Milk fat globules as a non-invasive source of mammary microRNA

Christine Leroux, INRA, Saint-Genès-Champanelle, France

D. Lago-Novais1,2*, K. Pawlowski1*, J.A.A. Pires1, C. Bevilacqua3, L. Mobuchon1,3, C. Boby1,Y. Faulconnier1, S. Bes1, P. Martin3, and C. Leroux1
1. INRA, VetAgroSup, Saint-Genès-Champanelle, France;
2. Universidade Federal da Bahia, Brazil;
3. INRA, AgroParisTech, Jouy-en-Josas, France
*: Equal contribution of the two first authors

Genomic mechanisms involved in the regulation of milk component synthesis and their secretion are not completely understood. MicroRNA (miRNA, small noncoding RNA) have been shown to influence mammary gland (MG) development and function. Samples for MG gene expression studies are usually obtained via invasive and expensive methods (biopsy or post-mortem) that limit high throughput and dynamic genomic analyses. Recently, milk fat globules (MFG) have been used to assess the mRNA content of the secretory mammary epithelium first in humans1,2 and in ruminants3,4, thus demonstrating that MFG may be an alternative to utilization of MG tissue as a source of mRNA representative of mammary epithelial cells for subsequent gene expression studies. However, the use of the bovine MFG as the source of miRNA has not been studied. The objective of this study was to assess MFG as a source of small RNAs to profile miRNA and to ensure that MFG miRNomes are representative of MG miRNA expression, by comparing the expression of candidate miRNA in MFG and MG sampled from mid-lactation Holstein cows.

Total RNA was extracted from MFG (n=6) and MG (n=6) using TRIzol (ThermoFisher, Inc, USA). Nine miRNA (miR-29a, miR-125b, miR-126, miR-141, miR-148a, miR-204, miR-223, miR-320a, miR-494), chosen on the base of their high abundance in the MG5,6. Their expression was quantified by RT-qPCR (ThermoFisher Inc, USA). The results are expressed as fold change of MFG relative to MG data and using U6 as internal reference. Statistical analyses were performed using a t-test (DataAssitTM software, ThermoFisher Inc) and P < 0.05 was considered significant. Target genes of studied miRNA and corresponding putative pathways were investigated using Pathway Studio software. Among the nine miRNA, two (miR-126 and miR-204) were not detected in MFG whereas they were abundant in MG, three were significantly more abundant in MG than in MFG: miR-29a, miR-125b, and miR-148a exhibiting a fold change value of 23.2, 13.9 and 8.7, respectively. Four studied miRNA were detected in both MFG and MG as equally expressed. Such results suggest different hypothesis. The first one is that there are selective mechanisms of miRNA transfer to milk fat. However, we cannot exclude the second hypothesis that miRNA lacking in MFG are not expressed in epithelial cells, but arising from other MG cell-types. Laser microdissection of mammary epithelial cells, currently in progress, will allow to corroborate the first or the second hypothesis. Among the biological processes targeted by the two miRNA detected exclusively in the MG tissue, there were the adherens junctions and cell-substrate adhesion which are involved in cellular structure. In addition, common biological processes were only enriched with miR-126 and miR-204. Among them, two were related to differentiation and related to regulation mechanisms. Together these biological processes related to miR-126 and miR-204 could be specific to MG cells. In conclusion, MFG can be used as non-invasive source of miRNA but do not reflect exactly the MG miRNome. Further research is warranted on the composition of MFG miRNome and on the modulation of their secretion in milk. A comprehensive study is currently in progress to compare miRNome from MG and MFG using bovine miRNA microarrays in dairy cows submitted to a nutrient restriction and inflammation protocol. A better knowledge of MFG’s miRNome is crucial before using miRNA from MFG as biomarkers of nutritional status or mammary inflammation in ruminants. Moreover, the MFG miRNome could increase the healthy quality of milk for the consumers. Indeed, the action of milk miRNA could be modulated by their packaging within MFG and/or extracellular vesicles which may play an important role for their transfer and function in consumers7. 1Maningat et al. (2007) J. of endocrinology 195: 503-511; 2Maningat et al. (2009) Physiological Genomics, 37: 12-22; 3Brenaut et al. (2012) J. of Dairy Science 95: 6130-6144; 4Canovas et al., (2014) Scientific Reports 4: 5297; 5Le Guillou et al. (2014) PloS ONE 9, e91938; 6Mobuchon et al. (2015) BMC genomics 16: 285; 7Alsaweed et al. (2015) J. of Cellular Biochemistry 116: 2397-2407

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