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. 2014 Feb 5;15(1):100.
doi: 10.1186/1471-2164-15-100.

Exploration of microRNAs in porcine milk exosomes

Ting Chen, Qian-Yun Xi, Rui-Song Ye, Xiao Cheng, Qi-En Qi, Song-Bo Wang, Gang Shu, Li-Na Wang, Xiao-Tong Zhu, Qing-Yan Jiang, Yong-Liang Zhang  1
Affiliations

Exploration of microRNAs in porcine milk exosomes

Ting Chen et al. BMC Genomics. .

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

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Figures

Figure 1
Figure 1
MiR-PC-86 and MiR-PC-263 directly regulate IGF-1R expression via 3’ UTR sites. (A) Schematic of IGF-1R mRNA and the luciferase reporter plasmids containing the miR-PC-86 and miR-PC-263 binding sites of IGF-1R mRNA. The 3’ UTR sites were inserted downstream of the luciferase reporter, as indicated. TCAGTGG was the predicted target site of miR-PC-86, GGATCTT was the predicted target site of miR-PC-263. (B) miR-PC-86 and miR-PC-263 sequences and predicted binding site between miR-PC-86 and miR-PC-263 and IGF-1R mRNA. IGF-1R mRNA has one putative binding site for miR-PC-86/ miR-PC-263 on the 3’ UTR. Twelve nucleotides TCAGTGGATCTT of IGF-1R 3’ UTR (underlined) were delete in order to disrupt the binding with miR-PC-86 and miR-PC-263 seed regions. (C) IPEC-J2 cells were transfected with each of the constructed plasmids, together with miR-PC-86/ miR-PC-263and Renilla luciferase reporter plasmid (*P < 0.05, n = 6).
Figure 2
Figure 2
Exosomes detected by TEM. The exosomes appeared as round or oval microvesicles (A, B), with a diameter of 50–150 nm and heavier density at the center than on the margin.
Figure 3
Figure 3
Milk-derived exosomes containing RNA. (A) Total RNA was extracted from porcine exosomes. M, 1, 2 and 3 represent the marker (DL 2000), RNA without any treatment, RNA treated with DNase I and RNA treated with RNase, respectively. (B) RNA sample analyzed by the Agilent Bioanalyzer 2100.
Figure 4
Figure 4
Length distribution of miRNAs reads from Solexa sequencing. A total of 4,964,542 clean reads were obtained, ranging from 10 to 32 nt, most of which were 18–25 nt in length (accounting for 74.89%).
Figure 5
Figure 5
Distribution of miRNA reads and top 10 miRNAs. (A) The distribution of miRNA reads showed that the top 10, top 20, top 50 and top 100 miRNAs accounted for 74.5%, 85.2% and 95.3% and 98.3% of total reads. (B) Cumulative proportions of top 10 miRNAs. MiR-193-3p ranked first, accounting for 29.6% of total reads.
Figure 6
Figure 6
MiRNAs detected randomly in porcine milk. (A) Known miRNAs from miRBase (18.0), from M to 14, respectively: marker (DL 2000), ssc-let-7e, ssc-miR-21, ssc-miR-206, ssc-let-7i, ssc-miR-140, ssc-miR-92b-5p, ssc-miR-22-3p, ssc-miR-28-5p, ssc-miR-205, ssc-miR-451, ssc-miR-125b, ssc-miR-9, ssc-let-7c and 5 s (control). (B) Top 15 predicted novel miRNAs, from M to 16, respectively: marker (DL 2000), P-m0227-5p, P-m0338-3p, P-m0105-3p, P-m0058-5p, P-m0281-5p, P-m0265-3p, P-m0279-5p, P-m0103-3p, P-m0113-3p, P-m0129-5p, P-m0355-5p, P-m0210-5p, P-m0070-3p, P-m0124-3p, P-m0186-5p and 5 s (control).
Figure 7
Figure 7
Expression of 15 predicted novel miRNAs in the sample pool detected by qRT-PCR. Trends in relative expression by qRT-PCR and counts from Solexa sequencing of miRNAs, except for PC-192, were consistent.
Figure 8
Figure 8
Distribution of 30 miRNA clusters. The number of base points near different bars indicates the number of clusters in the chromosome. The relative vertical dimension of the point on the bar represents the location of cluster. The label “number1 /number2” above every bar indicates the value of “pre-miRNAs/mature miRNA”.
Figure 9
Figure 9
5p and 3p arm expression of 46 pre-miRNAs. (A) 30 known pre-miRNAs. (B) 11 novel pre-miRNAs (representing 15 coupled 3p and 5p arm sequences).
Figure 10
Figure 10
Three distinctive pre-miRNAs identified in porcine milk exosomes. (A) The ssc-miR-27a precursor produced a 3p-arm miRNA sequence and a loop-derived small RNA. (B) The ssc-let-7e precursor produced a 5p-arm sequence, a 3p-arm miRNA sequence and a loop-derived small RNA. (C) The ssc-miR-30a precursor produced a 5p-arm sequence, a 3p-arm miRNA sequence and a loop-derived small RNA. In addition, ssc-moRNA-3, belonging to new type of miRNA termed moRNA, was found at the 5’ end of pre-miR-30.
Figure 11
Figure 11
MiR-363~20b~106a homologous or paralogous cluster and expression level. (A) The miR-363/92a/19b/20b cluster and its homologous cluster miR-363/92a/19b/20b/106a on chromosome X were separated by a 33.5 kb DNA fragment. (B) The miR-17/18a/19/92a cluster was located on chromosome 11. In the genome, miR-92a/19b showed three copies; miR-363 and miR-20b had two copies; while miR-17, miR-18a and miR-106a had one copy. (C) Expression of mature miRNAs produced from the miR-363~20b~106a cluster and miR-17~92 cluster.
Figure 12
Figure 12
Expression of miRNA families. (A) Expression of the let-7f family; let-7f-1, let-7a and let-7d formed a cluster, while miR-98 and let-7e formed a cluster. (B) Expression of miR-30 family; miR-30d clustered with miR-30b, and miR-30c clustered with miR-30e. (C) Expression of miR-181 family; miR-181a clustered with miR-181b, and miR-181c clustered with miR-181d. (D) MiR-222 and miR-221; miR-222 and miR-221 belonged to the same family and the same cluster. (E) MiR-27 family and miR-23 family; miR-27b and miR-23b formed a cluster in the genome. (F) Expression and clustering of remaining miRNA families; members 1, 2, 3 and 4 represent members of the indicated family. (G) Novel miRNA family with low expression. (H) Expression of the miR-148 family containing milk “marker” miRNAs.
Figure 13
Figure 13
MiRNAs targeting the IgA immune network. The top 20 miRNAs were analyzed, and 14 of them were found to participate in the IgA immune network, involving 20 target genes. Different colors and shapes represent various relationships between miRNAs and genes.
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