Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture

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Abstract

Background

Bovine milk contains not only a variety of nutritional ingredients but also microRNAs (miRNAs) that are thought to be secreted by the bovine mammary epithelial cells (BMECs). The objective of this study was to elucidate the production of milk-related miRNAs in BMECs under the influence of lactogenic hormones.

Results

According to a microarray result of milk exosomal miRNAs prior to cellular analyses, a total of 257 miRNAs were detected in a Holstein cow milk. Of these, 18 major miRNAs of interest in the milk were selected for an expression analysis in BMEC culture that was treated with or without dexamethasone, insulin, and prolactin (DIP) to induce a lactogenic differentiation. Quantitative polymerase chain reaction (qPCR) results showed that the expressions of miR-21–5p ( P = 0.005), miR-26a ( P = 0.016), and miR-320a ( P = 0.011) were lower in the DIP-treated cells than in the untreated cells. In contrast, the expression of miR-339a ( P = 0.017) in the cell culture medium were lower in the DIP-treated culture than in the untreated culture. Intriguingly, the miR-148a expression in cell culture medium was elevated by DIP treatment of BMEC culture ( P = 0.018). The medium-to-cell expression ratios of miR-103 ( P = 0.025), miR-148a ( P < 0.001), and miR-223 ( P = 0.013) were elevated in the DIP-treated BMECs, suggesting that the lactogenic differentiation-induced secretion of these three miRNAs in BMECs. A bioinformatic analysis showed that the miRNAs down-regulated in the BMECs were associated with the suppression of genes related to transcriptional regulation, protein phosphorylation, and tube development.

Conclusion

The results suggest that the miRNAs changed by lactogenic hormones are associated with milk protein synthesis, and mammary gland development and maturation. The elevated miR-148a level in DIP-treated BMECs may be associated with its increase in milk during the lactation period of cows.

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Most cited references 33

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Prediction of mammalian microRNA targets.

Benjamin Lewis, I-hung Shih, Matthew W Jones-Rhoades … (2003)

MicroRNAs (miRNAs) can play important gene regulatory roles in nematodes, insects, and plants by basepairing to mRNAs to specify posttranscriptional repression of these messages. However, the mRNAs regulated by vertebrate miRNAs are all unknown. Here we predict more than 400 regulatory target genes for the conserved vertebrate miRNAs by identifying mRNAs with conserved pairing to the 5' region of the miRNA and evaluating the number and quality of these complementary sites. Rigorous tests using shuffled miRNA controls supported a majority of these predictions, with the fraction of false positives estimated at 31% for targets identified in human, mouse, and rat and 22% for targets identified in pufferfish as well as mammals. Eleven predicted targets (out of 15 tested) were supported experimentally using a HeLa cell reporter system. The predicted regulatory targets of mammalian miRNAs were enriched for genes involved in transcriptional regulation but also encompassed an unexpectedly broad range of other functions.

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MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers.

Fang Xie, Janos Zempleni, Oliver R. Wood … (2014)

MicroRNAs (miRNAs) regulate genes in animals and plants and can be synthesized endogenously. In milk, miRNAs are encapsulated in exosomes, thereby conferring protection against degradation and facilitating uptake by endocytosis. The majority of bovine miRNAs have nucleotide sequences complementary to human gene transcripts, suggesting that miRNAs in milk might regulate human genes. We tested the hypotheses that humans absorb biologically meaningful amounts of miRNAs from nutritionally relevant doses of milk, milk-borne miRNAs regulate human gene expression, and mammals cannot compensate for dietary miRNA depletion by endogenous miRNA synthesis. Healthy adults (3 men, 2 women; aged 26-49 y) consumed 0.25, 0.5, and 1.0 L of milk in a randomized crossover design. Gene expression studies and milk miRNA depletion studies were conducted in human cell cultures and mice, respectively. For comparison, feeding studies with plant miRNAs from broccoli were conducted in humans. Postprandial concentration time curves suggest that meaningful amounts of miRNA (miR)-29b and miR-200c were absorbed; plasma concentrations of miR-1 did not change (negative control). The expression of runt-related transcription factor 2 (RUNX2), a known target of miR-29b, increased by 31% in blood mononuclear cells after milk consumption compared with baseline. When milk exosomes were added to cell culture media, mimicking postprandial concentrations of miR-29b and miR-200c, reporter gene activities significantly decreased by 44% and 17%, respectively, compared with vehicle controls in human embryonic kidney 293 cells. When C57BL/6J mice were fed a milk miRNA-depleted diet for 4 wk, plasma miR-29b concentrations were significantly decreased by 61% compared with miRNA-sufficient controls, i.e., endogenous synthesis did not compensate for dietary depletion. Broccoli sprout feeding studies were conducted as a control and elicited no detectable increase in Brassica-specific miRNAs. We conclude that miRNAs in milk are bioactive food compounds that regulate human genes. © 2014 American Society for Nutrition.

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Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products.

Xi Chen, Chao Gao, Haijin Li … (2010)

Recent baby formula milk powder contamination incidents have shown that the classic markers or standards in milk quality control are insufficient in identifying "manipulated" poor-quality milk. In the present study, we demonstrated for the first time that cow milk contains large amounts of microRNAs (miRNAs) and that the unique expression profile of milk-specific miRNAs can serve as a novel indicator and possible new standard for the quality control of raw milk and milk-related commercial products, such as fluid milk and powdered formula milk. First, using Solexa sequencing, we systematically screened miRNA expression in raw milk and identified a total of 245 miRNAs in raw milk. Unlike other classic biomarkers whose expression levels are nearly identical at different periods of lactation, individual miRNAs can be significantly altered during lactation process, implicating that miRNAs may be a more accurate indicator to reflect the quality alteration of milk. Second, using TaqMan probe-based miRNA quantitative RT-PCR, we further identified seven miRNAs that have a relatively consistent expression throughout the lactation process, and more importantly, the expression profile of these seven milk-specific miRNAs can serve as an ideal biomarker for discriminating poor-quality or "manipulated" milk from pure raw milk, as well as for the quality control of commercial milk products, such as fluid milk and powdered formula milk. Together, our findings provide a basis for understanding the physiological role of milk miRNAs and a new potential standard for determining the quality of raw milk or milk-related commercial products.

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Author and article information

Contributors

Susumu Muroya:

ORCID: http://orcid.org/0000-0002-2376-9352

+81 29 838 8686 , muros@affrc.go.jp

Journal

Journal ID (nlm-ta): J Anim Sci Biotechnol

Journal ID (iso-abbrev): J Anim Sci Biotechnol

Title: Journal of Animal Science and Biotechnology

Publisher: BioMed Central (London )

ISSN (Print): 1674-9782

ISSN (Electronic): 2049-1891

Publication date (Electronic): 17 February 2016

Publication date PMC-release: 17 February 2016

Publication date Collection: 2016

Volume: 7

Electronic Location Identifier: 8

Affiliations

[ ]Animal Products Research Division, NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901 Japan

[ ]Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori Japan

[ ]International Education and Research Center for Food and Agricultural Immunology (CFAI), Graduate School of Agricultural Science, Tohoku University, Aoba Sendai, Japan

Author information

Susumu Muroya http://orcid.org/0000-0002-2376-9352

Article

Publisher ID: 68

DOI: 10.1186/s40104-016-0068-x

PMC ID: 4756532

PubMed ID: 26889380

SO-VID: 21ee533b-823d-431c-b0bf-4763a0517457

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 19 September 2015

Date accepted : 4 February 2016

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;

Award ID: 25660220

Award Recipient : Susumu Muroya

Custom metadata

ScienceOpen disciplines: Animal science & Zoology

Keywords: bovine mammary epithelial cell,lactogenic differentiation,microrna,milk,secretion

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ScienceOpen disciplines: Animal science & Zoology

Keywords: bovine mammary epithelial cell, lactogenic differentiation, microrna, milk, secretion

Lactogenic hormones alter cellular and extracellular microRNA expression in bovine mammary epithelial cell culture

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Most cited references 33

Prediction of mammalian microRNA targets.

MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers.

Identification and characterization of microRNAs in raw milk during different periods of lactation, commercial fluid, and powdered milk products.

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