miRNAs derived from milk small extracellular vesicles inhibit porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration, and high mortality in neonatal piglets. It has caused huge economic losses to animal husbandry worldwide. Current commercial PEDV vaccines do not provide enough protection against variant and evolved virus strains. No specific drugs are available to treat PEDV infection. The development of more effective therapeutic anti-PEDV agents is urgently needed. Our previous study suggested that porcine milk small extracellular vesicles (sEV) facilitate intestinal tract development and prevent lipopolysaccharide-induced intestinal injury. However, the effects of milk sEV during viral infection remain unclear. Our study found that porcine milk sEV, which was isolated and purified by differential ultracentrifugation, could inhibit PEDV replication in IPEC-J2 and Vero cells. Simultaneously, we constructed a PEDV infection model for piglet intestinal organoids and found that milk sEV also inhibited PEDV infection. Subsequently, in vivo experiments showed that milk sEV pre-feeding exerted robust protection of piglets from PEDV-induced diarrhea and mortality. Strikingly, we found that the miRNAs extracted from milk sEV inhibited PEDV infection. miRNA-seq, bioinformatics analysis, and experimental verification demonstrated that miR-let-7e and miR-27b, which were identified in milk sEV targeted PEDV N and host HMGB1, suppressed viral replication. Taken together, we revealed the biological function of milk sEV in resisting PEDV infection and proved its cargo miRNAs, miR-let-7e and miR-27b, possess antiviral functions. This study is the first description of the novel function of porcine milk sEV in regulating PEDV infection. It provides a better understanding of milk sEV resistance to coronavirus infection, warranting further studies to develop sEV as an attractive antiviral.

Porcine milk exosome miRNAs protect intestinal epithelial cells against deoxynivalenol-induced damage.

Porcine milk exosomes play an important role in mother-infant communication. Deoxynivalenol (DON) is a toxin which causes serious damage to the animal intestinal mucosa. Our previous study showed porcine milk exosomes facilitate mice intestine development, but the effects of these exosomes to antagonize DON toxicity is unclear. Our in vivo results showed that milk exosomes attenuated DON-induced damage on the mouse body weight and intestinal epithelium growth. In addition, these exosomes could reverse DON-induced inhibition on cell proliferation and tight junction proteins (TJs) formation and reduce DON-induced cell apoptosis. In vitro, exosomes up-regulated the expression of miR-181a, miR-30c, miR-365-5p and miR-769-3p in IPEC-J2 cells and then down-regulated the expression of their targeting genes in p53 pathway, ultimately attenuating DON-induced damage by promoting cell proliferation and TJs and by inhibiting cell apoptosis. In conclusion, porcine milk exosomes could protect the intestine against DON damage, and these protections may take place through the miRNAs in exosomes. These results indicated that the addition of miRNA-enriched exosomes to feed or food could be used as a novel preventative measure for necrotizing enterocolitis.

Plant MIR167e-5p Inhibits Enterocyte Proliferation by Targeting ß-Catenin.

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. It is worth exploring whether plant miRNAs play a cross-kingdom regulatory role in animals. Herein, we found that plant MIR167e-5p regulates the proliferation of enterocytes in vitro. A porcine jejunum epithelial cell line (IPEC-J2) and a human colon carcinoma cell line (Caco-2) were treated with 0, 10, 20, and 40 pmol of synthetic 2'-O-methylated plant MIR167e-5p, followed by a treatment with 20 pmol of MIR167e-5p for 0, 24, 48, and 72 h. The cells were counted, and IPEC-J2 cell viability was determined by the MTT and EdU assays at different time points. The results showed that MIR167e-5p significantly inhibited the proliferation of enterocytes in a dose- and time-dependent manner. Bioinformatics prediction and a luciferase reporter assay indicated that MIR167e-5p targets ß-catenin. In IPEC-J2 and Caco-2 cells, MIR167e-5p suppressed proliferation by downregulating ß-catenin mRNA and protein levels. MIR167e-5p relieved this inhibition. Similar results were achieved for the ß-catenin downstream target gene c-Myc and the proliferation-associated gene PCNA. This research demonstrates that plant MIR167e-5p can inhibit enterocyte proliferation by targeting the ß-catenin pathway. More importantly, plant miRNAs may be a new class of bioactive molecules for epigenetic regulation in humans and animals.

Porcine Milk Exosome MiRNAs Attenuate LPS-Induced Apoptosis through Inhibiting TLR4/NF-κB and p53 Pathways in Intestinal Epithelial Cells.

Lipopolysaccharide (LPS) is a bacterial endotoxin that induces intestine inflammation. Milk exosomes improve the intestine and immune system development of newborns. This study aims to establish the protective mechanisms of porcine milk exosomes on the attenuation of LPS-induced intestinal inflammation and apoptosis. In vivo, exosomes prevented LPS-induced intestine damage and inhibited (p < 0.05) LPS-induced inflammation. In vitro, exosomes inhibited (p < 0.05) LPS-induced intestinal epithelial cells apoptosis (23% ± 0.4% to 12% ± 0.2%). Porcine milk exosomes also decreased (p < 0.05) the LPS-induced TLR4/NF-κB signaling pathway activation. Furthermore, exosome miR-4334 and miR-219 reduced (p < 0.05) LPS-induced inflammation through the NF-κB pathway and miR-338 inhibited (p < 0.05) the LPS-induced apoptosis via the p53 pathway. Cotransfection with these three miRNAs more effectively prevented (p < 0.05) LPS-induced cell apoptosis than these miRNAs individual transfection. The apoptosis percentage in the group cotransfected with the three miRNAs (14% ± 0.4%) was lower (p < 0.05) than that in the NC miRNA group (28% ± 0.5%), and also lower than that in each individual miRNA group. In conclusion, porcine milk exosomes protect the intestine epithelial cells against LPS-induced injury by inhibiting cell inflammation and protecting against apoptosis through the action of exosome miRNAs. The presented results suggest that the physiological amounts of miRNAs-enriched exosomes addition to infant formula could be used as a novel preventative measure for necrotizing enterocolitis.

Plant MIR156 regulates intestinal growth in mammals by targeting the Wnt/ß-catenin pathway.

MicroRNAs (miRNAs) are important negative regulators of genes involved in physiological and pathological processes in plants and animals. Recent studies have shown that miRNAs might regulate gene expression among different species in a cross-kingdom manner. However, the specific roles of plant miRNAs in animals remain poorly understood and somewhat. Herein, we found that plant MIR156 regulates proliferation of intestinal cells both in vitro and in vivo. Continuous administration of a high plant miRNA diet or synthetic MIR156 elevated MIR156 levels and inhibited the Wnt/ß-catenin signaling pathway in mouse intestine. Bioinformatics predictions and luciferase reporter assays indicated that MIR156 targets Wnt10b. In vitro, MIR156 suppressed proliferation by downregulating the Wnt10b protein and upregulating ß-catenin phosphorylation in the porcine jejunum epithelial (IPEC-J2) cell line. Lithium chloride and an MIR156 inhibitor relieved this inhibition. This research is the first to demonstrate that plant MIR156 inhibits intestinal cell proliferation by targeting Wnt10b. More importantly, plant miRNAs may represent a new class of bioactive molecules that act as epigenetic regulators in humans and other animals.

Exploration of long noncoding RNA in bovine milk exosomes and their stability during digestion in vitro.

Previous studies have demonstrated that bovine milk contains mRNA and microRNA that are largely encapsulated in milk-derived exosomes. However, little information is available about long noncoding RNAs (lncRNA) in bovine milk. Increasing evidence suggests that lncRNA are of particular interest given their key role in gene expression and development. We performed a comprehensive analysis of lncRNA in bovine milk exosomes by RNA sequencing. We used a validated human in vitro digestion model to investigate the stability of lncRNA encapsulated in bovine milk exosomes during the digestion process. We identified 3,475 novel lncRNA and 6 annotated lncRNA. The lncRNA shared characteristics with those of other mammals in terms of length, exon number, and open reading frames. However, lncRNA showed higher expression than mRNAs. We selected 12 lncRNA of high-expression abundance and identified them by PCR. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that lncRNA regulate immune function, osteoblastogenesis, neurodevelopment, reproduction, cell proliferation, and cell-cell communication. We also investigated the 12 lncRNA using quantitative real-time PCR to reveal their expression profiles in milk exosomes during different stages of lactation (colostrum 2 d, 30 d, 150 d, and 270 d); their resulting expression levels in milk exosomes showed variations across the stages. A digestion experiment showed that bovine milk exosome lncRNA was resistant to in vitro digestion with different digestive juices, including saliva, gastric juice, pancreatic juice, and bile juice. Taken together, these results show for the first time that cow milk contains lncRNA, and that their abundance varied at different stages of lactation. As expected, bovine milk exosomal lncRNA were stable during in vitro digestion. These findings provide a basis for further understanding of the physiological role of milk lncRNA.

Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells.

Milk-derived exosomes were identified as a novel mechanism of mother-to-child transmission of regulatory molecules, but their functions in intestinal tissues of neonates are not well-studied. Here, we characterized potential roles of porcine milk-derived exosomes in the intestinal tract. In vitro, treatment with milk-derived exosomes (27 ± 3 ng and 55 ± 5 ng total RNA) significantly promoted IPEC-J2 cell proliferation by MTT, CCK8, EdU fluorescence and EdU flow cytometry assays. The qRT-PCR and Western blot analyses indicated milk-derived exosomes (0.27 ± 0.03 µg total RNA) significantly promoted expression of CDX2, IGF-1R and PCNA, and inhibited p53 gene expression involved in intestinal proliferation. Additionally, six detected miRNAs were significantly increased in IPEC-J2 cell, while FAS and SERPINE were significantly down-regulated relative to that in control. In vivo, treated groups (0.125 µg and 0.25 µg total RNA) significantly raised mice' villus height, crypt depth and ratio of villus length to crypt depth of intestinal tissues, significantly increased CDX2, PCNA and IGF-1R' expression and significantly inhibited p53' expression. Our study demonstrated that milk-derived exosomes can facilitate intestinal cell proliferation and intestinal tract development, thus giving a new insight for milk nutrition and newborn development and health.