CHAPITRE III. COMMERCIAL DAIRY COW MILK MICRORNAS RESIST DIGESTION UNDER SIMULATED GASTROINTESTINAL
3.1. Avant-propos
Le manuscrit présenté dans ce chapitre et intitulé « Commercial dairy cow milk microRNAs
resist digestion under simulated gastrointestinal tract conditions » a été publié au Journal of Nutrition en
octobre 2016. J’en suis l’auteur principal et il est coécrit avec Chan Ho Chris Lee, Benoit Laffont, Patricia Savard, Jonathan Laugier, Eric Boilard, Caroline Gilbert, Ismail Fliss et Patrick Provost. J’ai effectué les expériences, l’analyse, l’interprétation des résultats et la révision de ce manuscrit, en collaboration avec les coauteurs. Ce projet de recherche a été supporté par les Instituts de recherche en santé du Canada (IRSC) [IG1-134171 et MOP-137081 via l’institut de Génétique, et PJT-165806]. Des modifications mineures du format ont été effectuées dans le manuscrit et la numérotation des figures a été adaptée à la facture de cette thèse.
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3.2. Résumé
Le lait contient des microARN encapsulés dans des vésicules extracellulaires (VE), le plus souvent des « exosomes », ce qui leur permet de résister à des conditions physico-chimiques habituellement délétères pour les acides nucléiques. Néanmoins, la biodisponibilité des microARN et des VE du lait reste à démontrer, notamment lors de la digestion. Pour pallier cette limite, nous avons injecté du lait bovin commercial dans le TIM-1, un appareil contrôlé par ordinateur qui simule in vitro la digestion humaine. Nous avons découvert que de nombreuses copies du bta-miR-223 et du bta-miR-125b (109-1010 copies/300 mL lait) étaient capables de résister à la digestion et que ces microARN étaient effectivement associés, en partie, à des exosomes. Néanmoins la majorité des microARN résistant à la digestion étaient co-isolés avec des particules, possiblement des VE, peu enrichies en marqueurs exosomaux (TSG101, ALIX et HSP70) et qui sédimentent à des vitesses plus faibles que les exosomes.
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3.3. Abstract
Background: MicroRNAs are small, gene-regulatory non-coding RNA species that are present in large amounts in milk, where they seem to be protected against degradative conditions, presumably because of their association with exosomes. Objective: Here, we monitored the relative stability of commercial dairy cow milk microRNAs, under the gastrointestinal (GI) tract conditions that prevail during digestion and examined their association with extracellular vesicles (EVs). Methods: We used a computer-controlled in vitro GI model (TIM-1) and analyzed, by quantitative PCR (qPCR), the level of two microRNAs within all GI tract compartments at different time points. MicroRNA-containing EVs within digested and non-digested samples were studied by immunoblotting, dynamic light scattering, qPCR and density measurements. Results: A large quantity of dairy milk bta-miR-223 and bta-miR-125b (~109-1010 copies/300 mL milk) withstood digestion under simulated GI tract conditions, with the stomach causing an important decrease in microRNA levels (30 to 64% of input in 30 min) compared to the other TIM-1 compartments. A high quantity of these two 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 positive for the exosomal markers Tumor susceptibility gene-101 (TSG101), 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 that are larger than exosomes and 2 to 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 strengthen 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.
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3.4. Introduction
MicroRNAs are small, 19 to 24-nucleotide (nt) non-coding RNA species generated by the ribonuclease III Dicer [1] and guiding Argonaute effector complexes to regulate ~60% of the genes in human [2]. Highly conserved within the clade of mammals [3, 4] and associated with numerous physiological and/or pathological processes, microRNAs are expressed in plants and animals that are part of the human diet. The provocative idea that microRNAs may cross the barrier between species through diet was initially explored by Zhang et al. [5], who reported on the uptake of exogenous plant (rice) microRNAs through food intake. Since then, an increasing number of studies have extended these observations on dietary microRNAs, also termed xenomiRs by Witwer et al. [6], to various food sources, including other plant microRNAs [7], chicken eggs [8] and cow milk [9], although not without some controversies [10-12]. While interspecies transfer of RNA (double-stranded RNA from bacteria to nematodes) had been reported 14 years earlier [13], a recent study has provided tangible support to this concept by demonstrating the transfer of microRNAs from a gastrointestinal (GI) parasitic nematode (Heligmosomoides polygyrus) to its mammalian (mouse) host [14].
Others and we have shown that microRNAs may be transferred between mammalian cells through the release (and uptake) of extracellular vesicles (EVs) known as microparticles (MP; < 1 μm in size). EVs may form a barrier against degradative components/conditions present in the extracellular milieu or various biological fluids, such as blood and maternal milk [15], and protect microRNAs from degradation, which would facilitate their transfer between cells [16], organs [17], and even between individuals [18].
Humans of all ages consume milk from various sources, mainly from their mother’s breast for newborns or dairy cows for adults, on a regular basis. The facts that (i) milk is a complete food for the newborns and highly consumed worldwide, (ii) microRNAs are highly conserved in mammals and may mediate the beneficial effects of dairy milk consumption in rheumatoid arthritis [19] or in immune functions [20], (iii) microRNAs contained in milk can be absorbed by human cells and mouse model organs [9, 21], and (iv) bovine milk microRNAs exhibit relatively high stability under degradative conditions [22], impelled us to characterize the kinetics of commercial dairy milk microRNAs during digestion under simulated GI tract conditions.
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We had previously shown that platelet-derived microRNAs could be transferred to endothelial cells [23], macrophages [24] or neutrophils [25] through EVs, which may protect microRNAs from degradation in the extracellular milieu (e.g. blood). The actual leading theory is that microRNAs present in milk are resilient to adverse conditions likely because they are contained within, and protected from degradation by, lipidic microvesicles, more specifically, exosomes [22, 26, 27]. Exosomes are small EVs (30-100 nm in diameter) produced by invagination of multivesicular bodies and released outside of the cells by fusion of the multivesicular bodies with the cellular membrane [28]. Well-established vehicle mediating the intercellular transfer of microRNAs [29, 30], exosomes were the initial focus of our investigations.
Reasoning that this type of EVs might help protect food-derived, or dietary, microRNAs from the degradative environment of the GI tract, which is a prerequisite for the absorption of microRNAs through the diet, we observed that dairy milk microRNAs are (i) relatively well protected from the biophysical, biochemical and enzymatic conditions of the GI tract, and (ii) associated with different types of EVs, including exosomes, which is likely to contribute to their bioavailability.