Clin Res Cardiol 101, Suppl 1, April 2012

V128 - Characterisation of circulating protein-bound miRNAs
 
L. Ghaeni1, J. Wagner2, D. Leistner3, S. De Rosa3, S. Fichtlscherer3, A. M. Zeiher3, S. Dimmeler4
 
1Med. Klinik III/Kardiologie, Universitätsklinikum Frankfurt am Main, Frankfurt am Main; 2Institut für Vascular Signalling, Klinikum der Goethe-Universität, Frankfurt am Main; 3Zentrum Innere Medizin III, Schwerpunkt Kardiologie, Universitätsklinikum Frankfurt am Main, Frankfurt am Main; 4Zentrum für Molekulare Medizin, Goethe Universität, Institut für Kardiovaskuläre Regeneration, Frankfurt am Main;
 
MicroRNAs (miRs) are small non-coding RNAs, which regulate multiple target genes by binding to mRNAs thereby inducing degradation or blocking protein translation. However, recent studies demonstrated that miRs also can be detected in a remarkably stable form in the circulating blood and may be suitable as biomarkers for various diseases. Cardiac or muscle derived miRs such as miR-133 or miR-499 are released into the circulation in patients with acute myocardial infarction. However, endothelial (miR-126, miR-92a) or smooth muscle enriched miRs (miR-145) were reduced in patients with stable coronary artery disease suggesting different mechanisms of release, degradation or up-take. Circulating miRs can be protected against degradation by lipid vesicles, RNA-binding proteins or lipoproteins.  Among the RNA-binding proteins, nucleophosmin (NPM1) was shown to bind miRs in cell culture studies whereas complexes of miRs with Argonaute2 (Ago2; the effector of target mRNA silencing by miRNAs) were detected in the circulating blood. To elucidate the regulation and potential biological relevance of protein-bound circulating miRs, we determined the quantitative contribution of protein-bound miRs to the entire miR pool and measured the regulation in patients with CAD.
Results: We established the RNA immonuprecipitation (RIP) method in endothelial cell culture. Therefore, miRNAs bound to the RNA-binding protein NPM1 or Ago2 were precipitated and quantified by real-time PCR. The immunoprecipitation was confirmed by Western blot analysis. We demonstrate that miR-92a and miR-126 were bound to Ago2, but not to NPM1 in endothelial cell extracts.
Next, we determined the binding of miRs to NPM1 and Ago2 in plasma from healthy volunteers. Circulating miR-92a, miR-126, and miR-145 were found in association with Ago2 in plasma samples. Using RIP, we detected 1.133 ± 0.029% of input of the total miR-92a in association with Ago2. Ago2-bound miR-126 levels were even lower (0.116 ± 0.015% input;). In NPMI immunoprecipitations, miR-92a and miR-126 were not detected, which is in accordance with the results obtained in endothelial cell culture.
Finally, we determined, whether the Ago2-bound miRs are differentially detected in patients with coronary artery disease (CAD) compared to healthy controls. However, the miR-92a and miR-126 levels detected in Ago2 RIPs were similar in patients with stable CAD compared to healthy controls (miR-92a: p=0.219; miR-126: p=0.343).
Conclusion: Our results demonstrate that Ago2-bound vascular miRs can be detected in circulating blood. miR-92a was more profoundly associated with Ago2 compared to miR-126 indicating a sequence specific regulation of miR export. However, the contribution of Ago2-bound miR-92a and miR-126 to the overall pool of circulating miRs is rather low (< 1.5 %) and is not regulated in patients with stable CAD.
 
Clin Res Cardiol 101, Suppl 1, April 2012
Zitierung mit Vortrags- oder Posternummer s.o.
DOI 10.1007/s00392-012-1100-6

http://www.abstractserver.de/dgk2012/ft/abstracts/V128.htm