Michael W Pfaffl, Benedikt Kirchner, Dominik Buschmann, Melanie Spornraft
TUM, Physiology, Germany
Exosomes are cell-derived vesicles that are present in many tissues and in all biological fluids, including blood, milk, urine, and sweat. Exosomes are proposed to have specialized functions and play a key role in cell-to-cell communication, e.g. in the immune system, in cancer progression, in intercellular signalling, and in cellular waste management. Consequently, there is a growing interest in molecular diagnostic and the clinical application. Very less information is available about the exosomes and their small RNA composition in biofluids such as milk and whether milk possesses its own defined small RNA profile.
First, an optimized RNA isolation method combined with small-RNA Seq was established to profile microRNAs (miRNAs) and a very emerging new class of small RNAs the piwi-interacting RNAs (piRNAs), in bovine plasma and whole blood. The presented data evaluation pipeline offered to analyze data regarding quality and allowed annotation and generation of readcount tables by successive reduction of complexity of data sets and aligning sequencing reads to reference databases.
Second, exosome isolation methods were established for high-yield and high-quality isolation from blood and milk. Exosomes were isolated by differential ultracentrifugation and density gradient centrifugation and further analyzed by western blotting for CD63 and MFGE8 as a general and milk-specific marker, respectively. To assess extracellular vesicles populations, Nanoparticle Tracking Analysis was used for the evaluation of particle count and diameter in distinct gradient fractions. Additionally, protein quantification (BCA assay) and RNA analysis (RT-qPCR) were also performed in exosome-containing fractions.
To generate a holistic overview of all present small RNAs Next Generation Sequencing (small RNA-Seq) was performed on whole blood, plasma, whole milk, and exosomes. Small RNA-Seq was performed using an Illumina HiSeq 2500 and subsequent data analysis was done independently, using either the GGS and GGA stations from Genomatix Software or using freely available python scripts and R-packages. To validate these findings key microRNAs were quantified via RT-qPCR for fold change comparisons. Experimentally validated mRNA targets for regulated microRNAs were taken from the Tarbase 6.0 database from Diana Lab and pathway analyses were generated using Genomatix Pathway System.
RNA Seq clearly showed that milk and exosomes possesses its own unique small RNA profile compared to blood. It highlights its dynamic changes during the first lactation days. Pathway analysis for affected targets revealed significant impact on cell cycle progression, cell adhesion, DNA repair, apoptosis, and oncogenic defense. This study underlines the potential role of microRNAs (and small RNAs in general) in mammary gland physiology and cell-to-cell communication. Milk microRNAs and exosomes seem essential for the mammary gland immune system, but also as an active modulator in the newborn calf.
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