CircEZH2 Regulates Milk Fat Metabolism through miR-378b Sponge Activity

SIMPLE SUMMARY: Heat stress has seriously threatened the performance and health of dairy cows and has become one of the most important factors restricting the development of the dairy industry. In our previous study, we found that heat stress markedly altered the expression patterns of circRNAs in dairy cow’s mammary gland tissue, and heat-induced circRNAs participated in the regulation of milk fat metabolism through competing endogenous RNA (ceRNA) networks. Therefore, we evaluated the roles of heat-induced circEZH2 in the regulation of milk fat metabolism in this study. In more detail, we found that circEZH2 affects the proliferation, apoptosis, and lipid metabolism of mammary gland epithelial cells, and successfully verified the targeting relationship of circEZH2-bta-miR378b-LPL and circEZH2-bta-miR378b-CD36. This experiment expands the basic data on the role of circRNA in milk fat regulation, and provides a theoretical basis for alleviating heat stress in dairy cows. ABSTRACT: 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.