miR-140调控奶牛乳腺脂肪酸代谢的分子机制研究

Investigation to elucidate the mechanism of fatty acid metabolism in bovine mammary gland are key to the scientific analysis of milk quality. Approaches such as miRNA expression profiling of mammary gland tissues in different lactation stages, secondary screening of prolactin-treated cells, and milk fatty acid correlation analysis and functional verification have shown that miR-140 expression is closely related to milk fatty acid metabolism. However, what the regulation of miR-140 expression is, and which fatty acids are regulated by miR-140 and the mechanism of regulation is still unclear. In view of this, miR-140 was selected as the research object. This project will investigate the molecular mechanisms by which prolactin regulates miR-140 expression by epigenetic regulation. This will elucidate the mode of miR-140 upstream regulation; The miR-140 knock-out/knock-in assays using CRISPR/ Cas9 technology will be performed on mammary epithelial cells to elucidate the regulatory role of miR-140 on mammary fatty acid metabolism. These results will clarify the function of miR-140; A fatty acid regulation network diagram of miR-140 and target gene LATS2 will be constructed to elucidate the miR-140 regulatory pathway. This will elucidate the pathway of miR-140 downstream regulation. This project seeks to accurately reveal the molecular mechanisms by which miR-140 can finely regulate the metabolism of milk fatty acids. This study will provide a theoretical basis to improve the milk quality.

研究奶牛乳腺脂肪酸代谢机理是解析牛乳品质的关键科学问题。申请人前期通过建立不同泌乳期奶牛乳腺组织miRNAs表达谱、PRL（催乳素）处理细胞二次筛选、进行目标miRNA与乳中脂肪酸构成的关联分析和功能验证，证实了miR-140与乳脂肪酸代谢密切相关。但miR-140表达受什么调控，通过何种机制调控脂肪酸代谢尚不清楚。鉴于此，本项目以miR-140为研究对象，探讨PRL通过表观遗传调控miR-140表达的分子机制，阐明miR-140上游调控方式；使用CRISPR/Cas9 技术在乳腺上皮细胞上进行miR-140敲除/敲入试验，验证miR-140对脂肪酸代谢的调控作用，明确miR-140功能；多层面构建miR-140与靶基因LATS2的脂肪酸调控网络图，明析miR-140下游调控途径。本项目旨在揭示miR-140调控牛乳脂肪酸代谢的分子机理，为改进牛乳品质提供理论依据。

脂肪酸在提高乳制品质量的过程中起着核心调节作用。脂肪酸的合成受到信号分子、基因表达和信号转导等诸多因素的调控，其中miRNAs是脂肪代谢的关键调控因子。同时，环状RNA（circRNA）也可以通过发挥miRNA海绵效应、调控RNA转录、与蛋白质相互作用和参与蛋白翻译等作用机制来对牛奶中脂肪酸的组成和表达进行调控。本项目的主要目的是研究circRNAs、miRNAs和mRNAs调控牛奶中脂肪酸的组成和表达的途径。.本项目进行期间，获得了以下实验结果：.1）miR-140调控奶牛乳腺上皮细胞中脂肪酸合成；. 2）circ09863通过结合miR-27a-3p调控奶牛乳腺脂肪酸代谢；. 3）circ01592通过结合miR-218调节奶牛乳腺上皮细胞不饱和脂肪酸代谢；. 4）circ11103与miR-128/PPARGC1A相互作用调节奶牛乳脂代谢；. 5）miR-106b通过ABCA1（ATP结合盒A亚家族成员1）调节奶牛乳腺上皮细胞乳脂代谢；. 6）miR-497通过LATS2调节奶牛乳腺上皮细胞脂肪酸合成。