Lactation-Related MicroRNA Expression Profiles Of Porcine Breast Milk Exosomes

To newborn mammals, breast milk is the primary source of nutrition and immune. Breast milk is rich in immunological components, can meet all the requirements of brain development, growth and immunology system. MicroRNAs (miRNAs) are widely exist in various kinds of body fluids and tissues, and are selectively packaged inside the exosomes. Exosome is a type of membrane vesicles, secreted by many kinds of cell types. These miRNAs packaged in exosomes could be actively delivered into the recipient cells, and regulate the expression of target genes and the function of recipient cells. In this study, exosomes are confirmed to present in Yorkshire breast milk observating with atomic force microscope (AFM), and we constructed8milk exosomal small RNA libraries from6typical time points (0day,3days,7days,14days,21days and28days after birth) across the entire lactation period (from newborn to28days after birth). The breast milk of0day and3days after birth were classed into colostrum, and the others were mature milk. Utilizing the method of deep sequencing, the expression profiles of these lactation-related exosomal miRNAs in porcine milk were analyzed,we screened mmune-related miRNAs are present and enriched in breast milk exosomes and are generally resistant to relatively harsh conditions. These immune-related miRNAs are present in higher numbers in the colostrum compared with the mature milk or the blood of colostrum-only fed piglets compared with the mature milk-only fed piglets. Taken together, We got the following results:By the method of high speed ultracentrifugation, we isolated abundant of exosome-like vesicles from porcine breast milk. The morphology and size of exosomes were observed by (AFM at the nanometer-scale. The size of these exosome-like vesicles following normal distribution, and the average width was approximate100nm and the height was about12nm. All the morphological characters of these exosome-like vesicles from porcine breast milk were similar with the reported exosomes in human saliva and breast milk.No18S and28S ribosomal RNA were found in the milk exosomes, but contain large number of small RNAs that were below300nt in length. Eight small RNA libraries were subjected to single-end sequencing in36nt reads using an Illumina Genome Analyzer Ⅱ. The analysis of sequencing data confirmed the enrichment of miRNAs in porcine breast milk exosomes. Of these mappable reads in the eight libraries, the overwhelming majority are21-24nt in length (91.97±3.18%). More than half of the reads are22nt in length (67.93±4.81%), followed by23nt (15.92±2.27%),24nt (4.71±1.65%), and21nt (3.42±1.77%), which are typical sizes of Dicer-processed products, confirming the presence of miRNAs in milk exosomes.The bioinformatics pipeline for miRNA discovery and expression profiling discovered180pre-miRNAs in the8exosomal small RNA libraries, of which140(77.78%) are known porcine pre-miRNAs and40are novel porcine pre-miRNAs. All of these40novel porcine pre-miRNAs are homologous to human pre-miRNAs deposited in miRBase18.0and could be mapped to the pig genome. These180pre-miRNAs encode237mature miRNAs, corresponding to234unique miRNAs.Based on the annotation of the Pathway Central Database (SABiosciences, MD, USA), among these1527known human pre-miRNAs,84(5.50%) are immune-related pre-miRNAs.Out of these84immune-related pre-miRNAs,58(69.05%) are present and enriched in each milk exosomal miRNA library (P＜10-16,χ2test). And in the8porcine milk exosomal miRNA libraries, the immune-related pre-miRNAs account for more than30%of the total pre-miRNAs, which was significantly higher than5.5%, the percentage in the human miRNA library. In the colostrum libraries (0day and3days after borth), there are more than52immune-related pre-miRNAs, significantly higher than40, the number of immune-related pre-miRNAs in the mature milk libraries (P<0.05).The enrichment of immune-related pre-miRNAs in the milk exosome suggested the important role of milk in the development of immune system.In the breast milk exosomal miRNA transcriptome, the majority of abundant miRNAs are from few miRNAs. The top10unique miRNAs with the highest expression level account for more than87.25%, by total counts, of all the234unique miRNAs. The unified set of top10unique miRNAs over six lactation stages corresponds to13kinds of unique miRNAs. Among these13miRNAs,7miRNAs (miR-148a-3p, miR-182-5p, miR-200c-3p, miR-25-3p, miR-30a-5p, miR-30d-5p, and miR-574-3p) are present in the top10miRNAs in all six libraries, which suggests essential roles in various immune and pathological conditions. The other6miRNAs (let-7a-1-5p, miR-30c-2-5p and-1-5p, miR-191-5p, miR-375-3p, miR-21-5p, and miR-27b-3p), which are present in the top10miRNAs in at least one of the6lactation stages, are all related to various immune and pathological responses. The miRNA expression profiles show typical lactation-specific pattern. The immune-related miRNAs exist in the colostrum are significantly higher than those in the mature milk. By quantitative analyzing, apart from miR-148a-3p, almost all the other12unique immune-related miRNAs exhibited higher abundances in the early lactation periods (0and3days) than in the later lactation periods (7,14,21and28days). And the expression level of miR-148a-3p, the top ranking miRNA across6lactation stages by counts, increased throughout the entire porcine lactation stage.All of these immune-related miRNAs have been shown to functionally target specific transcripts encoding cytokines, other immunological regulatory proteins, and immune response signaling pathway components.When the milk was subjected to various harsh conditions (low temperature, high temperature, low pH, multiple freeze-thaw cycles, and exogenous RNase), the exogenous synthetic worm-specific C. elegans miRNAs (cel-lin-4-5p, cel-miR-2-3p and cel-miR-39-5p) and Arabidopsis thaliana miRNA (ath-miR-159a-3p) were sharply degraded. However, the5endogenous breast milk exosome-derived high expression miRNAs (miR-148a-3p, miR-30c-5p, miR-574-3p, miR-21-5p and miR-27b-3p) exhibited resistance to degradation and high stability to harsh conditions. These results suggest that milk-derived miRNAs are selectively packaged into exosomes and exist in a remarkable stable form that is protected from endogenous RNase activity across various harsh conditions.After feeding with different milk, the colostrum-only and mature milk-only fed piglets have similar levels of7representing circulating metabolic indicators in their serum (P＞0.05). Nonetheless, the expression level of the above13immune-related abundant miRNAs in the serum changed significantly. Out of the13immune-related mRNAs, apart from miR-148a-3p, all the other12miRNAs exhibited higher abundance in the colostrum-fed piglets compared with the mature milk-fed piglets (P＜0.01). And the expression level of miR-148a-3p was significantly higher in the mature milk-fed piglets (P＜0.05). The expression pattern of above mentioned immune-related miRNAs in the serum of piglets kept the same trend as the results in the porcine colostrum and mature milk. It is reasonable to hypothesis that these immune-related exosomal miRNAs in breast milk may be transferred into the piglet body via the digestive tract under the protection of exosomes.The results in this study provide basic information for further research of the important roles of breast milk in the development of infant’s immune system. The study also proved that pig can be used as an ideal model for studying human lactation medicine and immune diseases.