College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China.
Animal Engineering Branch, Yangling Vocational and Technical College, Yangling, Shaanxi, China.
J Cell Physiol. 2020 May;235(5):4198-4216. doi: 10.1002/jcp.29370. Epub 2019 Oct 29.
Incremental proofs demonstrate that miRNAs, the essential regulators of gene expression, are implicated in various biological procedures, including mammary development and milk synthesis. Here, the role of miR-574-5p in milk synthesis, apoptosis, and proliferation of goat mammary epithelial cells (GMECs) are explored without precedent, and the molecular mechanisms for the impacts are elucidated. Small RNA libraries were constructed using GMECs transfected with miR-574-5p mimics and negative control followed by sequencing via Solexa technology. Overall, 332 genes were distinguishingly expressed entre two libraries, with 74 genes upregulated and 258 genes downregulated. This approach revealed mitogen-activated protein kinase kinase kinase 9 (MAP3K9), an upstream activator of MAPK signaling, as a differentially expressed unigene. miR-574-5p targeted seed sequences of the MAP3K9 3'-untranslated region and suppressed its messenger RNA (mRNA) and protein levels, correspondingly. GMECs with miR-574-5p overexpression and MAP3K9 inhibition showed increased cell apoptosis and decreased cell proliferation resulting from sustained suppression of MAPK pathways, while MAP3K9 elevation manifested the opposite results. miR-574-5p repressed the phosphorylation of members of protein kinase B (AKT)-mammalian target of rapamycin pathway via downregulating MAP3K9 and AKT3, resulting in reducing the secretion of β-casein and triglycerides in GMECs. Finally, according to the constructed circular RNA (circRNA) libraries and bioinformatics prediction approach, we selected circ-016910 and found it acted as a sponge for miR-574-5p and blocked its relevant behaviors to undertake biological effects in GMECs. The circRNA-miRNA-mRNA network facilitates further probes on the function of miR-574-5p in mammary development and milk synthesis.
递增的证据表明，miRNAs 是基因表达的重要调控因子，参与多种生物学过程，包括乳腺发育和乳汁合成。在这里，miR-574-5p 在羊奶腺上皮细胞 (GMEC) 的乳汁合成、凋亡和增殖中的作用是前所未有的，其影响的分子机制也得到了阐明。用 miR-574-5p 模拟物和阴性对照转染 GMECs 构建小 RNA 文库，然后通过 Solexa 技术进行测序。总的来说，两个文库之间有 332 个基因表达差异显著，其中 74 个基因上调，258 个基因下调。这种方法揭示了丝裂原活化蛋白激酶激酶激酶 9 (MAP3K9)，即 MAPK 信号的上游激活物，作为一个差异表达的基因。miR-574-5p 靶向 MAP3K9 3'非翻译区的种子序列，相应地抑制其信使 RNA (mRNA) 和蛋白水平。miR-574-5p 过表达和 MAP3K9 抑制的 GMECs 表现出细胞凋亡增加和增殖减少，这是由于 MAPK 通路持续抑制所致，而 MAP3K9 升高则表现出相反的结果。miR-574-5p 通过下调 MAP3K9 和 AKT3 抑制蛋白激酶 B (AKT)-雷帕霉素靶蛋白通路的磷酸化，导致 GMECs 中 β-酪蛋白和甘油三酯的分泌减少。最后，根据构建的环状 RNA (circRNA) 文库和生物信息学预测方法，我们选择了 circ-016910，并发现它作为 miR-574-5p 的海绵，阻断其相关行为，从而在 GMECs 中发挥生物学效应。circRNA-miRNA-mRNA 网络有助于进一步研究 miR-574-5p 在乳腺发育和乳汁合成中的功能。
