Ningxia Key Laboratory of Ruminant Molecular and Cellular Breeding, School of Agriculture, Ningxia University, Yinchuan, China.
Yinchuan Animal Husbandry Technology Promotion Service Center, Yinchuan, China.
Reprod Domest Anim. 2024 May;59(5):e14567. doi: 10.1111/rda.14567.
In this study, Holstein dairy cows raised in Ningxia were selected as the research object. Mammary epithelial cells (BMECs) were extracted from the milk of eight Holstein cows with significantly different milk fat expression rates and transcribed for sequencing. Bioinformatics analysis was used to analyse the correlation of fat milk percentage, and the critical 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. The tissue difference of miR-2285f Holstein cows was quantitatively analysed 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 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 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 MAP2K2 protein in BMECs at the translation level. Bta-miR-2285f can promote cell proliferation and slow cell apoptosis by regulating MAP2K2. Bta-miR-2285f can promote triglyceride (TAG) and lipid droplet accumulation in mammary epithelial cells by targeting MAP2K2. Bta-miR-2285f can regulate protein levels of fat milk marker gene PPARG by targeting MAP2K2. In conclusion, miR-2285f can target the expression of the MAP2K2 gene, promote the proliferation of dairy mammary epithelial cells, inhibit cell apoptosis and regulate the milk fat metabolism in dairy mammary epithelial cells. The results of this study revealed the function of miR-2285f in regulating the differential expression of fat milk in Holstein dairy cows at the cellular level. They provided a theoretical and experimental basis for analysing the regulation network of milk fat synthesis of Holstein dairy cows and the molecular breeding of dairy cows.
本研究以宁夏地区荷斯坦奶牛为研究对象，提取 8 头乳脂率差异显著荷斯坦奶牛的乳汁中的乳腺上皮细胞（BMECs）进行转录测序，并进行生物信息学分析，分析脂肪乳率的相关性，筛选出关键调控乳脂的微小 RNA-2285f。预测靶基因结合位点，利用 293T 细胞和乳腺上皮细胞作为 miRNA 和靶基因模型进行体外功能验证。通过转染 miR-2285f 模拟物和抑制剂，定量分析 miR-2285f 在荷斯坦奶牛组织中的差异。检测（双荧光素酶报告基因检测）和实时定量 PCR（定量实时 PCR，qRT-PCR）、甘油三酯（TAG）检测、脂质滴油红 O 检测、Western Blot 检测、Edu 和流式细胞术，研究 miR-2285f 及其靶基因 MAP2K2 对荷斯坦奶牛乳脂代谢的分子调控作用。构建 map2k2-3'utr 的野生型载体和突变型载体，进行双荧光素酶报告实验验证 MAP2K2 是 miR-2285f 的靶基因之一。根据 qRT-PCR 和 Western Blot 分析，miR-2285f 主要在 BMECs 中转录水平调节 MAP2K2 蛋白的表达。Bta-miR-2285f 可以通过调节 MAP2K2 促进细胞增殖和减缓细胞凋亡。Bta-miR-2285f 可以通过靶向 MAP2K2 促进乳腺上皮细胞中甘油三酯（TAG）和脂质滴的积累。Bta-miR-2285f 可以通过靶向 MAP2K2 调节脂肪乳标记基因 PPARG 的蛋白水平。综上所述，miR-2285f 可以靶向 MAP2K2 基因的表达，促进奶牛乳腺上皮细胞的增殖，抑制细胞凋亡，调节奶牛乳腺上皮细胞的乳脂代谢。本研究结果揭示了 miR-2285f 在细胞水平上调节荷斯坦奶牛乳脂差异表达的功能，为分析荷斯坦奶牛乳脂合成调控网络和奶牛分子育种提供了理论和实验依据。
