Bta-miR-2285f Regulates Milk Fat Synthesis And Validates Its Function In Holstein Dairy Cows

In this study,Holstein dairy cows raised in Ningxia were selected as the research object,and mammary epithelial cells(BMECs)extracted from the milk of 8 Holstein dairy cows with extremely different milk fat percentage were selected for transcriptional sequencing.Bioinformatics analysis was used to analyze the correlation of milk fat percentage,and the key miR-2285f regulating milk fat was screened out.The target gene binding sites were predicted,and 293T cells and mammary epithelial cells were used as miRNA and target gene models for functional verification in vitro.Tissue difference of miR-2285f Holstein cows was quantitatively analyzed by transfecting miR-2285f mimic and inhibitor.assay(dual luciferase reporter gene assay)and quantitative real-time PCR(quantitative real-time PCR,qRT-PCR),triglyceride(TAG)detection,oil red O detection of lipid droplets,Western Blot assay,EDU,CCK-8 and Flow cytometry,The molecular regulatory effects of miR-2285f and target gene MAP2K2 on milk fat metabolism of Holstein dairy cows were studied.The main research results are as follows:(1)As a bovine specific miRNA,bta-miR-2285f had a high expression level in mammary tissues of dairy cows,which was significantly different from other tissues(P<0.05),and 172 intersection target genes of miR-2285f were predicted to be significantly enriched in milk fat metabolism related pathways such as mTOR signaling pathway.(2)The wild-type vector and mutant vector of MAP2K2-3’utr were constructed and double luciferase reporting experiments were conducted to verify that MAP2K2 was one of the target genes of miR-2285f.According to qRT-PCR and Western Blot analysis,miR-2285f mainly regulates the expression of MAP2KA protein in BMECs at the translation level.(3)bta-miR-2285f can promote cell proliferation and slow cell apoptosis by regulating MAP2K2.(4)bta-miR-2285f can promote the level of triglyceride(TAG)and lipid droplet accumulation in mammary epithelial cells by targeting MAP2K2.(5)Overexpression of bta-miR-2285f significantly up-regulated the mRNA expression of milk fat synthesis related genes ELOVL6,SLC27A6,PPARG and FASN,while inhibition of bta-miR-2285f down-regulated the expression of milk fat marker genes.(6)bta-miR-2285f can regulate the protein level of milk fat marker gene PPARG by targeting MAP2K2.In conclusion,miR-2285f can target the expression of MAP2K2 gene,promote the proliferation of dairy mammary epithelial cells,inhibit cell apoptosis,and regulate the regulation of milk fat in dairy mammary epithelial cells.The results of this study revealed the function of miR-2285f in the regulation of milk fat ghosting of Holstein dairy cows at the cellular level,and provided theoretical and experimental basis for the analysis of the regulation network of milk fat synthesis of Holstein dairy cows and the molecular breeding of dairy cows.