, Boilard E, Gilbert C, Fliss I, Provost P. (2016) Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions. J Nutr 146(11):2206-2215. [article]

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