Unique microRNA profiles revealed in different fractions of human milk and maternal blood during lactation
ABSTRACT: 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  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')  the sample data tables contain raw data.
SUBMITTER: Donna Savigni Foteini HassiotouDonna T GeddesJohn NewnhamChin-Tat LaiMohammed AlsaweedPeter E Hartmann
Project description:This study examines circulating plasma miRNAs of adult human subjects before and after bovine milk intake. We sought to confirm and expand findings previously reported by Baier, et al., J Nutr, 2014, PMID 25122645, using the same samples. Plasma samples were provided by the Zempleni lab (see Baier, et al., J Nutr, 2014, PMID 25122645) and had been taken from five healthy donors prior to milk intake (T0) and at three hours post-intake (T3). miRNA profiles were generated for each sample by OpenArray (Thermo Fisher).
Project description:Human milk (HM) is rich in miRNAs, which are thought to contribute to infant protection and development. We used deep sequencing to profile miRNAs in the cell and lipid fractions of HM obtained post-feeding from 10 lactating women in month 2, 4, and 6 postpartum. In both HM fractions, 1,195 mature known miRNAs were identified, which were positively associated with the cell (p=0.048) and lipid (p=0.010) content of HM. An additional 5,167 novel miRNA species were predicted, of which 235 were high-confidence miRNAs expressed in ≥3 samples with total reads of >20. HM cells contained more known miRNAs than HM lipids (1,136 and 835 respectively, p<0.001), with 146 of the common species upregulated in HM cells and 133 in HM lipids (p<0.05). Further, the profile of the novel miRNAs was very different between the two HM fractions, with the majority conserved in the cell fraction and being mother-specific. However, out of the 776 known miRNA species commonly present in the two HM fractions, 63.9% were similarly expressed (p>0.05), with great similarities in the profile of top 20 known miRNAs. These were largely similar also between the three lactation stages examined, as were the total miRNA concentration, and the number and overall expression of the known miRNAs commonly present in the two HM fractions (p>0.05). Yet, approximately a third of known miRNAs were differentially expressed during the first 6 months of lactation (p<0.05), with more pronounced miRNA upregulation seen in month 4. These findings indicate that although the total miRNA concentration of HM cells and lipids provided to the infant does not change in the first 6 months of lactation, the miRNA composition is somewhat altered, particularly in month 4 compared to months 2 and 6. This may reflect the remodeling of the gland in response to infant feeding patterns, which usually change after exclusive breastfeeding, and thus adaptation to infant needs. Overall design: Human milk samples were collected from 10 women longitudinally, at month 2, 4 and 6 postpartum. Total RNA and miRNA were extracted and quantified from cell and lipid fractions. Next-generation sequencing using Illumina HiSeq 2000 was performed on 45 samples (30 milk cells, and 15 lipid samples) to profile mature miRNAs in the breast milk cellular and lipid fractions longitudinally. Filtered (clean) reads were aligned to the latest version of miRBase 21.0. Also, mirdeep software was used to predict novel miRNAs. DESeq and Linear mixed effect models and ANOVA were used to compare miRNA expression levels between samples.
Project description: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 Overall design: Freshly expressed breastmilk samples were collected from 10 mothers on month 2 of lactation. Total RNA and miRNA content were quantified in pre- and post-feed samples from each mother. Next generation sequencing using illumina HiSeq 2000 was performed on 20 samples (one sample pre-feed and one sample post-feed from each mother) to profile mature miRNAs in the breastmilk cellular fraction. Filtered (clean) reads were aligned to the latest version of miRBase 21.0. Also, mireap software was used to predict novel miRNAs. Linear mixed effect models and ANOVA were used to compare miRNA expression levels between samples.
Project description:Human milk is highly recommended for infant during the first six month of life by World Healthy Organisation (WHO). Human milk is not only rich in macro-nutritional components, but also rich in cells and molecules. MicroRNAs are small non-coding RNAs, which enriched in human milk. These molecules are vital in enormous biological and cellular functions including immune system and in response to infections. By using deep sequencing method, 770,374,554 raw reads were generated from all samples (n=26). Then, filter analysis was done to remove 81,091,772 (10.5%), and 689,282,782 clean reads (89.5%) were considered as clean reads, which was retained for the subsequent bioinformatics analysis. Annotation and matching reads to miRBase revealed1780 mature known microRNAs identified in human milk cells and lipids derived from healthy, cold and other infection types mothers. In particular, 1680 known microRNAs were determined in infected mothers (n=14), while 1,606 known microRNAs in healthy mothers (n=12). Of these known microRNAs, 453 microRNAs were differentially expressed (p<0.05) between healthy and infected samples. The majority of the highly expressed miRNAs in all samples, in particular top 20 microRNAs, were also differentially expressed between healthy and infections. Further, 592 novel mature microRNA sequences were predicted, with only 65,878 total reads. Amongst the total reads of the novel microRNAs, top 20 novel miRNAs were found to contributed in 73.3% (total reads 48,295) of the total reads (65,878). Overall design: Human milk samples were collected from 11 mothers in healthy (n=6) and in infection (n7) conditions. Human milk samples were fractionated to obtain cell and lipid fractions. Total RNA and miRNA were extracted and quantified from cell and lipid fractions. Next generation sequencing using illumina HiSeq 2000 was used to profile microRNAs in 26 samples, where 13 cells and 13 lipids. Filtered (clean) reads were aligned to the latest version of miRBase 21.0. Also, mirdeep software was used to predict novel miRNAs. DESeq and Linear mixed effect models and ANOVA were used to compare miRNA expression levels between samples and conditions
Project description:This study examines circulating PBMC miRNAs of adult human subjects before and after bovine milk intake. We sought to confirm and expand findings previously reported by Baier, et al., J Nutr, 2014, PMID 25122645, using the same samples. PBMC RNA samples were provided by the Zempleni lab (see Baier, et al., J Nutr, 2014, PMID 25122645) and had been taken from five healthy donors prior to milk intake (T0) and at six hours post-intake (T6). miRNA profiles were generated for each sample by OpenArray (Thermo Fisher).
Project description:Milk is an indispensable source of infant nutrition in all mammals, made up of complex constituents needed for infant nourishment and immunity. Comparison of miRNA profiles between infected and non-infected/control Holstein cattle could provide important information on the differences in composition of milk at the level of miRNA, if any, and broaden our perspective and understanding of the effect of pathology on cellular compositions and functions. In the present study we therefore analyzed the expression profiles of bovine milk exosomal miRNAs during S. uberis infection and identified 328 known miRNAs and 82 high-confidence miRNA candidates by deep sequencing. The top 10 miRNAs in both control and infected replicates accounted for approximately 80% of total counts, which were predicted to target 605 genes using two computational approaches. In addition, 15 significantly differentially expressed miRNAs were identified between control and infected replicates during S. uberis infection, and a total of 1,852 unique genes were predicted to be targeted by these miRNAs. Ingenuity Canonical Pathways and Diseases and Biological Function analyses using IPA indicated that the identified miRNAs targets mainly enriched in regulating the innate and adaptive immune responses in newborns, as well as infant growth and development. The characterization of these miRNAs could contribute to a better understanding of the molecular mechanisms involved in lactation physiology and milk imparted immune function in the dairy cattle. Overall design: Three exosomal small RNA libraries were constructed from S. uberis infected bovine breast milk at 2 days post-infection (dpi), and correspondingly three libraries from bovine milk prior to infection were used as controls. They were named control (1, 2 and 3) and infection (1, 2 and 3), respectively.
Project description:Breast milk is a complex liquid that enriched in immunological components and affect the development of the infant immune system. Exosomes, the membranous vesicles of endocytic origin, are ubiquitously in various body fluids which can mediate intercellular communication. MicroRNAs (miRNAs), a well-defined group of non-coding small RNAs, in human breast milk are packaged inside exosomes. Here, we present the identification of miRNAs in human breast milk exosomes using deep sequencing technology. We found that the immune-related miRNAs are enriched in breast milk exosomes, and are resistant to the general harsh conditions. Four small RNA libraries in human breast milk exosomes from four healthy women (30 +/- 0.9 years old, primiparity) when the infant were aged at 60 days were sequenced.
Project description:MicroRNAs (miRNAs) are small, endogenous, non-protein coding RNAs that are an important means of post-transcriptional gene regulation. Deletion of Dicer, a key miRNA processing enzyme, is embryonic lethal in mice, and tissue-specific Dicer deletion results in developmental defects. Using a conditional knockout model, we generated mice lacking Dicer in the adrenal cortex. These Dicer knockout (KO) mice exhibited perinatal mortality and failure of the adrenal cortex during late gestation between embryonic day 16.5 (E16.5) and E18.5. Further study of Dicer KO adrenals demonstrated a significant loss of Sf1 expressing cortical cells that was histologically evident as early as E16.5 coincident with an increase in p21 and cleaved-caspase 3 staining in the cortex. However, peripheral cortical proliferation persisted in KO adrenals as assessed by anti-PCNA staining. To further characterize the embryonic adrenals from Dicer KO mice, we performed microarray analyses for both gene expression and miRNA on purified RNA isolated from control and KO adrenals of E15.5 and E16.5 embryos. Consistent with the absence of Dicer and the associated loss of miRNA-mediated mRNA degradation, we observed an up-regulation of a small subset of adrenal transcripts in Dicer KO mice, most notably the transcripts coded by the genes Nr6a1 and Acvr1c. Indeed, several miRNAs, including let-7, miR-34c, and miR-21 that are predicted to target these genes for degradation, were also markedly down-regulated in Dicer KO adrenals. Together these data suggest a role for miRNA mediated regulation of a subset of genes that are essential for normal adrenal growth and homeostasis. Adrenals from control and Dicer KO litter mates were pooled separately from 4 individual litters, resulting in a total of 4 control (cre-) and 4 Dicer KO biological
Project description:Frozen blocks of human embryonic limb tissue were sectioned and laser capture microdissected to collect three human chodrocyte tissues - precursor, differentiated and hypertrophic chondrocytes.Total RNA including miRNAs was isolated and analyzed using Applied Biosystems OpenArray® Real-Time PCR system. RT-PCR miRNA expression profiling. miRNA expression from three chondrocyte sub types - precursor, differentiated and hypertrophic chondrocytes were analyzed using TaqMan® OpenArray® Human MicroRNA Panel.
Project description:The breast milk plays a crucial role in shaping the initial intestinal microbiota and mucosal immunity of the infant. Interestingly, breastfeeding has proven to be protective against the early onset of immune-mediated diseases including type 1 diabetes (T1D). Studies have shown that exosomes from human breast milk (HM) are enriched in immune-modulating miRNAs suggesting that exosomal miRNAs transferred to the infant could play a critical role in the development of the infant’s immune system. In this study, we extracted exosome exosomal microRNAs (exomiRs) from breast milk of type 1 diabetic and healthy lactating mothers, in order to identify any differences in the exomiR content between the two groups