Abstract：
MicroRNAs (miRNAs) are important post-transcriptional factors involved in the regulation of gene expression and play crucial roles in biological processes related to milk fat metabolism. Our previous study revealed that miR-19a expression was significantly higher in the mammary epithelial cells of high-milk fat cows than in those of low-milk fat cows. However, the precise molecular mechanisms underlying these differences remain unclear. In this study, we found a high expression of miR-19a in the mammary tissues of dairy cows. The regulatory effects of miR-19a on bovine mammary epithelial cells (BMECs) were analyzed using cell counting kit-8 and 5-ethynyl-2\'-deoxyuridine assays, which demonstrated that miR-19a significantly inhibited BMEC proliferation. Transfection of the miR-19a mimic into BMECs significantly upregulated the expression of milk fat marker genes LPL, SCAP, and SREBP1, promoting triglyceride (TG) synthesis and lipid droplet formation, whereas the miR-19a inhibitor exhibited the opposite function. TargetScan and miRWalk predictions revealed that synaptotagmin 1 (SYT1) is a target gene of miR-19a. A dual luciferase reporter gene assay, RT-qPCR, and western blot analyses revealed that miR-19a directly targets the 3\'-untranslated region (UTR) of SYT1 and negatively regulates SYT1 expression. Functional validation revealed that overexpression of SYT1 in BMECs significantly downregulated the expression of LPL, SCAP, and SREBP1, and inhibited TG synthesis and lipid droplet formation. Conversely, the knockdown of SYT1 had the opposite effect. Altogether, miR-19a plays a crucial role in regulating the proliferation and differentiation of BMECs and regulates biological processes related to TG synthesis and lipid droplet formation by suppressing SYT1 expression. These findings provide a strong foundation for further research on the functional mechanisms underlying milk fat metabolism in dairy cows.
摘要：
微小RNA(microRNAs,miRNAs)是参与基因表达调控的重要转录后因子,在乳脂代谢相关的生物过程中发挥重要作用。我们之前的研究表明，miR-19a在高乳脂奶牛的乳腺上皮细胞中的表达明显高于低乳脂奶牛。然而,这些差异背后的确切分子机制仍不清楚.在这项研究中,我们发现miR-19a在奶牛乳腺组织中高表达。使用细胞计数试剂盒-8和5-乙炔基-2'-脱氧尿苷测定法分析miR-19a对奶牛乳腺上皮细胞（BMECs）的调节作用，这表明miR-19a显著抑制BMEC增殖。将miR-19a模拟物转染到BMECs中显著上调乳脂标记基因LPL的表达,SCAP,和SREBP1，促进甘油三酯（TG）合成和脂滴形成，而miR-19a抑制剂表现出相反的功能。TargetScan和miRWalk预测显示突触结合蛋白1(SYT1)是miR-19a的靶基因。双荧光素酶报告基因检测,RT-qPCR,和蛋白质印迹分析显示miR-19a直接靶向SYT1的3'-非翻译区(UTR)并负调控SYT1表达。功能验证表明，SYT1在BMECs中的过表达显著下调LPL的表达，SCAP,和SREBP1，并抑制TG合成和脂滴形成。相反，SYT1的敲低具有相反的效果。总之,miR-19a在调节BMECs的增殖和分化中起关键作用，并通过抑制SYT1表达来调节与TG合成和脂滴形成相关的生物学过程。这些发现为进一步研究奶牛乳脂代谢的功能机制奠定了坚实的基础。
