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 β-catenin. In IPEC-J2 and Caco-2 cells, MIR167e-5p suppressed proliferation by downregulating β-catenin mRNA and protein levels. MIR167e-5p relieved this inhibition. Similar results were achieved for the β-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 β-catenin pathway. More importantly, plant miRNAs may be a new class of bioactive molecules for epigenetic regulation in humans and animals.

Summer temperatures are generally high in Southern China, and cows are likely to suffer a heat stress reaction. Heat stress will have a negative impact on the performance of dairy cows; however, the mechanism by which high temperature affects lactation is not clear. CircRNA is a type of non-coding RNA discovered in recent years, which performs a crucial function in many biological activities. However, the effects of circRNA on lactation function of dairy cows under heat stress is unknown. The present study aimed to explore the expression levels of circRNA in the mammary gland tissue of cows under heat stress. Firstly, we collected blood and milk samples of summer and winter cows and evaluated lactation performance using serum indicators, milk production, and milk composition. Incorporating the calculation of the temperature and humidity index, we conformed the heat stress status of cows in summer. Heat stress increased the concentration of HSP70 and decreased the concentration of SOD and PRL. Heat stress not only reduced milk yield but also affected milk quality, with milk lactose and milk protein decreasing with increased temperature. The analysis of the fatty acid composition in summer milk found significantly reduced concentrations of unsaturated fatty acids, especially long-chain unsaturated fatty acids. Sequencing of the cow’s mammary gland transcriptome revealed that compared to the appropriate temperature (ST) group, the heat stress (HS) group had a total of 2204 upregulated and 3501 downregulated transcripts. GO enrichment and KEGG pathway analysis showed that these genes were mainly related to milk fat metabolism. In addition, 19 upregulated and 19 downregulated circRNA candidates were found in response to heat stress. We used Pearson’s test to establish the correlation of circRNA-mRNA and identified four pairs of circRNA-miRNA networks between four circRNAs, six miRNAs, and the CD36 gene. In this study, we revealed the possible role of circRNAs in lactation of dairy cows and identified that circRNA-miRNA-mRNA networks might exist in the cow’s mammary glands, providing valuable experience for dairy lactation and milk quality.

Moringa oleifera is a tropical plant with high nutritional and medicinal value. Recent studies have reported its remarkable effects in inflammatory, antioxidative, and anti‐diabetes modulations, but there was no significant report on its role in animal breeding. In this study, we investigated the effects of dietary Moringa oleifera leaf (MOL) on reproductive performances in mice. We studied the reproductive performance of mice for six consecutive gestations. Mice fed with 4% MOL diet showed improved litter size, litter birth weight, and litter survivals until weaning age compared to control mice fed with normal diet (p < 0.05). Mice fed with MOL diet did not change weight and organ coefficients. Serum malondialdehyde (MDA) concentrations in both male and female mice were significantly decreased by dietary MOL (p < 0.05), but glutathione peroxidase (GSH‐PX) and superoxide dismutase (SOD) were unchanged. For male, dietary MOL significantly reduced sperm abnormality rate (p < 0.05) and Bcl2‐associated X protein (Bax) expression in testis (p < 0.05), but did not affect serum testosterone and the expression levels of androgen receptor (AR), phosphoglycerate kinase 2 (Pgk2), protamine2 (Prm2), and B cell leukemia/lymphoma 2 (Bcl2) in testis. For female, dietary MOL did not change serum estradiol and the expressions of estrogen receptor beta (ERβ), Bcl2, Bax, and vascular endothelial growth factor receptor (VEGFR) in ovary. In summary, MOL increased litter size and antioxidant ability, reduced the rate of sperm abnormality and the expression of Bax. Therefore, MOL may serve as a functional feed addictive for improving animal reproductive performance.

Skeletal muscle-fat interaction is essential for maintaining organismal energy homeostasis and managing obesity by secreting cytokines and exosomes, but the role of the latter as a new mediator in inter-tissue communication remains unclear. Recently, we discovered that miR-146a-5p was mainly enriched in skeletal muscle-derived exosomes (SKM-Exos), 50-fold higher than in fat exosomes. Here, we investigated the role of skeletal muscle-derived exosomes regulating lipid metabolism in adipose tissue by delivering miR-146a-5p. The results showed that skeletal muscle cell-derived exosomes significantly inhibited the differentiation of preadipocytes and their adipogenesis. When the skeletal muscle-derived exosomes co-treated adipocytes with miR-146a-5p inhibitor, this inhibition was reversed. Additionally, skeletal muscle-specific knockout miR-146a-5p (mKO) mice significantly increased body weight gain and decreased oxidative metabolism. On the other hand, the internalization of this miRNA into the mKO mice by injecting skeletal muscle-derived exosomes from the Flox mice (Flox-Exos) resulted in significant phenotypic reversion, including down-regulation of genes and proteins involved in adipogenesis. Mechanistically, miR-146a-5p has also been demonstrated to function as a negative regulator of peroxisome proliferator-activated receptor γ (PPARγ) signaling by directly targeting growth and differentiation factor 5 (GDF5) gene to mediate adipogenesis and fatty acid absorption. Taken together, these data provide new insights into the role of miR-146a-5p as a novel myokine involved in the regulation of adipogenesis and obesity via mediating the skeletal muscle-fat signaling axis, which may serve as a target for the development of therapies against metabolic diseases, such as obesity.

Secretory immunoglobulin A (SIgA) plays an important role in gut acquired immunity and mucosal homeostasis. Breast milk is the irreplaceable nutritional source for mammals after birth. Current studies have shown the potential functional role of milk-derived small extracellular vesicles (sEVs) and their RNAs cargo in intestinal health and immune regulation. However, there is a lack of studies to demonstrate how milk-derived sEVs affect intestinal immunity in recipient. In this study, through in vivo experiments, we found that porcine milk small extracellular vesicles (PM-sEVs) promoted intestinal SIgA levels, and increased the expression levels of polymeric immunoglobulin receptor (pIgR) both in mice and piglet. We examined the mechanism of how PM-sEVs increased the expression level of pIgR in vitro by using a porcine small intestine epithelial cell line (IPEC-J2). Through bioinformatics analysis, dual-luciferase reporter assays, and overexpression or knockdown of the corresponding non-coding RNAs, we identified circ-XPO4 in PM-sEVs as a crucial circRNA, which leads to the expression of pIgR via the suppression of miR-221-5p in intestinal cells. Importantly, we also observed that oral administration of PM-sEVs increased the level of circ-XPO4 and decreased the level of miR-221-5p in small intestine of piglets, indicating that circRNAs in milk-derived sEVs act as sponge for miRNAs in recipients. This study, for the first time, reveals that PM-sEVs have a capacity to stimulate intestinal SIgA production by delivering circRNAs to receptors and sponging the recipient’s original miRNAs, and also provides valuable data for insight into the role and mechanism of animal milk sEVs in intestinal immunity.