Xenobiotic tracers uncover the gastrointestinal passage, cellular uptake and Ago2 loading of milk microRNAs in neonates

Background and aims: Exclusive breastfeeding is the best source of nutrition for most infants, but it is not always possible. Enteral nutrition influences intestinal gene regulation and the susceptibility for inflammatory bowel disorders, such as necrotizing enterocolitis (NEC). In modern neonatology it is observed that lactase activity increases during intestinal maturation, but formula-fed infants exhibit lower activity levels than milk-fed infants. Since human breast milk has a high miRNA content in comparison to other body fluids, it is controversially discussed whether they could influence gene regulation in term and preterm neonates and thus might vertically transmit developmental relevant signals. Methods: Following their cross-species profiling via miRNA deep-sequencing we utilized dietary xenobiotic taxon-specific milk miRNA as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells (HIEC-6) using Ad5-mediated miRNA-gene transfer. Results: Mammals have in common a large number of milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced intact bovine-specific miRNAs from formula-nutrition in human preterm stool and 9 days after the onset of enteral feeding in intestinal cells of preterm piglets. Few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed enrichment of the xenobiotic cel-miR in blood serum and in Ago2-immunocomplexes from intestinal biopsies. This points to a transmissibility of milk miRNA signals. We performed Adenovirus type 5-based miRNA-gene transfer into HIEC-6 and examined predicted bovine milk miRNA targets on the protein and transcriptome levels. Conclusions: Results suggest that milk miRNAs could influence gene expression in intestinal epithelia of neonates under special conditions in vitro.