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Different miRNA contents between mammary epithelial cells and milk fat globules: a random or a targeted process?

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Abstract

MicroRNAs (miRNAs) are small noncoding RNAs present in milk-derived extracellular vesicles and milk fat globules (MFG). Nucleic acid content between the lactating mammary tissue (MT) and MFG are quite similar but discrepancies exist in their miRNA content. Our objective was to identify the origin of these discrepancies, and to evaluate the existence of a possible mechanism sorting miRNAs that will or will not be exported from the mammary epithelial cells (MECs) in bovine MFG. miR-125b-5p, miR-126-3p, miR-141-3p, and miR-204-5p, chosen on the basis of their abundance in the MT, were quantified using RT-qPCR in lactating cow MT, MFG, and laser capture-microdissected MECs. Two miRNAs (miR-125b-5p and miR-141-3p) were detected in the MT as well as in MFG and MECs. miR-204-5p was detected only in the MT, suggesting that it is very likely expressed in a cell type other than MECs. miR-126-3p was detected both in the MT and in MECs but not in MFG, suggesting a targeting mechanism for miRNAs in MECs. These results highlights differences in miRNA content between MECs and MFG may be due to a possibly not random mechanism for loading MFG with miRNA cargos that could involve a variable distribution in MECs or a sorting mechanism.

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Abbreviations

Ct:

Threshold cycle

EV:

Extracellular vesicle

MT:

Mammary tissue

MFG:

Milk fat

MEC:

Mammary epithelial cell

miRNA:

MicroRNAs

nt:

Nucleotides

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Acknowledgements

We thank D. Roux and the staff at Ruminant Research Unit of Theix (UERT, 63122 Saint Genes-Champanelle, France) for the management of the animals, Chiara Simonetti for her help in the laser capture microdissected MEC sample preparation, and Jose Pires for his helpful discussion.

Funding

This work received a financial support from the PHASE (Physiologie Animale & Systèmes d’Elevage) Department of INRA. Daiane Lago-Novais was awarded a grant from the Brazilian Ministry of Science and Technology “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES). Karol Pawlowski grant was provided by GISA meta-program of INRA (Ruminflame project).

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Author notes

  1. Karol Pawlowski and Daiane Lago-Novais have contributed equally to this work.

Authors and Affiliations

  1. UMR1213 Herbivores, INRAE, Université Clermont-Auvergne, VetAgro Sup, 63122, Saint Genès Champanelle, France

    Karol Pawlowski, Daiane Lago-Novais, Lenha Mobuchon, Yannick Faulconnier, Céline Boby & Christine Leroux

  2. Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland

    Karol Pawlowski

  3. Programa de Pós Graduação em Zootecnia, Universidade Federal da Bahia (UFBA), Av. Adhemar de Barros 500, CEP 40170-110, Salvador, BA, Brazil

    Daiane Lago-Novais & Gleidson Carvalho

  4. UMR1313 Génétique Animale et Biologie Intégrative, AgroParisTech, Université Paris-Saclay, INRAE, 78350, Jouy-en-Josas, France

    Claudia Bevilacqua, Lenha Mobuchon, Nicolas Crapart & Patrice Martin

  5. Plateforme @BRIDGE, INRAE, Domaine de Vilvert, 78350, Jouy-en-Josas, France

    Claudia Bevilacqua & Nicolas Crapart

  6. Excilone, 78990, Elancourt, France

    Nicolas Crapart

Authors
  1. Karol Pawlowski
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  2. Daiane Lago-Novais
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  3. Claudia Bevilacqua
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  4. Lenha Mobuchon
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  5. Nicolas Crapart
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  6. Yannick Faulconnier
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  8. Gleidson Carvalho
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  9. Patrice Martin
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  10. Christine Leroux
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Contributions

CL, KP and DLN conceived, designed, and initiated the project. KP and and DLN performed RT-qPCR analyses. CB, and NC performed laser microdissection. CL, KP and PM wrote the original draft. LM, YF, CB, GC helped to revise the manuscript. CL supervised the project. All authors read and approved the final version.

Corresponding author

Correspondence to Christine Leroux.

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Conflict of interest

The authors declare no competing interests.

Ethical approval

The experiment was conducted at the INRAE Herbipole experimental farm (https://doi.org/10.15454/1.5572318050509348E12). Animal procedures were performed in compliance with Regional Animal Care Committee guidelines of the “Comité d’Ethique en Matière d’Expérimentation Animale-Auvergne” (CEMEA: Auvergne), French Ministry of Agriculture and European Union guidelines for animal research C2EA-02. All mammary biopsies were performed with relevant national legislation and were done by an accredited person (No. of certification: 63–20). Cows were fed ad libitum. The cows were housed in stalls with free access to water.

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11033_2020_5787_MOESM1_ESM.docx

Supplementary file1 (DOCX 40 kb)—Supplementary Table 1: Primers used for RT-qPCR analyses. Supplemental Table 2: RT-qPCR data from mammary tissue (MT) and milk fat globule (MFG). Raw Ct mean are indicated by biological matrix type (MT and MFG). Supplementary Table 3: RT-qPCR data from mammary tissue (MT) and mammary epithelial cells (MEC) obtained by laser capture microdissection. Raw Ct mean are indicated by biological matrix type (MT and MFG).

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Pawlowski, K., Lago-Novais, D., Bevilacqua, C. et al. Different miRNA contents between mammary epithelial cells and milk fat globules: a random or a targeted process?. Mol Biol Rep 47, 8259–8264 (2020). https://doi.org/10.1007/s11033-020-05787-8

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  • DOI: https://doi.org/10.1007/s11033-020-05787-8

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