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Small RNA sequencing of human milk cells reveals numerous known and novel miRNAs, with no effects of milk removal

ABSTRACT: Very little is known about miRNAs found in breastmilk cells, which also reflect the cells of the lactating mammary epithelium. Our hypothesis is that breastmilk cells are richer in miRNA compared to other milk fractions, such as skim milk. Further, the effects of milk removal by the infant on milk cell miRNA content and/or composition have not been investigated. Breastmilk cells conserved higher miRNA content than previously published lipid and skim fractions of breastmilk as well as other known sources of miRNA in humans. Specifically, 1,467 known mature miRNAs were identified and a further 1996 novel miRNAs, of which 89 were highly expressed. As previously shown, post-feed milk contained more cells than pre-feed milk, and the same was observed for miRNA content. However, no statistically significant difference was found in the expression of the total known and novel miRNAs between pre- and post-feed milk (p=0.76), although 27 known miRNAs and 1 novel miRNA were higher expressed in post-feed milk. As expected, samples richer in viable cells contained more known miRNAs (p = 0.01). Functional analysis of the top 10 most highly expressed known miRNAs showed that they may be potentially involved in crucial roles for the infant, including body fluid balance, thirst, appetite, immune responses, and development. In conclusion, breastmilk is highly rich in miRNA which may play important functions in the breastfed infant and the lactating breast. Milk removal by the infant can influence the total miRNA content of breastmilk, similar to its cell and fat content, but the miRNA composition remains constant

ORGANISM(S): Homo sapiens

PROVIDER: GSE71098 | GEO | 2016/10/04

SECONDARY ACCESSION(S): PRJNA290361

REPOSITORIES: GEO

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Project description:Human milk (breastmilk) is much more than nutrition for the infant, containing an array of regulatory agents with immunoprotective and developmental functions. Amongst those, microRNAs (miRNAs) have recently been identified, with their properties, roles, origin and distribution in breastmilk as well as in the mammary gland being still undetermined. In this study, we examined the miRNA profile of different fractions of human milk (cells and lipids) using the OpenArray system (Applied Biosystems, 770 miRNA species measured per sample) and compared it with maternal peripheral blood mononuclear cells (PBMCs) and plasma. Although PBMCs were the richest group in miRNA species, plasma showed very low expression pattern. Thus, the human milk fractions (cells, lipid) and skim milk (not being investigated in this study) were found to conserve higher levels of miRNAs than blood in general. Specifically, human milk cell miRNA quantity was found relatively close to PBMCs, and higher than milk lipids. Correlation and clustering analyses indicated that miRNA expression and types of milk cells were highly similar to those in lipids. Milk miRNAs showed a slight correlation to PBMCs, so PBMCs potentially are not contributing to milk miRNAs. Plasma was different to all other three groups in miRNA content and expression pattern. Further, two infant formulae (a plant-based and a cow milk-based) were compared to human milk and found to contain significantly fewer miRNA species than human milk cells and lipids (p>0.001). Taken together with previous studies on miRNAs, our findings demonstrate that human milk is one of the richest sources of miRNAs among human body fluids. As a non-invasive and plentiful source of miRNAs, human milk could be used as a disease biomarker for the mammary gland, with potential in assessing lactation performance. Finally, gene target and pathways analyses identified several target mRNAs regulated by miRNAs found to be abundant in breastmilk. Given the recently identified stability and function of food-derived miRNAs in regulating mammalian genes, we propose that breastmilk is a rich source of miRNA ingested by the infant during the first months of life, and which potentially contribute to early infant development. 10 exclusively breastfeeding dyads were recruited. 10 whole milk and 10 whole blood samples were collected and fractionated to obtain 10 milk cells, 10 milk lipid, 10 mononeucleoted blood cells (PBMCs), and 10 plasma. In addition to the above 40 samples, 2 infant formula were profiled. 4 different extraction kits were used, miRNeasy mini Kit for human milk cell and PBMC samples. miRCURY RNA Isolation-Biofluids Kit for human milk lipid samples and both infant formulae. mirVana PARIS Kit for plasma samples. NanoDrop 2000 and Bioanalyzer 2100 were used to determine concentration and purity of the extracted miRNA from all samples (n=42). miRNA OpenArray panel system (Life Technologies, CA, USA) was used to profile 754 human mature miRNAs in samples. RNU48, RNU44 and U6 rRNA were used as housekeeping controls for normalisation. ath-miR159a was used as a negative control for human samples. GeneGO and Ingenuity Pathway Analysis were used to determine biological pathways. Please note that normalization of miRNAs was done in R but without generating deltaCT values, thus [1] only the list of normalized miRNA with Ct vlaue between 8 and 29 and that detected in at least 4 samples out of 10 analysed in each group is provided ('normalized_miRNAs_list.txt') [2] the sample data tables contain raw data.

Project description:Effect of breed in mid lactation Holstein (H) and Montbéliarde (M) cows on mammary glande miRNA profile. Genetic polymorphisms are known to influence milk production and composition. However, genomic mechanisms involved in the genetic regulation of milk component synthesis are not completely understood. MicroRNAs (miRNA) regulate gene expression. The objective of the present study was to compare mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, with different dairy performances. Milk, fat, protein, and lactose yields were lower in Montbéliarde than in Holstein cows. MiRNomes obtained using RNA-Seq technology from the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) lactating cows revealed 623 distinct expressed miRNAs, among which 596 were known and 27 were predicted miRNAs. The comparison of their abundance in the mammary gland of Holstein versus Montbéliarde cows showed 22 differentially expressed miRNAs (Padj ≤ 0.05). Among them, 11 presented a fold change ≥2, with 2 highly expressed miRNAs (miR-100 and miR-146b). Without taking into account the fold change, the differential miRNA with the highest abundance was miR-186, which is known to inhibit cell proliferation and epithelial-to-mesenchymal transition. Data mining showed that the 17 differentially expressed miRNAs with more than 20 reads on average, regulate mammary gland plasticity and may be related to the observed differences in milk production between Holstein and Montbéliarde, which are two breeds with different mammogenic potential. Some of the 17 miRNAs could potentially target mRNAs involved in signaling pathways (such as mTOR) and in lipid metabolism, thereby suggesting that they could influence milk composition. In conclusion, we showed differences in mammary gland miRNomes of two dairy bovine breeds. These differences suggest a potential role of miRNAs in mammary gland plasticity and in milk component synthesis related to milk production and composition.

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Small RNA sequencing of human milk cells reveals numerous known and novel miRNAs, with no effects of milk removal

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