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Exploration of microRNAs in porcine milk exosomes.

Chen T, Xi QY, Ye RS, Cheng X, Qi QE, Wang SB, Shu G, Wang LN, Zhu XT, Jiang QY, Zhang YL - BMC Genomics (2014)

Bottom Line: Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component.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.

View Article: PubMed Central - PubMed

Affiliation: Guandong Provincial Key Lab of Agro-Animal Genomics And Molecular Breeding, College of Animal Science, ALLTECH-SCAU Animal Nutrition Control Research Alliance, National Engineering Research Center For Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China. Zhangyl@scau.edu.cn.

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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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.
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Fig11: 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.

Mentions: According to criteria for classification in miRbase, pre-miRNAs located on a chromosome with an interval of less than 10 kb are defined as belonging to an miRNA cluster. In our analysis, 30 (including 11 novel and 19 known) (Figure 8) clusters were detected (Table 5). Among all miRNAs clusters, there were several pre-miRNAs with intervening sequences of less than 1 kb, including 10 known clusters (miR-99b/let-7e/125a, miR-24-2/27b/23b, miR-99a/let-7c, miR-29b/29a, miR-221/222, miR-98/let-7f, miR-181c/d, miR-363/92a/19b-2/106a, miR-363/92a/19b-2, miR-181b-1/181a-1 and miR-17/18a/19b-1/92a-1) and 4 novel miRNAs clusters (cluster 3, 9, 12, 22). We identified a typical polycistronic miRNA cluster, miR-363/92a/19b-2/20b, on chromosome X. Interestingly, the homologous cluster, miR-363/92a/19b-2/20b/106a on chromosome X, was located 33.5 kb downstream of miR-363/92a/19b-2/20b (Figure11A, C), and a paraologous cluster miR-17/18a/19b-1/92a-1 was found on chromosome 11 (Figure 11B, C). The organization of miRNA precursors in the genome may account for variable levels of expression and regulation of mature miRNAs.Table 5


Exploration of microRNAs in porcine milk exosomes.

Chen T, Xi QY, Ye RS, Cheng X, Qi QE, Wang SB, Shu G, Wang LN, Zhu XT, Jiang QY, Zhang YL - BMC Genomics (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4008308&req=5

Fig11: 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.
Mentions: According to criteria for classification in miRbase, pre-miRNAs located on a chromosome with an interval of less than 10 kb are defined as belonging to an miRNA cluster. In our analysis, 30 (including 11 novel and 19 known) (Figure 8) clusters were detected (Table 5). Among all miRNAs clusters, there were several pre-miRNAs with intervening sequences of less than 1 kb, including 10 known clusters (miR-99b/let-7e/125a, miR-24-2/27b/23b, miR-99a/let-7c, miR-29b/29a, miR-221/222, miR-98/let-7f, miR-181c/d, miR-363/92a/19b-2/106a, miR-363/92a/19b-2, miR-181b-1/181a-1 and miR-17/18a/19b-1/92a-1) and 4 novel miRNAs clusters (cluster 3, 9, 12, 22). We identified a typical polycistronic miRNA cluster, miR-363/92a/19b-2/20b, on chromosome X. Interestingly, the homologous cluster, miR-363/92a/19b-2/20b/106a on chromosome X, was located 33.5 kb downstream of miR-363/92a/19b-2/20b (Figure11A, C), and a paraologous cluster miR-17/18a/19b-1/92a-1 was found on chromosome 11 (Figure 11B, C). The organization of miRNA precursors in the genome may account for variable levels of expression and regulation of mature miRNAs.Table 5

Bottom Line: Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component.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.

View Article: PubMed Central - PubMed

Affiliation: Guandong Provincial Key Lab of Agro-Animal Genomics And Molecular Breeding, College of Animal Science, ALLTECH-SCAU Animal Nutrition Control Research Alliance, National Engineering Research Center For Breeding Swine Industry, South China Agricultural University, Guangzhou 510642, China. Zhangyl@scau.edu.cn.

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.

Show MeSH
Related in: MedlinePlus