Late Breaking Oral Abstract Australasian Extracellular Vesicles Conference 2018

Origin, biogenesis and bio-distribution of milk-borne extracellular microRNAs (#41)

Veronica Barragán-Borrero 1 , Daniel Van Leeuwen 1 , Olivier Voinnet 2 , Bogdan Mateescu 1
  1. Group RNA silencing and extracellular RNAs, RNA biology laboratory, Swiss Federal Institute of Technology Zurich,, Switzerland
  2. RNA biology laboratory, Swiss Federal Institute of Technology Zurich, Switzerland

Extracellular RNAs (exRNAs), stabilized within protein complexes or extracellular vesicles (EVs), not only emerged as promising circulating biomarkers, but also as potential new players in cell-to-cell communication in mammals, although their functional relevance in vivo remains controversial. Here we provide evidence that mammary gland and milk, represent excellent model systems to unambiguously explore exRNA and EVs biogenesis, bio-distribution and potential uptake in physiological conditions. 

First, we show that milk carries relatively high level of RNAs, including full length rRNA, mRNA and microRNAs (miRNAs), and also specific cytoplasmic RNA silencing factors, such as Argonautes (AGOs), all primarily concentrated in milk fat globules (MFGs). Second, Using AGOs knockout mouse, we demonstrate that AGO1, AGO3 or AGO4 proteins are not essential for miRNA stabilization and packaging in milk, thereby suggesting an AGO2 dependent process or functional redundancy between AGO2 factors. Lactocyte-specific expression of tagged- AGO2 in mouse, and small RNA sequencing comparison of AGO2-bound miRNAs in lactocytes and in produced milk allowed us to investigate selective sorting of miRNA in milk, and represent the first report of loaded-miRNA profiling in a biofluid and its coupled producing cell type in vivo. Third, using specific miRNA knockout mice, and lactocyte-specific expression of membrane-bound fluorescent proteins followed by high-resolution flow cytometry detection in the gut, we report the first bio-distribution map of milk EVs and of two abundant milk miRNAs (miR-22 and miR-21) along the intestine of day 3/6/9 suckling pups. Our results demonstrate high correlation between milk-born EVs and miRNAs, and an age-dependent clearance/stabilization of milk miRNAs. Importantly, we could not detect milk-borne miR-21 or miR-22 in peripheral organs, suggesting inefficient uptake and/or systemic transfer from mouse neonate intestine. 

In conclusion, by combining biochemistry, genetic and cell biology approaches, our study represents one of the most advanced description of the biogenesis mechanisms and biodistribution of physiological extracellular miRNA in vivo, Building on these innovative approaches, we are currently investigating the biogenesis and function of milk EVs and exRNAs within mammary gland (MG) duct during lactogenic and involution phase.