Husseini, Z. | Search bioRxiv

bioRxiv2026

Investigating dietary microRNA stability and function using a transgenic milk model with unique microRNA sequences

Milk microRNAs are believed to play gene regulatory functions in the consumers cells. Milk from different species is enriched in microRNAs predicted to influence immunity, metabolism, and intestinal homeostasis. For milk microRNAs to regulate gene expression in the consumer, they must survive digestion and be present at sufficient levels to influence intestinal cells and potentially beyond-intestinal cells. Milk microRNAs are proposed to be protected from degradation through their association with milk extracellular vesicles (EVs), which might also deliver them to cells. Studies on milk microRNA oral transfer and tissue bioavailability are limited by interspecies sequence homology, making it difficult to distinguish endogenous from exogenous microRNAs. Here, we used a transgenic (TG) cow model expressing four unique microRNA sequences (AmiRs) in its milk to study their association with milk EVs, their resistance to in vitro digestion, and AmiR uptake and regulatory activity in vitro. We confirmed the presence of the four milk EV populations in raw wild-type (WT) and TG cow milk, similar to those previously reported in commercial (pasteurized) cow milk, and confirmed their association with AmiRs and classical milk microRNAs. AmiRs showed differential resistance to simulated adult digestion. In vitro uptake studies showed a modest gene regulatory effect of AmiRs in Caco-2 cells incubated with TG milk EVs. The intent of using this model was to perform in vitro analysis which could lay the groundwork for later in vivo bioavailability studies, taking advantage of the uniqueness of the AmiRs sequences and bypassing the limitation of microRNA sequence homology. HighlightsO_LINew milk EV populations identified previously in commercial bovine milk were also identified in raw milk, indicating that they are not merely the result of processing C_LIO_LIThe routinely discarded EVs (12K and 35K) seem to be preferentially enriched with microRNAs in raw cow milk as was previously shown for commercial cow milk C_LIO_LIAmiRs in transgenic milk resist differentially to simulated digestion C_LI

molecular biology↗

bioRxiv2026

Human milk contains a heterogeneous population of EVs and microRNAs that resist simulated digestion

Mothers milk is known for its crucial roles in infant health and development. Probably the most commonly studied effects of milk are those on an infants intestinal barrier, metabolism, and immunity. While these functions of milk were mostly attributed to its protein and fat content, recent evidence points to a potential role of milk microRNAs in these processes. MicroRNAs are small non-coding RNAs that can fine-tune gene expression at the post-transcriptional level. Human milk (HM) is rich in microRNAs, which are mainly found associated with milk extracellular vesicles (EVs). HM microRNAs are proposed to transfer from mother to infant via breastfeeding and execute gene regulatory functions in infant cells. For microRNAs to be able to act as "genetic programmers" rather than mere nutritional molecules, they should resist digestion in the infants gastrointestinal tract. Milk EVs are believed to protect microRNAs against degradation and facilitate their delivery to the cells. Here, we used two lots of pasteurized HM that were originally destined for human milk banks. We showed that HM contains different populations of EVs with different physicochemical properties, similar to those previously identified in commercial bovine milk. We also showed that these EVs, which are often discarded, contain the majority of HM microRNAs. Finally, we showed that three highly abundant milk microRNAs resisted differentially to infants simulated digestion conditions, with a relatively small number of microRNAs surviving a two-hour digestion. Milk microRNA copy numbers surviving digestion may be too low to influence gene expression in infant cells. HighlightsO_LIPasteurized HM contains heterogeneous populations of EVs. C_LIO_LIThese EVs associate with the majority of HM microRNAs. C_LIO_LIDifferent microRNAs show varying stability during infant digestion. C_LIO_LIThe copy number of milk miR-148a-3p surviving digestion might be too low to influence gene expression in infant cells. C_LI

molecular biology↗