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Circulating miRNAs. isomiRs and small RNA clusters in human plasma and breast milk

ABSTRACT: Circulating small RNAs. including miRNAs but also isomiRs and other RNA species. have the potential to be used as non-invasive biomarkers for communicable and non-communicable diseases. This study aims to characterize miRNAs. isomiRs and small RNA clusters in biofluids. to compare their profiles. and to help in the establishment of standard guidelines. For this purpose. RNA from plasma and breast milk samples from 15 healthy postpartum mothers was extracted. Small RNA libraries were prepared with the NEBNext® small RNA library preparation kit and sequenced in an Illumina HiSeq2000 platform. After an initial quality control. miRNAs. isomiRs and clusters of small RNAs were annotated using seqBuster/seqCluster framework. The average amount of extracted RNA was 81 ng/mL [standard deviation (SD): 41] and 3985 ng/mL (SD: 3767) for plasma and breast milk. respectively. Mean number of good quality reads was 4.04 million (M) (40.01% of the reads) in plasma and 12.5M (89.6%) in breast milk. One thousand one hundred eighty two miRNAs. 74.317 isomiRs and 1.053 small RNA clusters that included piwi-interfering RNAs (piRNAs). tRNAs. small nucleolar RNAs (snoRNA) and small nuclear RNAs (snRNAs) were detected. Samples grouped by biofluid. with 308 miRNAs. 4.737 isomiRs and 778 small RNA clusters differentially detected. In summary. plasma and milk showed a completely different small RNA profile. In both. miRNAs. piRNAs. tRNAs. snRNAs. and snoRNAs were identified. confirming the presence of non-miRNA species in biofluids

ORGANISM(S): Homo sapiens

PROVIDER: GSE107524 | GEO | 2018/03/14

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.

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Circulating miRNAs. isomiRs and small RNA clusters in human plasma and breast milk

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