Screening And Functional Validation Of Differentially Expressed MiRNAs For Milk Fat Percentage In Holstein Cows

Milk fat is an important substance that constitutes milk and is a key reference indicator for milk quality evaluation.Milk fat metabolism in dairy cows is influenced by multiple factors,and its trait performance is closely related to genetic factors.Therefore,in this study,eight Holstein cows with extreme differences in milk fat percentage in Ningxia region were selected and their milk was collected to isolate mammary epithelial cells(BMECs)for transcriptome sequencing.Key miR-19a and miR-1343-3p,which regulate milk fat metabolism,were screened using bioinformatics analysis,their target genes were predicted and cellular functions were validated.The targeting regulatory mechanisms of miR-19a with SYT1 and miR-1343-3p with PPP1R16B were verified by dual luciferase reporter assay,differential quantitative analysis,miRNA mimic and inhibitor,mRNA overexpression and interference,cell proliferation,milk lipid index assay,and protein immunoblotting to further reveal their effects on milk lipid metabolism.The main findings are as follows:(1)Transcriptome sequencing results showed that a total of 34 differentially expressed miRNAs(16 up-regulated and 18 down-regulated)were identified in the comparison group of high milk fat and low milk fat,including 33 known miRNAs and 1 newly identified miRNA.Candidate target genes of differential miRNAs were significantly enriched for functional entries in intracellular signaling,single organism processes,kinase binding and phosphotransferase activity,and enriched for TNF signaling,MAPK signaling,Ras signaling,Rap1 signaling pathway and oxytocin signaling,which are pathways related to milk lipid metabolism.Real-time fluorescence quantitative PCR verified that the expression trends of miRNA and mRNA were consistent with the sequencing results.(2)The interaction network between differentially expressed miRNAs and differentially expressed mRNAs identified a total of 16 miRNA-mRNA interaction pairs,eight of which had negative regulatory relationships.The differential target mRNAs were enriched in several biological function entries such as protein kinase A binding,phosphatidylinositol phosphodiesterase activity,mRNA catabolism and positive regulation of insulin secretion.miRNA-mRNAs were correlated with milk fat rate,and miR-1343-3p was found to be significantly negatively correlated with milk fat rate,and miR-370 and miR-2285cb were significantly positively the correlation was found to be significant.Tissue expression profile analysis of miR-1343-3p showed the highest expression in breast tissue,followed by higher expression in uterine and ovarian tissues.(3)The protein interaction network revealed that the PPP1R16B has a regulatory relationship with different subunits of the same family of serine/threonine protein phosphatases and is involved in the regulation of prolactin,PI3K/AKT and insulin signaling pathways.Dual luciferase reporter assays demonstrated that PPP1R16B is a target gene of miR-1343-3p.CCK-8 and Edu cell proliferation assays showed that miR-1343-3p promotes proliferation of mammary epithelial cells and PPP1R16B inhibits cell proliferation.Oil red O staining,BODIPY staining and triglyceride assay demonstrated that miR-1343-3p inhibited triglyceride synthesis,which in turn inhibited milk lipid synthesis and lipid droplet accumulation.Overexpression of PPP1R16B gene promoted triglyceride synthesis and consequently lipid droplet accumulation in mammary epithelial cells.(4)Protein interaction network revealed that SYT1 interacts with genes such as secretion and transport related hormone proteins CHGB and VAMP1,thereby affecting lipid synthesis and metabolism.Tissue expression profiling analysis of miR-19a showed the highest expression in breast tissue,followed by ovary.Dual luciferase reporter assay demonstrated that SYT1 was the target gene of miR-19a.CCK-8 and Edu cell proliferation assays showed that miR-19a inhibited the proliferation of mammary epithelial cells and overexpression of SYT1 gene promoted cell proliferation.Oil red O staining,BODIPY staining,triglyceride assay and changes in expression of milk lipid marker genes demonstrated that miR-19a promoted triglyceride synthesis,which in turn promoted milk lipid synthesis and lipid droplet accumulation.Overexpression of SYT1 gene inhibited triglyceride synthesis and consequently lipid droplet accumulation in mammary epithelial cells.In summary,this study identified miR-19a-SYT1 and miR-1343-3p-PPP1R16B as key factors regulating mammary epithelial cell proliferation and lipid droplet deposition.The above results explored the functional mechanisms of milk lipid metabolism in Holstein cows in Ningxia region at miRNA and mRNA levels,and provided a theoretical basis for molecular breeding of selected high quality and high yielding cows.