Key Laboratory of Ruminant Molecular and Cellular Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan, 750021, China.
Sci Rep. 2024 Apr 20;14(1):9117. doi: 10.1038/s41598-024-59948-x.
Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased β-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.
牛奶蛋白含量是评估牛奶质量和营养价值的重要指标。越来越多的证据表明，microRNAs（miRNAs）在奶牛泌乳中发挥着重要作用，但关于植物源性外源性 miRNAs（xeno-miRNAs）在乳蛋白合成中的跨界调控作用，特别是其潜在的分子机制，人们知之甚少。本研究旨在探讨苜蓿衍生的外源性 miRNAs 对奶牛乳腺上皮细胞（BMEC）增殖和乳蛋白合成的调控机制。我们之前的研究表明，苜蓿 miR159a（mtr-miR159a，xeno-miR159a）在苜蓿中高度表达，与低蛋白奶奶牛相比，高蛋白奶奶牛的血清和乳清中 mtr-miR159a 的丰度显著降低。在这项研究中，通过实时定量 PCR（qRT-PCR）检测 mRNA 表达，通过酶联免疫吸附试验（ELISA）评估酪蛋白含量。使用细胞计数试剂盒 8（CCK-8）测定法、5-乙炔基-2'-脱氧尿苷（EdU）染色、western blot 和流式细胞术检测细胞增殖和凋亡。双荧光素酶报告基因检测用于确定 xeno-miR159a 对 Protein Tyrosine Phosphatase Receptor Type F（PTPRF）的调节作用。我们发现，xeno-miR159a 过表达抑制 BMEC 的增殖并促进细胞凋亡。此外，xeno-miR159a 过表达降低了 BMEC 中β-酪蛋白的丰度，增加了α-酪蛋白和κ-酪蛋白的丰度。双荧光素酶报告基因检测结果证实 PTPRF 是 xeno-miR159a 的靶基因。这些结果为苜蓿衍生的 miRNAs 调节 BMEC 增殖和乳蛋白合成的机制提供了新的见解。
