MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers

Abstract

Background: MicroRNAs (miRNAs) regulate genes in animals and plants and can be synthesized endogenously. In milk, miRNAs are encapsulated in exosomes, thereby conferring protection against degradation and facilitating uptake by endocytosis. The majority of bovine miRNAs have nucleotide sequences complementary to human gene transcripts, suggesting that miRNAs in milk might regulate human genes.

Objectives: We tested the hypotheses that humans absorb biologically meaningful amounts of miRNAs from nutritionally relevant doses of milk, milk-borne miRNAs regulate human gene expression, and mammals cannot compensate for dietary miRNA depletion by endogenous miRNA synthesis.

Methods: Healthy adults (3 men, 2 women; aged 26-49 y) consumed 0.25, 0.5, and 1.0 L of milk in a randomized crossover design. Gene expression studies and milk miRNA depletion studies were conducted in human cell cultures and mice, respectively. For comparison, feeding studies with plant miRNAs from broccoli were conducted in humans.

Results: Postprandial concentration time curves suggest that meaningful amounts of miRNA (miR)-29b and miR-200c were absorbed; plasma concentrations of miR-1 did not change (negative control). The expression of runt-related transcription factor 2 (RUNX2), a known target of miR-29b, increased by 31% in blood mononuclear cells after milk consumption compared with baseline. When milk exosomes were added to cell culture media, mimicking postprandial concentrations of miR-29b and miR-200c, reporter gene activities significantly decreased by 44% and 17%, respectively, compared with vehicle controls in human embryonic kidney 293 cells. When C57BL/6J mice were fed a milk miRNA-depleted diet for 4 wk, plasma miR-29b concentrations were significantly decreased by 61% compared with miRNA-sufficient controls, i.e., endogenous synthesis did not compensate for dietary depletion. Broccoli sprout feeding studies were conducted as a control and elicited no detectable increase in Brassica-specific miRNAs.

Conclusion: We conclude that miRNAs in milk are bioactive food compounds that regulate human genes.

Conflict of interest statement

Author disclosures: S. R. Baier, C. Nguyen, F. Xie, J. R. Wood, and J. Zempleni, no conflicts of interest.

© 2014 American Society for Nutrition.

Figures

FIGURE 1

Plasma time curves of miR-29b (A), miR-1 (negative control; A), and miR-200c (B) after a milk meal in healthy adults. Values are means, n = 5. SEMs were omitted for clarity (compare with Table 1). miR, microRNA.

FIGURE 2

Effects of milk microRNAs on the abundance of microRNAs in human peripheral blood mononuclear cells. Values are means ± SEMs, n = 5. *Different from hour 0, P < 0.05. miR, microRNA.

FIGURE 3

Effects of milk microRNAs on gene expression in humans. Activities of microRNA reporter genes in HEK-293 cells (A). Expression of endogenous RUNX2 and ZEB1 genes in PBMCs from healthy adults (B). Values are means ± SEMs; n = 4 (LUC-miR-29b), n = 6 (LUC-miR-200c), and n = 5 (PBMCs). *Different from vehicle, P < 0.05. HEK, human embryonic kidney; LUC, luciferase; miR, microRNA; PBMC, peripheral blood mononuclear cell; RUNX2, runt-related transcription factor 2; ZEB1, zinc finger E-box binding homeobox 1.