Collagen V alpha 2 as a direct target of miR-143 in arteriogenesis
T. Hammerschick1, K. Troidl2, K. T. Preissner1, S. Fischer1 (1Giessen, Germany, 2Bad Nauheim, Germany)
Time: 11:00 - 12:00
Objective: Arteriogenesis describes the formation of collateral arteries from preexisting small vessels, that involves the proliferation of endothelial and smooth muscle cells as well as the recruitment of leukocytes. This process is induced by physical forces, most importantly fluid shear stress (FSS). It was shown that FSS leads to a change in the expression pattern of several microRNAs (miRs) in growing collateral arteries; especially miR-143 is highly expressed. Accordingly, a miR-143 knockdown resulted in the inhibition of the growth of collateral arteries in mice. Collagen V alpha 2 (COLVA2) was identified by us as a candidate target gene of miR-143 carrying the predicted target sequence and being downregulated in growing collaterals.
Methods: To confirm the direct interaction between miR-143 and COLVA2 mRNA, a Dual Luciferase Reporter Assay was performed by cloning the COLVA2-3’UTR-sequence into the miR-specific plasmid vector psiCHECK^TM-2.
Results: Co-transfection of this plasmid for 48 h together with an artificial miR-143 construct (but not with the scrambled miRNA-construct) into endothelial cells (Ea.hy926; express only low amounts of miR-143) resulted in increased miR-143 expression while luciferase activity decreased. These data indicate that miR-143 modulates COLVA2 expression by directly targeting the 3'UTR-region of the COLVA2 mRNA. Accordingly, 48 h co-transfection of the plasmid with anti-miR-143 (but not with the scrambled anti-miRNA) in murine vascular smooth muscle cells or NIH/3T3-fibroblasts respectively, which both express high amounts of miR-143, induced the knockdown of miR 143 and upregulated the luciferase expression.
Conclusion: These findings demonstrate that miR-143 directly regulates COLVA2 expression, which may play a decisive role in vessel and tissue remodeling during arteriogenesis.