2017 Cardiovascular Research Day Abstract Book

101
Identification of a novel lipoprotein microRNA carrier in plasma
Wanying Zhu 1 • Danielle L. Michell 1 • Ryan M. Allen 1 • Kasey C. Vickers 1
Vanderbilt University Medical
Staff
Recently, multiple groups have detected of microRNAs (miRNA) in plasma by high-throughput
sequencing. Circulating sRNAs are likely protected from RNases in plasma through their association
with lipid carriers. Our lab previously reported that lipoproteins (HDL and LDL) transport miRNAs
in plasma within potential intercellular communication networks. Based on these previous studies,
we sought to define the distribution of miRNAs across lipid carriers in plasma.
Methods: To specifically precipitate carriers, whole plasma and pre-purified HDL (density-gradient
ultracentrifugation) treated with lipid removal agents (LRA and Cleanascite) for the separation of
lipid-associated miRNAs. Plasma samples from humans and mice were fractionated using sizeexclusion
chromatography (FPLC, Superdex 200 Increase in triplicate). Each fraction was assessed
for total cholesterol, triglycerides, protein, phospholipids, and markers for lipoproteins and
extracellular vesicles by western blotting. Carrier sizes in each fraction were quantified using
dynamic light scattering. Total RNA was isolated from whole fractions and separated contents, lipid
pellets and supernatants, after centrifugation. Real-time PCR and high-throughput sequencing were
used to quantify miRNAs.
Results: Results demonstrated that Cleanascite efficiently removed lipids from plasma without
damaging lipoproteins, as determined by the lack of phospholipids and apolipoproteins in the
supernatants. Candidate miRNAs (miR-24-3p, miR-26a-5p, miR-146a-5p, miR-92a-3p, and miR-
223-3p) were detected in fractions corresponding to HDL and LDL based on the distribution of
cholesterol and triglycerides. Strikingly, candidate miRNAs also entirely separated with lipids in
fractions that contained low cholesterol and high phospholipid content with a mass corresponding
to very small HDL. These fractions were found to contain apoA-I and spherical particles