Milk Exosomal miRNA Research Articles

Sheep (Ovis aries) Milk Exosomal miRNAs Attenuate Dextran Sulfate Sodium-Induced Colitis in Mice via TLR4 and TRAF-1 Inhibition.

Mammalian milk exosomal miRNAs play an important role in maintaining intestinal immune homeostasis and protecting epithelial barrier function, but the specific miRNAs and whether miRNA-mediated mechanisms are responsible for these benefits remain a matter of investigation. This study isolated sheep milk-derived exosomes (sheep MDEs), identifying the enriched miRNAs in sheep MDEs, oar-miR-148a, and oar-let-7b as key components targeting TLR4 and TRAF1, which was validated by a dual-luciferase reporter assay. In dextran sulfate sodium-induced colitis mice, administration of sheep MDEs alleviated colitis symptoms, reduced colonic inflammation, and systemic oxidative stress, as well as significantly increased colonic oar-miR-148a and oar-let-7b while reducing toll-like receptor 4 (TLR4) and TNF-receptor-associated factor 1 (TRAF1) level. Further characterization in TNF-α-challenged Caco-2 cells showed that overexpression of these miRNAs suppressed the TLR4/TRAF1-IκBα-p65 pathway and reduced IL-6 and IL-12 production. These findings indicate that sheep MDEs exert gastrointestinal anti-inflammatory effects through the miRNA-mediated modulation of TLR4 and TRAF1, highlighting their potential in managing colitis.

Risk of Fat Mass- and Obesity-Associated Gene-Dependent Obesogenic Programming by Formula Feeding Compared to Breastfeeding.

It is the purpose of this review to compare differences in postnatal epigenetic programming at the level of DNA and RNA methylation and later obesity risk between infants receiving artificial formula feeding (FF) in contrast to natural breastfeeding (BF). FF bears the risk of aberrant epigenetic programming at the level of DNA methylation and enhances the expression of the RNA demethylase fat mass- and obesity-associated gene (FTO), pointing to further deviations in the RNA methylome. Based on a literature search through Web of Science, Google Scholar, and PubMed databases concerning the dietary and epigenetic factors influencing FTO gene and FTO protein expression and FTO activity, FTO's impact on postnatal adipogenic programming was investigated. Accumulated translational evidence underscores that total protein intake as well as tryptophan, kynurenine, branched-chain amino acids, milk exosomal miRNAs, NADP, and NADPH are crucial regulators modifying FTO gene expression and FTO activity. Increased FTO-mTORC1-S6K1 signaling may epigenetically suppress the WNT/β-catenin pathway, enhancing adipocyte precursor cell proliferation and adipogenesis. Formula-induced FTO-dependent alterations of the N6-methyladenosine (m6A) RNA methylome may represent novel unfavorable molecular events in the postnatal development of adipogenesis and obesity, necessitating further investigations. BF provides physiological epigenetic DNA and RNA regulation, a compelling reason to rely on BF.

Human Breast Milk Exosomal miRNAs are Influenced by Premature Delivery and Affect Neurodevelopment.

This study investigates the exosomal microRNA (miRNA) profiles of term and preterm breast milk, including the most abundant and differentially expressed (DE) miRNAs, and their impact on neurodevelopment in infants. Mature milk is collected from the mothers of term and preterm infants. Using high-throughput sequencing and subsequent data analysis, exosomal miRNA profiles of term and preterm human breast milk (HBM) are acquired and it is found that the let-7 and miR-148 families are the most abundant miRNAs. Additionally, 23 upregulated and 15 downregulated miRNAs are identified. MiR-3168 is the most upregulated miRNA in preterm HBM exosome, exhibiting targeting activity toward multiple genes involved in the SMAD and MAPK signaling pathways and playing a crucial role in early neurodevelopment. Additionally, the effects of miR-3168 on neurodevelopment is confirmed and it is determined that it is an essential factor in the differentiation of neural stem cells (NSCs). This study demonstrates that miRNA expression in breast milk exosomes can be influenced by preterm delivery, thereby potentially impacting neurodevelopment in preterm infants.

Breed-Related Differential microRNA Expression and Analysis of Colostrum and Mature Milk Exosomes in Bamei and Landrace Pigs.

Breast milk, an indispensable source of immunological and nutrient components, is essential for the growth and development of newborn mammals. MicroRNAs (miRNAs) are present in various tissues and body fluids and are selectively packaged inside exosomes, a type of membrane vesicle. Milk exosomes have potential regulatory effects on the growth, development, and immunity of newborn piglets. To explore the differences in milk exosomes related to the breed and milk type, we isolated exosomes from colostrum and mature milk from domestic Bamei pigs and foreign Landrace pigs by using density gradient centrifugation and then characterized them by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Furthermore, the profiles and functions of miRNAs in the two types of pig milk exosomes were investigated using miRNA-seq and bioinformatics analysis. We identified a total of 1081 known and 2311 novel miRNAs in pig milk exosomes from Bamei and Landrace pigs. These differentially expressed miRNAs (DE-miRNAs) are closely associated with processes such as cell signaling, cell physiology, and immune system development. Functional enrichment analysis showed that DE-miRNA target genes were significantly enriched in endocytosis, the T cell receptor signaling pathway, and the Th17 cell differentiation signaling pathway. The exosomal miRNAs in both the colostrum and mature milk of the two pig species showed significant differences. Based on related signaling pathways, we found that the colostrum of local pig breeds contained more immune-system-development-related miRNAs. This study provides new insights into the possible function of milk exosomal miRNAs in the development of the piglet immune system.

Milk exosomal microRNA profiling identified miR-375 and miR-199-5p for regulation of immune response during subclinical mastitis of crossbred cattle.

The dairy industry has experienced significant economic losses as a result of mastitis, an inflammatory disease of cows, including both subclinical and clinical cases. Milk exosome microRNAs have gained attention due to their stable and selective wrapping nature, offering potential for the prognosis and diagnosis of bovine mastitis, the most common pathological condition of the mammary gland. In the present investigation, the microRNA profile of milk exosomes was explored using high-throughput small RNA sequencing data in sub-clinical mastitic and healthy crossbred Vrindavani cattle. In both groups, 349 microRNAs were identified, with 238 (68.19%) microRNAs co-expressed; however, 35 and 76 distinct microRNAs were found in subclinical mastitic and healthy cattle, respectively. Differential expression analysis revealed 11 microRNAs upregulated, and 18 microRNAs were downregulated in sub-clinical mastitic cattle. The functional annotation of the target genes of differentially expressed known and novel microRNAs including bta-miR-375, bta-miR-199-5p and bta-miR-12030 reveals their involvement in the regulation of immune response and inflammatory mechanisms and could be involved in development of mastitis. The analysis of milk exosomal miRNAs cargos hold great promise as an approach to study the underlying molecular mechanisms associated with mastitis in high milk producing dairy cattle. Concurrently, the significantly downregulated miR-375 may upregulate key target genes, including CTLA4, IHH, IRF1, and IL7R. These genes are negative regulators of immune response pathways, which could be associated with impaired inflammatory mechanisms in mammary cells. According to the findings, bta-miR-375 could be a promising biomarker for the development of mastitis in dairy cattle.

Influence of Estrus on Dairy Cow Milk Exosomal miRNAs and Their Role in Hormone Secretion by Granulosa Cells.

Estrus is crucial for cow fertility in modern dairy farms, but almost 50% of cows do not show the behavioral signs of estrus due to silent estrus and lack of suitable and high-accuracy methods to detect estrus. MiRNA and exosomes play essential roles in reproductive function and may be developed as novel biomarkers in estrus detection. Thus, we analyzed the miRNA expression patterns in milk exosomes during estrus and the effect of milk exosomes on hormone secretion in cultured bovine granulosa cells in vitro. We found that the number of exosomes and the exosome protein concentration in estrous cow milk were significantly lower than in non-estrous cow milk. Moreover, 133 differentially expressed exosomal miRNAs were identified in estrous cow milk vs. non-estrous cow milk. Functional enrichment analyses indicated that exosomal miRNAs were involved in reproduction and hormone-synthesis-related pathways, such as cholesterol metabolism, FoxO signaling pathway, Hippo signaling pathway, mTOR signaling pathway, steroid hormone biosynthesis, Wnt signaling pathway and GnRH signaling pathway. Consistent with the enrichment signaling pathways, exosomes derived from estrous and non-estrous cow milk both could promote the secretion of estradiol and progesterone in cultured bovine granulosa cells. Furthermore, genes related to hormonal synthesis (CYP19A1, CYP11A1, HSD3B1 and RUNX2) were up-regulated after exosome treatment, while exosomes inhibited the expression of StAR. Moreover, estrous and non-estrous cow-milk-derived exosomes both could increase the expression of bcl2 and decrease the expression of p53, and did not influence the expression of caspase-3. To our knowledge, this is the first study to investigate exosomal miRNA expression patterns during dairy cow estrus and the role of exosomes in hormone secretion by bovine granulosa cells. Our findings provide a theoretical basis for further investigating milk-derived exosomes and exosomal miRNA effects on ovary function and reproduction. Moreover, bovine milk exosomes may have effects on the ovaries of human consumers of pasteurized cow milk. These differential miRNAs might provide candidate biomarkers for the diagnosis of dairy cow estrus and will assist in developing new therapeutic targets for cow infertility.

Milk exosomes elicit a potent anti-viral activity against dengue virus

BackgroundExosomes are nano-sized vesicles secreted by various cells into the intra and extracellular space and hence is an integral part of biological fluids including milk. In the last few decades, many research groups have proved the potential of milk exosomes as a sustainable, economical and non-immunogenic drug delivery and therapeutic agent against different pathological conditions. However, its anti-viral properties still remain to be unearthed.MethodsHere, we have been able to isolate, purify and characterize the milk derived exosomes from Cow (CME) and Goat (GME) and further studied its antiviral properties against Dengue virus (DENV), Newcastle Disease Virus strain Komarov (NDV-K) and Human Immunodeficiency Virus (HIV-1) using an in-vitro infection system.ResultsTEM, NTA and DLS analysis validated the appropriate size of the isolated cow and goat milk exosomes (30–150 nm). Real-time PCR and immunoblotting results confirmed the presence of several milk exosomal miRNAs and protein markers. Our findings suggest that GME significantly decreased the infectivity of DENV. In addition, we confirmed that GME significantly reduces DENV replication and reduced the secretion of mature virions. Furthermore, heat inactivation of GME did not show any inhibition on DENV infection, replication, and secretion of mature virions. RNase treatment of GME abrogates the anti-viral properties indicating direct role of exosomes in DENV inhibition. In addition GME inhibited the infectivity of NDV-K, but not HIV-1, suggesting that the GME mediated antiviral activity might be virus specific.ConclusionThis study demonstrates the anti-viral properties of milk exosomes and opens new avenues for the development of exosome-based therapies to treat viral diseases.Graphical

Comparison of porcine milk microRNA expression in milk exosomes versus whole swine milk and prediction of target genes.

Milk exosomal microRNAs (miRNAs) are important for postnatal growth and immune system maturation in newborn mammals. The functional hypothesis of milk exosomal miRNAs and their potential bioavailability in milk to newborn mammals were investigated. Briefly, 37 exosomal miRNAs were upregulated compared to miRNAs found outside the exosomes. Among these miRNAs, ssc-miR-193a-3p expression was upregulated 1467.35 times, while ssc-miR-423-5p, ssc-miR-551a, ssc-miR-138, ssc-miR-1 and ssc-miR-124a were highly concentrated and upregulated 13.58–30.06 times. Moreover, these miRNAs appeared to be relevant for cell development and basic physiological processes of the immune system. Following the analysis of target gene prediction and related signalling pathways, 9262 target genes were mainly concentrated in three signalling pathways: metabolic pathways, pathways in cancer, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathways. Among 9262 target genes, more than 20 miRNAs were enriched in exosomes, such as methyl CpG binding protein 2 (MECP2) and glycogen synthase 1 (GYS1). After determining the miRNA localization-, distribution- and function-related metabolism, we found that these exosomes were specifically concentrated miRNA target genes and they were interrelated with cell development and basic cell functions, such as metabolism and immunity. It is speculated that miRNAs in milk can influence offspring via milk exosomes.

Identification and bioinformatics analysis of differentially expressed milk exosomal microRNAs in milk exosomes of heat-stressed Holstein cows.

In summer, heat stress is one of the primary reasons for the compromised health and low milk productivity of dairy cows. Hyperthermia affects milk synthesis and secretion in the mammary glands of dairy cows. As molecules for intercellular communication, milk-derived exosomes carry genetic material, proteins, and lipids, playing a crucial role in mammary tissue growth and milk synthesis in dairy cows. The aim of this study was to explore the milk exosomal miRNA profile of heat-stressed and normal Holstein cows. We isolated and identified milk exosomes to screening for differentially expressed miRNAs using small RNA sequencing. Then, TargetScan and miRanda algorithms were used to predict the putative targets of the differentially expressed miRNAs, whereas GO and KEGG pathway enrichment analyses were performed for the differentially expressed miRNA-target genes. Our results showed that 215 miRNAs were significantly differentially expressed in heat-stressed milk exosomes, of which one was upregulated and 214 were significantly downregulated. GO and KEGG enrichment analyses indicated that differentially expressed miRNAs might play a role in apoptosis, autophagy, and the p38 MAPK pathway. qRT-PCR assay verified that the expression of miRNAs was consistent with the sequencing results, warranting further verification of their specific targets of action. In conclusion, changes in the miRNA expression profile of milk exosomes indicated the role of exosomal miRNAs in regulating heat stress resistance and apoptosis in dairy cows. Our results suggested that milk-derived exosomal miRNAs could increase mammary gland resistance to heat stress, thereby enhancing milk synthesis in dairy cows.

Yak milk–derived exosomal microRNAs regulate intestinal epithelial cells on proliferation in hypoxic environment

Intestinal epithelial cells (IEC) act as an important intestinal barrier whose function can be impaired upon induction by hypoxia. Although intestinal barrier injuries are preventable by milk-derived exosomal microRNAs (miRNAs), the underlying mechanism remains poorly understood. This study aimed to characterize the effect of yak and cow milk-derived exosomal miRNA on the barrier function of IEC-6 under hypoxic conditions, and explore the mechanism of yak milk exosomal miRNA to relieve the hypoxia stress. First, by Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) sequencing, the miRNA expression was systematically screened, and differential expression of 130 miRNAs was identified with 51 being upregulated and 79 downregulated in yak and cow milk-derived exosomes. Furthermore, the top 20 miRNAs that had a relatively consistent high expression in yak milk exosome were identified, and bta-miR-34a was found to be an effective regulator for alleviating hypoxic injury of IEC-6. In vitro assay of the role of bta-miR-34a on survival of IEC-6 in hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) confirmed its effectiveness to significantly increase IEC-6 survival up to 13% for 12 h, and up to 9.5% for 24 h. Investigation on the regulatory relationship between bta-miRNA-34a and the hypoxia-inducible factor/apoptosis signaling pathway provided insights into the possible mechanisms by which bta-miR-34a activated the hypoxia-inducible factor and apoptosis signaling pathway, thus promoting IEC-6 survival. The results of this study suggest an important relationship between miRNA expression and intestine barrier integrity, which facilitated further understanding of the physiological function of yak and cow milk exosomal miRNAs, as well as mechanisms of hypoxia-driven epithelial homeostasis.

Bovine Milk Exosome and miRNA-2478 Enhances Insulin Sensitivity by Upregulating Insulin Receptor and Promotes Glycogen Synthesis in HepG2 Cells

Background: Milk and milk products are a sort of mandatory component of diet world-wide. Milk exosome and its bioactive content especially miRNA has been linked with several metabolic regulatory pathways and diseases. However, there is lack of convincing experimental validation. Recently, we have profiled cow milk exosomal miRNAs and in silico analysis identified insulin receptor (INSR) as potential target of miRNA-2478 which one of the candidate miRNA is encapsulated in cow milk exosomes. Down-regulation of insulin receptor (INSR) in peripheral tissues including liver results in the development of pathogenesis of Type II diabetes. However, role of miRNA-2478 in regulation of insulin signaling has not been studied so far. The main aim of the study was to decipher the role of milk exosomal miR-2478 on its target insulin receptor in HepG2 cells. Methods: The effect of miR-2478 mimic on INSR mRNA and protein expression, glycogen content and enrichment of RNA polymerase II at insulin gene promoter in HepG2 cells was analysed. Findings: Results of the current study showed that there was increased expression of insulin receptor gene at both mRNA and protein level with increased abundance of bta-miR-2478 in HepG2 cells. Similarly, glycogen content was also found to be increased in bta-miR-2478 transfected cells. Results of ChIP assay also showed enrichment of RNA polymerase on insulin receptor promoter in miR-2478 transfected cells as compared to control. Interpretation: In conclusion, bovine milk exosomes and miR-2478 enhanced insulin sensitivity through increased expression of insulin receptor and glycogen synthesis in HepG2 cells. Funding Statement: None. Declaration of Interests: All authors declare they have no conflict of interest. Ethics Approval Statement: Cow (Sahiwal) milk was collected from the healthy and early lactating animals of ICAR-National Dairy Research Institute, Karnal as per the Institute Animal Ethics Guidelines.

Milk exosome-derived miRNAs from water buffalo are implicated in immune response and metabolism process

BackgroundBuffalo milk is rich in various nutritional components and bioactive substances that provide more essential health benefits to human body. Recently, exosome identified in the breast milk has been reported as a neotype nutrient and can mediate intercellular communication with exosomal miRNAs. In the present study, we therefore hypothesized that exosome-derived miRNAs from buffalo milk would play the potential physiological importance of consumption of buffalo milk.ResultsWe isolated exosomes from buffalo and cow milk samples that were obtained at mid-lactation period, and the exosomal miRNA profiles were then generated using miRNA-seq. In addition, miRNAomes of pig, human and panda milk exosomes were downloaded from GEO database. Finally, a total of 27 milk exosomal miRNA profiles that included 4 buffalo, 4 cow, 8 pig, 4 human and 7 panda were analyzed using the miRDeep2 program. A total of 558 unique miRNA candidates existed across all species, and the top 10 highly expressed miRNA were evolutionarily conserved across multiple species. Functional analysis revealed that these milk enriched miRNAs targeted 400 putative sites to modulate disease resistance, immune responsiveness and basic metabolism events. In addition, a total of 32 miRNAs in buffalo milk were significantly up-regulated compared with non-buffalo milks, while 16 were significantly down-regulated. Of interest, functional analysis showed that up-regulated miRNAs were mainly related to host metabolism processes, while the predicted functions of down-regulated miRNAs were enriched in immune response.ConclusionIn this study, we explored the exosomal miRNAome differences between milks of different animals, expanding the theoretical basis for potential applications of the miRNA-containing vesicles.

Milk exosomal miRNAs: potential drivers of AMPK-to-mTORC1 switching in \u03b2-cell de-differentiation of type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) steadily increases in prevalence since the 1950’s, the period of widespread distribution of refrigerated pasteurized cow’s milk. Whereas breastfeeding protects against the development of T2DM in later life, accumulating epidemiological evidence underlines the role of cow’s milk consumption in T2DM. Recent studies in rodent models demonstrate that during the breastfeeding period pancreatic β-cells are metabolically immature and preferentially proliferate by activation of mechanistic target of rapamycin complex 1 (mTORC1) and suppression of AMP-activated protein kinase (AMPK). Weaning determines a metabolic switch of β-cells from a proliferating, immature phenotype with low insulin secretion to a differentiated mature phenotype with glucose-stimulated insulin secretion, less proliferation, reduced mTORC1- but increased AMPK activity. Translational evidence presented in this perspective implies for the first time that termination of milk miRNA transfer is the driver of this metabolic switch. miRNA-148a is a key inhibitor of AMPK and phosphatase and tensin homolog, crucial suppressors of mTORC1. β-Cells of diabetic patients return to the postnatal phenotype with high mTORC1 and low AMPK activity, explained by continuous transfer of bovine milk miRNAs to the human milk consumer. Bovine milk miRNA-148a apparently promotes β-cell de-differentiation to the immature mTORC1-high/AMPK-low phenotype with functional impairments in insulin secretion, increased mTORC1-driven endoplasmic reticulum stress, reduced autophagy and early β-cell apoptosis. In contrast to pasteurized cow’s milk, milk’s miRNAs are inactivated by bacterial fermentation, boiling and ultra-heat treatment and are missing in current infant formula. Persistent milk miRNA signaling adds a new perspective to the pathogenesis of T2DM and explains the protective role of breastfeeding but the diabetogenic effect of continued milk miRNA signaling by persistent consumption of pasteurized cow’s milk.

Oral Delivery of miRNA With Lipidic Aminoglycoside Derivatives in the Breastfed Rat.

ContextSpecific targeting of endogenous miRNAs which are involved in epigenetics, may help understanding homeostasis with therapeutic benefits. We use new biologically inspired vehicles consisting of lipoaminoglycosides to deliver in vivo mir-320-3p, a known human breast milk exosomal miRNA, or its antagomiR.Materials and MethodsFour lipoaminoglycosides were screened for cytotoxicity and their biophysical properties. 1-h breast-restricted rats received single-oral treatment of either the lipoaminoglycoside Dioleyl-Succinyl Paromomycin (DOSP) complexed with miRNA or antagomiR, or of control medium at the light on (ZeitGeber Time: ZT-0H) or off (ZT-12H). Glycemia, triglycerides, cholesterol, free-fatty acid were assayed at 0, 4, 8, and 12 h post-treatment. In the stomach, small intestine, liver, plasma, adipose tissue, plexus choroid, and cortex, relevant miRNA with precursors and mRNA (polr3d, hspb6, c-myc, stat1, clock, bmal1, per1, npas2, sirt1-6, and cyclinD1) were quantified by q-PCR. Expression of POLR3D and HSPB6 proteins were analyzed in stomach and liver by Western blot. Immunoprecipitations with anti-AGO1 and 2 were performed on nuclear and cytoplasmic fractions of gastric cells along with detection of miRNA-320-3p in nucleoli. Chromatin ImmunoPrecipitation with anti-Trimethyl-histone-3-Lys-4 and Lys-27 detecting the polr3d promoter and miR-320-3p, were performed for all groups.ResultsSelected DOSP (diameter: 80–200 nm) did not alter gastric extracellular vesicle secretion a few hours after intake. The miR-320-3p was mainly found in gastric or small intestinal cells, reaching the blood and liver in low amount. We have found significant up-regulation of polr3d mRNA (ANOVA, p < 0.0001) at ZT-20H for the miR-320-3p-supplemented group and a higher expression of POLR3D for antagomiR group (ANOVA, p < 0.05). We had a low accumulation of miR-320-3p at ZT-20H in nucleoli, without stat1 evolution. Delivering a high amount of miRNA or antagomiR disrupts RNA-Induced Silencing Complexes in cytoplasm triggering some transfer of extracellular molecules into nuclei with alteration of immune complexes on the polr3d promoter (with a higher amount found in the K4 histone-3-me3 immune complexes at ZT-20H).ConclusionExtracellular miRNAs embedded in DOSP have a rapid impact on RNAi and on nuclear chromatin complexes depending on the daily rhythm. An integrative view of the impact of extracellular miRNA on physiology will improve assaying epigenetic manipulations following nutritional stress.

Genome-wide microRNA profiling of bovine milk-derived exosomes infected with Staphylococcus aureus.

Bovine milk is rich in exosomes, which contain abundant miRNAs and play important roles in the regulation of neonatal growth and development of adaptive immunity. Here, we analyzed miRNA expression profiles of bovine milk exosomes from three healthy and three mastitic cows, and then six miRNA libraries were constructed. Interestingly, we detected no scRNAs and few snRNAs in milk exosomes; this result indicated a potential preference for RNA packaging in milk exosomes. A total of 492 known and 980 novel exosomal miRNAs were detected, and the 10 most expressed miRNAs in the six samples accounted for 80-90% of total miRNA-associated reads. Expression analyses identified 18 miRNAs with significantly different expression between healthy and infected animals; the predicted target genes of differentially expressed miRNAs were significantly enriched in immune system process, response to stimulus, growth, etc. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including inflammatory, immune, and cancer pathways. Our survey provided comprehensive information about milk exosomes and exosomal miRNAs involved in mastitis. Moreover, the differentially expressed miRNAs, especially miR-223 and miR-142-5p, could be considered as potential candidates for mastitis.

Milk miRNAs encapsulated in exosomes are stable to human digestion and permeable to intestinal barrier in vitro

Milk exosomal miRNAs are bioactive nucleic acids. However, their stability against the digestive processes and bioavailability to the cellular functions is still controversial. In the present study, a novel in vitro digestion model has been used to investigate the stability of miRNAs encapsulated in the cow milk exosomes against the digestive processes, along with their uptake and trans-epithelial transport by intestinal epithelial cells. Milk exosomal miRNAs were found resistant to in vitro digestion with different digestive juice including saliva, gastric, bile and pancreatic juice. Milk exosomes were internalized by in vitro cultured intestinal epithelial cells in linear pattern until 2h. Moreover, trans-epithelial transport of bovine milk exosomal miRNAs across the Caco-2 monolayer indicated their potential to cross the intestinal barrier. Overall, the results demonstrated that cow milk exosomes protect miRNAs against harsh digestive processes and they are able to cross the intestinal barrier to reach blood circulation for cellular function.

Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cellculture.

BackgroundBovine milk contains not only a variety of nutritional ingredients but also microRNAs (miRNAs) that are thought to be secreted by the bovine mammary epithelial cells (BMECs). The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones.ResultsAccording to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin (DIP) to induce a lactogenic differentiation. Quantitative polymerase chain reaction (qPCR) results showed that the expressions of miR-21–5p (P = 0.005), miR-26a (P = 0.016), and miR-320a (P = 0.011) were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a (P = 0.017) in the cell culture medium were lower in the DIP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture (P = 0.018). The medium-to-cell expression ratios of miR-103 (P = 0.025), miR-148a (P < 0.001), and miR-223 (P = 0.013) were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development.ConclusionThe results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.