Differential miRNAs analysis of colostrum and normal milk exosomes in Bamei and Landrace pigs

Breast milk is an indispensable source of nutrition for the growth and development of newborn mammals. The exosomes in breast milk have potential regulatory effects on the growth, development, and immunity of newborn piglets. We isolated two types of exosomes from porcine colostrum and normal milk by density gradient centrifugation and characterized them by transmission electron microscopy (TEM) and particle size analysis. Furthermore, the expression profiles of miRNAs in the two types of exosomes were investigated by miRNA-Seq and bioinformatics analysis. The sequencing results identified a total of 480 known miRNAs, 601 conservative miRNAs, and 2311 novel miRNAs in porcine milk exosomes from Bamei and Landrace pigs. The differential miRNA target genes were significantly enriched in the endocytosis, T-cell receptor signaling pathway and Th17 cell differentiation signaling pathways. Differentially expressed miRNAs (DE-miRNAs) were closely associated with processes such as cell signaling, cell physiology, and immune system development. Differential expression profiles of miRNAs in milk exosomes from different lactation stages in two breeds of pigs identified some immune-related miRNA-mRNA negative regulatory networks and KEGG pathways. Our findings provide a theoretical basis for further studies on the functions of miRNAs in milk exosomes.母乳是新生哺乳动物生长发育不可或缺的营养来源。母乳中的外泌体(exosomes)对新生仔猪的生长、发育与免疫具有潜在调控作用。本研究通过密度梯度离心法从猪初乳与常乳中分离得到两种外泌体，并借助透射电子显微镜(TEM)与粒径分析对其进行了表征。此外，本研究通过miRNA测序(miRNA-Seq)与生物信息学分析，探究了这两种外泌体中微小RNA(miRNA)的表达谱。测序结果显示，在八眉猪与长白猪的母乳外泌体中，共鉴定出480种已知miRNA、601种保守miRNA以及2311种新型miRNA。差异miRNA的靶基因显著富集于内吞作用、T细胞受体信号通路以及Th17细胞分化信号通路中。差异表达miRNA(DE-miRNAs)与细胞信号转导、细胞生理以及免疫系统发育等过程密切相关。对两个猪品种不同泌乳阶段母乳外泌体中的miRNA差异表达谱进行分析，鉴定出若干免疫相关的miRNA-mRNA负调控网络及KEGG通路。本研究结果为后续探究母乳外泌体中miRNA的功能提供了理论依据。