Figure 1.
Evidence of miRNA-loaded exosomes in porcine breast milk.
(A) 3D AFM image of isolated milk exosomes. (B) The line profile of AFM image for a milk exosome. X- and Y-axes are the width and height of a porcine milk exosome, respectively. (C) RNA from porcine breast milk exosomes was detected using Agilent Bioanalyzer 2100.
Figure 2.
Distribution of shared pre-miRNAs among miRBase (Version 18.0) entries for human (1,527) and pig (228), as well as 180 pre-miRNAs identified in porcine breast milk exosomes.
Out of 180 pre-miRNAs in porcine breast milk exosomes, 140 are known porcine pre-miRNAs and 40 are novel porcine pre-miRNAs, which encoding 237 mature miRNAs, corresponding to 234 unique miRNAs.
Figure 3.
Lactation-related miRNA expression profiles.
(A) Distribution of lactation-related pre-miRNAs. Out of 1,527 pre-miRNAs deposited in miRBase 18.0, 84 (5.50%) pre-miRNAs have been designated as immunopathology-related pre-miRNAs, based on annotation in the Pathway Central database (SABiosciences, MD, USA). These immune-related pre-miRNAs are enriched in each milk exosomal miRNA library. χ2 test (**p<10−16): Numbers of immunopathology-related miRNAs and others detected in milk exosomes compared with the total entries in miRBase 18.0. The three biological replicates at 0 days are denoted by 0d-1, -2 and -3, respectively. (B) Hierarchical clustering and heat map matrix of pairwise Spearman correlations of the counts of 234 unique miRNAs between eight exosomal miRNA libraries. (C) Top 10 unique miRNAs with the highest expression levels in milk exosomal miRNA libraries. Plot of the unique miRNAs versus their % in total counts of all unique miRNAs for each library. The dashed vertical lines represent the accumulative % of the top 10 unique miRNAs in total counts of all unique miRNAs. Seven miRNAs that are present in the top 10 miRNAs in all six libraries are connected by lines.
Figure 4.
Lactation-related expression patterns of 13 abundant immune-related miRNAs.
The data are normally distributed (Kolmogorov-Smirnov test, p>0.05). Pearson correlation was used to determine the relation of miRNAs expression changes between the q-PCR and the deep sequencing approaches. Values are means±SD.
Figure 5.
Differences of circulating metabolic indicators and immune-related miRNAs in serum between the colostrum-only and the mature milk-only fed piglets.
(A) Seven representative circulating indicators of metabolism. (B) Thirteen well-characterized immune-related miRNAs. The data are normally distributed (Kolmogorov-Smirnov test, p>0.05). The statistical significance was calculated by Student’s t-test (n = 6 per group). Values are means±SD. CF: colostrum-only feeding; MF: mature milk-only feeding.