In this study, we evaluated the roles of heat-induced circEZH2 in the regulation of milk fat metabolism. CircEZH2 overexpression increased HC11 cell proliferation and decreased apoptosis. These changes were accompanied by increased expression of proliferation marker proteins (PCNA, Cyclin D, and Cyclin E) and the anti-apoptotic protein Bcl2, while expression of the pro-apoptotic proteins Bax and cleaved-caspase was reduced. SiRNA-mediated silencing of EZH2 in HC11 cells had the opposite effects. CircEZH2 overexpression promoted the uptake of a fluorescent fatty acid (Bodipy) as well as expression of the fatty acid transport-related protein CD36, lipolysis-related protein LPL, and unsaturated fatty acid metabolism-related proteins FADS1 and SCD1. Dual luciferase reporter assays verified the targeting relationship of the two ceRNA networks, circEZH2-miR378b-LPL and circEZH2-miR378b-CD36. This information provides further clarification of the role of circRNAs in milk fat regulation in addition to a theoretical basis for alleviating the effects of heat stress on milk production by dairy cows.

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.