Disentangling the regulatory role of microRNA of milk exosomes in mastitis of dairy cows

The aim of the research was to evaluate the effect of the mastitis in lactating cows on the differential expressed miRNA in milk exosomes. On the basis of somatic cell count and differential somatic cell counts in the milk, 10 cows were healthy (group G), 11 cows were at risk of mastitis (Group Y) and 11 cows with subclinical mastitis (group R). The exosomes were isolated from milk samples using an isoelectric precipitation and ultrcentrifugation and total RNA extracted. After library preparation, RNA was sequenced to 50 nucleotide long single reads and mapped against Btau_5.0.1. The final number miRNA considered for the analysis was 225. The differentially expressed (DE, p<0.05) miRNA were 38, 18 and 13 for the G_Y, Y_R and G_R comparisons, respectively. The target genes of DE miRNA were 2195 for the G_Y comparison, 713 for the G_R comparison and 1313 for the Y_R comparison. The comparison of enriched pathways obtained for the target genes, highlighted 56 DE pathways for the G_R and 57 for the G_Y comparison. For the G_Y network, the main miRNA were bta-mir-2415-3p, bta-mir-2431-3p and bta-mir-6517, bta-mir-339a, bta-mir-1343-3p, bta-mir-2407, bta-mir-328, bta-mir-1306. For the G_R pairwise comparison, the most relevant miRNAs were bta-mir-339a, bta-mir-1247-5p and bta-mir-1306, bta-mir-345-5p, bta-mir-320a and bta-mir-2388-3p. Among the significantly KEGG enriched pathways, NF-KB Signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, Adherens junction, Leukocyte transendothelial migration were counted for G_Y. For G_R, the NF-KB Signaling pathway was not enriched.Interconnections among miRNA and target genes in relation to KEGG pathways showed that a regulation of relevant genes and pathways involved in the immune functions affect the expression of miRNa in the milk exosome.

本研究旨在探讨泌乳奶牛乳腺炎对牛奶外泌体（exosomes）中差异表达微小RNA（miRNA）的影响。基于牛奶中的体细胞计数与差异体细胞计数，将受试奶牛分为三组：健康组（G组）10头、乳腺炎易感组（Y组）11头、亚临床乳腺炎组（R组）11头。本研究采用等电沉淀结合超速离心法从牛奶样本中分离外泌体，并提取总RNA。完成文库构建后，对RNA进行50核苷酸单端测序，并将测序reads比对至牛参考基因组Btau_5.0.1。本研究最终纳入分析的微小RNA共225个。在G_Y、Y_R与G_R三组比对中，差异表达（DE，p<0.05）的微小RNA分别为38个、18个和13个。差异表达微小RNA的靶基因数量在G_Y比对中为2195个，G_R比对中为713个，Y_R比对中为1313个。对靶基因的富集通路进行比对分析后，G_R与G_Y比对分别凸显出56条和57条差异富集通路。在G组与Y组的调控网络中，核心微小RNA包括bta-mir-2415-3p、bta-mir-2431-3p、bta-mir-6517、bta-mir-339a、bta-mir-1343-3p、bta-mir-2407、bta-mir-328及bta-mir-1306。在G组与R组的两两比对中，核心关联微小RNA包括bta-mir-339a、bta-mir-1247-5p、bta-mir-1306、bta-mir-345-5p、bta-mir-320a及bta-mir-2388-3p。在京都基因与基因组百科全书（KEGG）富集的显著通路中，G_Y组涉及NF-κB信号通路、T细胞受体信号通路、TNF信号通路、黏着连接及白细胞跨内皮迁移通路。而G_R组未富集到NF-κB信号通路。微小RNA与靶基因之间的关联以及其与KEGG通路的关联分析表明，免疫功能相关基因与通路的调控可影响牛奶外泌体中微小RNA的表达。