Shen Binglei, Zhang Liying, Lian Chuanjiang, Lu Chunyan, Zhang Yonghong, Pan Qiqi, Yang Runjun, Zhao Zhihui
College of Animal Science, Jilin University, 5333 Xi'an Road, Changchun 130062, China.
College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China.
Int J Mol Sci. 2016 Feb 17;17(2):200. doi: 10.3390/ijms17020200.
Milk fat is a key factor affecting milk quality and is also a major trait targeted in dairy cow breeding. To determine how the synthesis and the metabolism of lipids in bovine milk is regulated at the miRNA level, primary mammary epithelial cells (pMEC) derived from two Chinese Holstein dairy cows that produced extreme differences in milk fat percentage were cultured by the method of tissue nubbles culture. Small RNA libraries were constructed from each of the two pMEC groups, and Solexa sequencing and bioinformatics analysis were then used to determine the abundance of miRNAs and their differential expression pattern between pMECs. Target genes and functional prediction of differentially expressed miRNAs by Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis illustrated their roles in milk fat metabolism. Results show that a total of 292 known miRNAs and 116 novel miRNAs were detected in both pMECs. Identification of known and novel miRNA candidates demonstrated the feasibility and sensitivity of sequencing at the cellular level. Additionally, 97 miRNAs were significantly differentially expressed between the pMECs. Finally, three miRNAs including bta-miR-33a, bta-miR-152 and bta-miR-224 whose predicted target genes were annotated to the pathway of lipid metabolism were screened and verified by real-time qPCR and Western-blotting experiments. This study is the first comparative profiling of the miRNA transcriptome in pMECs that produce different milk fat content.
乳脂肪是影响牛奶品质的关键因素，也是奶牛育种的主要目标性状。为了确定在miRNA水平上牛乳中脂质的合成与代谢是如何调控的，采用组织块培养法培养了来自两头乳脂率差异极大的中国荷斯坦奶牛的原代乳腺上皮细胞（pMEC）。从两组pMEC中分别构建小RNA文库，然后利用Solexa测序和生物信息学分析来确定miRNA的丰度及其在pMEC之间的差异表达模式。通过基因本体论和京都基因与基因组百科全书分析对差异表达miRNA的靶基因和功能进行预测，阐明了它们在乳脂肪代谢中的作用。结果表明，在两组pMEC中总共检测到292个已知miRNA和116个新miRNA。已知和新miRNA候选物的鉴定证明了细胞水平测序的可行性和敏感性。此外，两组pMEC之间有97个miRNA存在显著差异表达。最后，通过实时定量PCR和蛋白质免疫印迹实验筛选并验证了3个miRNA，包括预测靶基因注释到脂质代谢途径的bta-miR-33a、bta-miR-152和bta-miR-224。本研究首次对产生不同乳脂肪含量的pMEC中的miRNA转录组进行了比较分析。
