bta-miR-224 regulates milk fat metabolism by targeting FABP4 in bovine mammary epithelial cells

Abstract

Milk fat is produced and secreted by the mammary gland, which is mainly regulated by diet and gene-molecule network. Therefore, understanding the molecular mechanism of milk fat synthesis is of practical significance for improving milk quality. Fatty acid-binding protein 4 (FABP4) is a candidate messenger RNA (mRNA) closely linked to milk fat metabolism obtained from transcriptomic analysis of mammary epithelial cells of cows in the pre-existing high- and low-milk-fat groups, and its expression pattern and function are still unclear. The qRT-PCR results depicted that FABP4 was highly expressed in bovine mammary epithelial cells (BMECs) in breast tissues and the high milk fat group. Subsequently, the regulatory effects of FABP4 on BMECs were analyzed by CCK8, EdU, and flow cytometry, and the results demonstrated that FABP4 inhibited the proliferation and viability of BMECs and promoted their apoptosis. Next, the effect of FABP4 on milk lipid metabolism was explored. pEGFP-N1-FABP4 was transfected into BMECs, and FABP4 upregulated the expression levels of the milk lipid marker genes XDH, PPARG, and ACSS2, and promoted the formation of triglycerides (TGs), cholesterol, lipid droplets, and β-casein. Strong interactions between FABP4 and PPARG were identified using STRING prediction. Western blotting revealed that FABP4 interacted with PPARG to promote PPARG expression, while the opposite result was observed after interfering with FABP4. The gene regulation of microRNA (miRNA) is essential for fatty acid metabolism and synthesis. Predicted by website and combined with pre-miRNA transcriptome sequencing results, we hypothesized that FABP4 might be the target gene of bta-miR-224. The results of the dual-luciferase reporter gene and qRT-PCR revealed that bta-miR-224 negatively regulated FABP4 expression by targeting the 3′-UTR of FABP4. By exploring the function of bta-miR-224, we observed that bta-miR-224 mimics downregulated the expression of the milk fat marker genes AGPAT6, ACSS2, and XDH and inhibited TG synthesis and lipid droplet secretion. However, the bta-miR-224 inhibitor depicted the opposite results. In conclusion, FABP4 plays a crucial role in regulating BMEC proliferation and differentiation. Bta-miR-224 targeting FABP4 may promote biological processes such as TG synthesis and lipid droplet formation through PPARG, which lays a solid foundation for further analysis of the functional mechanism of milk lipid metabolism in dairy cows from a miRNA-mRNA perspective.