Commercial Dairy Cow Milk microRNAs Resist Digestion under Simulated Gastrointestinal Tract Conditions.
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 x 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.
The Journal of nutrition