Ineffective delivery of diet-derived microRNAs to recipient animal organisms

Abstract

Cross-kingdom delivery of specific microRNAs to recipient organisms via food ingestion has been reported recently. However, it is unclear if such delivery of microRNAs occurs frequently in animal organisms after typical dietary intake. We found substantial levels of specific microRNAs in diets commonly consumed orally by humans, mice, and honey bees. Yet, after ingestion of fruit replete with plant microRNAs (MIR156a, MIR159a, and MIR169a), a cohort of healthy athletes did not carry detectable plasma levels of those molecules. Similarly, despite consumption of a diet with animal fat replete in endogenous miR-21, negligible expression of miR-21 in plasma or organ tissue was observed in miR-21 -/- recipient mice. Correspondingly, when fed vegetarian diets containing the above plant microRNAs, wild-type recipient mice expressed insignificant levels of these microRNAs. Finally, despite oral uptake of pollen containing these plant microRNAs, negligible delivery of these molecules was observed in recipient honeybees. Therefore, we conclude that horizontal delivery of microRNAs via typical dietary ingestion is neither a robust nor a frequent mechanism to maintain steady-state microRNA levels in a variety of model animal organisms, thus defining the biological limits of these molecules in vivo.

Keywords: cross-kingdom delivery; diet; ecology; honey bee; human; microRNA; mouse; non-coding RNA; nutrition; plant.

Figures

Figure 1. Substantial levels of miRNAs in oral diets consumed by humans, mice, and honey bees. (A) MiRNAs conserved in the plant kingdom were present in substantial quantity in ripened fruits, while miR-21, a conserved miRNA in the animal kingdom, was present in a common dietary source of meat (ham). & denotes that miR-21 was not detectable in fruit products, and this absence of signal was significantly different (p < 0.05) than copy number of MIR156a, MIR159a or MIR169a in a given diet, based on ANOVA and post-hoc testing. # denotes that plant miRNAs were not detectable in ham, and this absence of signal was significantly different (p < 0.05) than copy number of miR-21. (B) Conserved plant miRNAs were expressed at high levels in a custom vegetarian diet and a custom diet with soy, while miR-21 was present in a custom diet carrying animal lard and casein. & denotes that miR-21 was not detectable in the vegetarian or soy diets, and this absence of signal was significantly different (p < 0.05) than copy number of MIR156a, MIR159a or MIR169a in a given diet. # denotes that MIR-159a and MIR169a were not detectable in the casein and lard diet, and * denotes that copy number of miR-21 was significantly different (p < 0.05) than copy number of MIR156a, MIR159a and MIR169a in that diet. (C) Conserved plant miRNAs were present in substantial quantity in pollen, a primary food source for the honey bee, and in honey stored by honey bees for later consumption. In all panels, each food source was sampled and analyzed three independent times (n = 3), and error bars, which are small but present on each column, reflect SEM.

Figure 2. Negligible steady-state expression of diet-derived miRNAs in plasma or organ tissue. (A) Oral ingestion of a lard diet replete with miR-21 did not lead to a steady-state increase of miR-21 in plasma or organ tissue in miR-21 −/− recipient mice (n = six mice per group). (B) In spite of vegetarian diets (vegetarian, black bar; soy, gray bar) containing endogenous MIR156a, MIR159a and MIR169a as compared with a diet with lower levels of plant miRNAs (lard, hatched bar), minimal expression of MIR156a in plasma was observed (left graph) with negligible steady-state expression in organ tissue (right graph) of wild-type recipient mice (n = five mice per group). Notably, in these preparations, MIR159a and MIR169a were not detectable. (C) Unprocessed avocado ingestion increases the expression of conserved plant miRNAs in the stomach contents of wild-type mice. (D) Ingestion of fresh avocado does not result in substantial steady-state expression of MIR156a in plasma (left graph) or organs (right graph) in wild-type recipient mice (n = four mice). Again, in these preparations, MIR159a and MIR169a were not detectable. (E) (Right graph) The honey bee miRNA let7 was expressed in the abdomen of both nurses and foragers. Higher levels of let7 were observed incidentally in nurses as compared with foragers. (Left graph) In spite of oral uptake of pollen and honey/nectar replete with MIR156a, MIR159a and MIR169a, negligible levels of MIR156a were detected in the abdominal tissue of recipient honey bees (n = three nurses; n = four foragers). MIR159a and MIR169a were undetectable in these tissues. In all panels, error bars reflect SEM; * signifies p < 0.05; NS signifies p ≥ 0.05. # denotes that the expression of a given miRNA was either undetectable or at the level of minimal detection. For plasma, normalized copy number is expressed per μl plasma. For animal organs, copy number is expressed per 10 pg of total RNA. For stomach contents, normalized copy number is expressed per total gastric material harvested per mouse.