The biosynthesis of milk fat affects both the technological properties and organoleptic quality of milk and dairy products. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that inhibit the expression of their mRNA targets and are involved in downstream signaling pathways that control several biological processes, including milk fat synthesis. miR-34b is a member of the miR-34 miRNA cluster, which is differentially expressed in the mammary gland tissue of dairy cows during lactation and dry periods. Previous studies have indicated miR-34b is a potential candidate gene that plays a decisive role in regulating milk fat synthesis; therefore, it is important to focus on miR-34b and investigate its regulatory effect on the biosynthesis of milk fat in bovine mammary epithelial cells (BMECs). In this study, elevated miR-34b levels reduced milk fat synthesis, upregulated 1,999 genes, and downregulated 2,009 genes in BMECs. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes suggested that miR-34b may play an inhibitory role in milk fat synthesis via the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway by reducing phosphorylation levels. Notably, the mTOR activator MHY1485 rescued the inhibitory effect of miR-34b. Furthermore, we demonstrated that retinoic acid-induced protein 14 (RAI14) is a target of miR-34b via TargetScan and immunofluorescence assays. RAI14 mRNA and protein levels were significantly decreased by the miR-34b mimic and increased by the miR-34b inhibitor. Moreover, the reduction in RAI14 levels led to the inhibition of the Akt/mTOR signaling pathway. Overall, our results identified a miR-34b-RAI14-Akt/mTOR regulatory network, while also providing a theoretical basis for the molecular breeding of dairy cows.

中文翻译：

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Bta-miR-34b 通过 Akt/mTOR 信号通路靶向牛乳腺上皮细胞中的 RAI14 来控制乳脂生物合成


乳脂的生物合成影响牛奶和乳制品的技术特性和感官质量。 MicroRNA (miRNA) 是内源性小非编码 RNA，可抑制其 mRNA 靶标的表达，并参与控制多种生物过程（包括乳脂合成）的下游信号传导通路。 miR-34b是miR-34 miRNA簇的成员，在泌乳期和干奶期的奶牛乳腺组织中差异表达。前期研究表明miR-34b是一个潜在的候选基因，在调节乳脂合成中起决定性作用；因此，关注miR-34b并研究其对牛乳腺上皮细胞（BMEC）乳脂生物合成的调节作用非常重要。在这项研究中，BMEC 中 miR-34b 水平升高会减少乳脂合成，上调 1,999 个基因，下调 2,009 个基因。此外，京都基因和基因组百科全书（KEGG）对差异表达基因的分析表明，miR-34b可能通过蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路通过减少乳脂合成而发挥抑制作用。磷酸化水平。值得注意的是，mTOR 激活剂 MHY1485 挽救了 miR-34b 的抑制作用。此外，我们通过 TargetScan 和免疫荧光测定证明视黄酸诱导蛋白 14 (RAI14) 是 miR-34b 的靶标。 miR-34b 模拟物显着降低了 RAI14 mRNA 和蛋白质水平，而 miR-34b 抑制剂则显着增加了 RAI14 mRNA 和蛋白质水平。此外，RAI14 水平的降低导致 Akt/mTOR 信号通路受到抑制。 总体而言，我们的结果确定了miR-34b-RAI14-Akt/mTOR调控网络，同时也为奶牛的分子育种提供了理论基础。