Immune-related microRNAs are abundant in breast milk exosomes

Abstract

Breast milk is a complex liquid rich in immunological components that affect the development of the infant's immune system. Exosomes are membranous vesicles of endocytic origin that are found in various body fluids and that can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, are packaged inside exosomes in human breast milk. Here, we identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes using deep sequencing technology. We found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) are presented and enriched in breast milk exosomes (P < 10(-16), χ(2) test). In addition, compared with exogenous synthetic miRNAs, these endogenous immune-related miRNAs are more resistant to relatively harsh conditions. It is, therefore, tempting to speculate that these exosomal miRNAs are transferred from the mother's milk to the infant via the digestive tract, and that they play a critical role in the development of the infant immune system.

Keywords: breast milk; deep sequencing.; exosome; immune-related miRNAs.

Figure 1

Length distribution and frequency percent of mappable reads. Values are means ± s.d.

Figure 2

Distribution and characterization of miRNAs in breast milk exosomes. (A) Distribution of pre-miRNAs. Out of 1,424 pre-miRNAs deposited in miRBase 17.0 (blue circle), 87 (6.11%) pre-miRNAs have been designated as immune-related pre-miRNAs based on annotation in the Pathway Central database (SABiosciences, MD, USA). Immune-related pre-miRNAs (59 of 452, 13.05%) are enriched in breast milk exosomes (red circle). χ² test: Number of immune-related miRNAs and others detected in breast milk exosomes compared with the total entries in miRBase 17.0. (B) Top ten most highly expressed unique miRNAs. Plot of the unique miRNAs (starting from the miRNA with the highest counts, x-axis) versus their cumulative % of the total counts from 602 unique miRNAs detected in breast milk exosomes for each small RNA library. Values are means ± s.d. The dashed horizontal line at 62.3% represents the level of the top ten unique miRNAs and the % of individual miRNAs is marked by the gray line. (C-E) The expression profile of selected miRNAs in breast milk exosomes. (C) 59 immune-related pre-miRNAs; (D) miR-17-92 cluster and paralogous clusters, and (E) ten well-characterized tissue-specific pre-miRNAs.

Figure 2

Distribution and characterization of miRNAs in breast milk exosomes. (A) Distribution of pre-miRNAs. Out of 1,424 pre-miRNAs deposited in miRBase 17.0 (blue circle), 87 (6.11%) pre-miRNAs have been designated as immune-related pre-miRNAs based on annotation in the Pathway Central database (SABiosciences, MD, USA). Immune-related pre-miRNAs (59 of 452, 13.05%) are enriched in breast milk exosomes (red circle). χ² test: Number of immune-related miRNAs and others detected in breast milk exosomes compared with the total entries in miRBase 17.0. (B) Top ten most highly expressed unique miRNAs. Plot of the unique miRNAs (starting from the miRNA with the highest counts, x-axis) versus their cumulative % of the total counts from 602 unique miRNAs detected in breast milk exosomes for each small RNA library. Values are means ± s.d. The dashed horizontal line at 62.3% represents the level of the top ten unique miRNAs and the % of individual miRNAs is marked by the gray line. (C-E) The expression profile of selected miRNAs in breast milk exosomes. (C) 59 immune-related pre-miRNAs; (D) miR-17-92 cluster and paralogous clusters, and (E) ten well-characterized tissue-specific pre-miRNAs.

Figure 2

Distribution and characterization of miRNAs in breast milk exosomes. (A) Distribution of pre-miRNAs. Out of 1,424 pre-miRNAs deposited in miRBase 17.0 (blue circle), 87 (6.11%) pre-miRNAs have been designated as immune-related pre-miRNAs based on annotation in the Pathway Central database (SABiosciences, MD, USA). Immune-related pre-miRNAs (59 of 452, 13.05%) are enriched in breast milk exosomes (red circle). χ² test: Number of immune-related miRNAs and others detected in breast milk exosomes compared with the total entries in miRBase 17.0. (B) Top ten most highly expressed unique miRNAs. Plot of the unique miRNAs (starting from the miRNA with the highest counts, x-axis) versus their cumulative % of the total counts from 602 unique miRNAs detected in breast milk exosomes for each small RNA library. Values are means ± s.d. The dashed horizontal line at 62.3% represents the level of the top ten unique miRNAs and the % of individual miRNAs is marked by the gray line. (C-E) The expression profile of selected miRNAs in breast milk exosomes. (C) 59 immune-related pre-miRNAs; (D) miR-17-92 cluster and paralogous clusters, and (E) ten well-characterized tissue-specific pre-miRNAs.

Figure 2

Distribution and characterization of miRNAs in breast milk exosomes. (A) Distribution of pre-miRNAs. Out of 1,424 pre-miRNAs deposited in miRBase 17.0 (blue circle), 87 (6.11%) pre-miRNAs have been designated as immune-related pre-miRNAs based on annotation in the Pathway Central database (SABiosciences, MD, USA). Immune-related pre-miRNAs (59 of 452, 13.05%) are enriched in breast milk exosomes (red circle). χ² test: Number of immune-related miRNAs and others detected in breast milk exosomes compared with the total entries in miRBase 17.0. (B) Top ten most highly expressed unique miRNAs. Plot of the unique miRNAs (starting from the miRNA with the highest counts, x-axis) versus their cumulative % of the total counts from 602 unique miRNAs detected in breast milk exosomes for each small RNA library. Values are means ± s.d. The dashed horizontal line at 62.3% represents the level of the top ten unique miRNAs and the % of individual miRNAs is marked by the gray line. (C-E) The expression profile of selected miRNAs in breast milk exosomes. (C) 59 immune-related pre-miRNAs; (D) miR-17-92 cluster and paralogous clusters, and (E) ten well-characterized tissue-specific pre-miRNAs.

Figure 2

Distribution and characterization of miRNAs in breast milk exosomes. (A) Distribution of pre-miRNAs. Out of 1,424 pre-miRNAs deposited in miRBase 17.0 (blue circle), 87 (6.11%) pre-miRNAs have been designated as immune-related pre-miRNAs based on annotation in the Pathway Central database (SABiosciences, MD, USA). Immune-related pre-miRNAs (59 of 452, 13.05%) are enriched in breast milk exosomes (red circle). χ² test: Number of immune-related miRNAs and others detected in breast milk exosomes compared with the total entries in miRBase 17.0. (B) Top ten most highly expressed unique miRNAs. Plot of the unique miRNAs (starting from the miRNA with the highest counts, x-axis) versus their cumulative % of the total counts from 602 unique miRNAs detected in breast milk exosomes for each small RNA library. Values are means ± s.d. The dashed horizontal line at 62.3% represents the level of the top ten unique miRNAs and the % of individual miRNAs is marked by the gray line. (C-E) The expression profile of selected miRNAs in breast milk exosomes. (C) 59 immune-related pre-miRNAs; (D) miR-17-92 cluster and paralogous clusters, and (E) ten well-characterized tissue-specific pre-miRNAs.

Figure 3

Characterization of breast milk miRNAs. The expression changes of a spiked-in C. elegans miRNA and of the top ten most highly expressed unique miRNAs from the small RNA libraries across various harsh conditions. Total RNA was extracted and then analyzed by q-PCR. Breast milk was (A) incubated at 26°C for 0.5, 1, 2, 4, 8 or 24 hours, (B) subjected to six freeze-thaw cycles at -20°C, (C) treated with RNase A and T1 for 60 min at 37°C, and (D) incubated at 100°C for 10 min.

Figure 3

Characterization of breast milk miRNAs. The expression changes of a spiked-in C. elegans miRNA and of the top ten most highly expressed unique miRNAs from the small RNA libraries across various harsh conditions. Total RNA was extracted and then analyzed by q-PCR. Breast milk was (A) incubated at 26°C for 0.5, 1, 2, 4, 8 or 24 hours, (B) subjected to six freeze-thaw cycles at -20°C, (C) treated with RNase A and T1 for 60 min at 37°C, and (D) incubated at 100°C for 10 min.

Figure 3

Characterization of breast milk miRNAs. The expression changes of a spiked-in C. elegans miRNA and of the top ten most highly expressed unique miRNAs from the small RNA libraries across various harsh conditions. Total RNA was extracted and then analyzed by q-PCR. Breast milk was (A) incubated at 26°C for 0.5, 1, 2, 4, 8 or 24 hours, (B) subjected to six freeze-thaw cycles at -20°C, (C) treated with RNase A and T1 for 60 min at 37°C, and (D) incubated at 100°C for 10 min.

Figure 3

Characterization of breast milk miRNAs. The expression changes of a spiked-in C. elegans miRNA and of the top ten most highly expressed unique miRNAs from the small RNA libraries across various harsh conditions. Total RNA was extracted and then analyzed by q-PCR. Breast milk was (A) incubated at 26°C for 0.5, 1, 2, 4, 8 or 24 hours, (B) subjected to six freeze-thaw cycles at -20°C, (C) treated with RNase A and T1 for 60 min at 37°C, and (D) incubated at 100°C for 10 min.