Chen Ting, Xi Qian-Yun, Ye Rui-Song, Cheng Xiao, Qi Qi-En, Wang Song-Bo, Shu Gang, Wang Li-Na, Zhu Xiao-Tong, Jiang Qing-Yan, Zhang Yong-Liang
Guandong Provincial Key Lab of Agro-Animal Genomics And Molecular Breeding, College of Animal Science, ALLTECH-SCAU Animal Nutrition Control Research Alliance, National Engineering Research Center For Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China.
BMC Genomics. 2014 Feb 5;15(1):100. doi: 10.1186/1471-2164-15-100.
Breast milk contains complex nutrients and facilitates the maturation of various biological systems in infants. Exosomes, membranous vesicles of endocytic origin found in different body fluids such as milk, can mediate intercellular communication. We hypothesized that microRNAs (miRNAs), a class of non-coding small RNAs of 18-25 nt which are known to be packaged in exosomes of human, bovine and porcine milk, may play important roles in the development of piglets.
In this study, exosomes of approximately 100 nm in diameter were isolated from porcine milk through serial centrifugation and ultracentrifugation procedures. Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component. Solexa sequencing showed a total of 491 miRNAs, including 176 known miRNAs and 315 novel mature miRNAs (representing 366 pre-miRNAs), which were distributed among 30 clusters and 35 families, and two predicted novel miRNAs were verified targeting 3'UTR of IGF-1R by luciferase assay. Interestingly, we observed that three miRNAs (ssc-let-7e, ssc-miR-27a, and ssc-miR-30a) could be generated from miRNA-offset RNAs (moRNAs). The top 10 miRNAs accounted for 74.5% (67,154 counts) of total counts, which were predicted to target 2,333 genes by RNAhybrid software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using DAVID bioinformatics resources indicated that the identified miRNAs targeted genes enriched in transcription, immunity and metabolism processes, and 14 of the top 20 miRNAs possibly participate in regulation of the IgA immune network.
Our findings suggest that porcine milk exosomes contain a large number of miRNAs, which potentially play an important role in information transfer from sow milk to piglets. The predicted miRNAs of porcine milk exosomes in this study provide a basis for future biochemical and biophysical function studies.
母乳含有复杂的营养成分，有助于婴儿各种生物系统的成熟。外泌体是一种在内吞起源的膜性囊泡，存在于不同的体液中，如乳汁，可介导细胞间通讯。我们推测，微小RNA（miRNA），一类18 - 25个核苷酸的非编码小RNA，已知其被包装在人、牛和猪乳汁的外泌体中，可能在仔猪发育中发挥重要作用。
在本研究中，通过连续离心和超速离心程序从猪乳中分离出直径约为100nm的外泌体。从外泌体中提取总RNA，发现5S核糖体RNA是主要的RNA成分。Solexa测序显示共有491个miRNA，包括176个已知miRNA和315个新的成熟miRNA（代表366个前体miRNA），它们分布在30个簇和35个家族中，并且通过荧光素酶测定验证了两个预测的新miRNA靶向IGF - 1R的3'UTR。有趣的是，我们观察到三个miRNA（ssc - let - 7e、ssc - miR - 27a和ssc - miR - 30a）可以从miRNA偏移RNA（moRNA）产生。前10个miRNA占总数的74.5%（67,154个计数），通过RNAhybrid软件预测它们靶向2,333个基因。使用DAVID生物信息学资源进行的基因本体论和京都基因与基因组百科全书（KEGG）途径分析表明，鉴定出的miRNA靶向富含转录、免疫和代谢过程的基因，并且前20个miRNA中的14个可能参与IgA免疫网络的调节。
我们的研究结果表明，猪乳外泌体含有大量miRNA，它们可能在从母猪乳汁到仔猪的信息传递中发挥重要作用。本研究中预测的猪乳外泌体miRNA为未来的生化和生物物理功能研究提供了基础。
