The distribution of small RNAs in milk and their functional relation to mammary gland physiology

Michael W Pfaffl1, Benedikt Kirchner1, Alexander Hahn2, Vladimir Benes3
1Physiology Weihenstephan, Technical University Munich, Germany; 2Genomatix Software GmbH, Munich, Germany; 3EMBL Genomics Core Facility (GeneCore), Heidelberg, Germany

Small RNAs, in particular microRNAs, regulate gene expression by post transcriptional binding and thereby suppressing protein translation. They are present in most eukaryotic cells and play an important role in nearly all physiological and regulative processes. Small RNAs were detected in various extracellular locations such as blood plasma and urine. However, very less information is available about the small RNA composition in biofluids such as milk and whether milk possesses its own defined small RNA profile differing from blood. To generate a holistic overview of all present small RNAs in bovine blood and milk and to identify shifts in their profiles, small RNA NGS was performed on whole blood and milk samples during the progressing lactation period. Small RNA-Sequencing was performed using an Illumina HiSeq and subsequent data analysis was done independently using either the GGS and GGA stations from Genomatix Software GmbH (Munich, Germany) or using freely available python scripts and R-packages (Bioconductor). First focus was on the dynamic regulation of microRNAs in milk in comparison to blood. Significantly regulation of miRNAs between different tissues and lactation stages was defined by a fold change of expression of at least 2-fold and a Benjamini-Hochberg adjusted p-value of less than 0.05. To validate these findings key microRNAs were quantified via RT-qPCR for fold change comparisons. Experimentally validated mRNA targets for regulated miRNAs were taken from the Tarbase 6.0 database from Diana Lab (Athens, Greece) and pathway analyses were generated using GePS (Genomatix Pathway System). RNA-sequencing clearly showed that milk possesses its own unique small RNA profile compared to blood and highlights its changes during the lactation period, with focus on the colostrum phase. Pathway analysis for affected targets revealed heavy influences on cell cycle progression, cell adhesion, DNA repair, apoptosis, and oncogenic defense. This underlines the potential role of microRNAs in mammary gland and milk not only for the mammary immune system, but also as an active modulator of gene expression in newborn calf.

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