Abstract 798: Role of MicroRNA-Regulating Enzymes Dicer and Drosha for Endothelial Cell Biology
MicroRNAs (miR) are small noncoding RNAs that regulate gene expression by binding to the cellular transcript leading to translational repression or degradation of the target mRNA. Dicer and Drosha are the miR processing enzymes, which are required for the maturation of miR. Increasing evidence indicates that miR have distinct expression profiles and play crucial roles, e.g. in cardiogenesis and stem cell expansion. However, the role of miR in endothelial cell (EC) biology is entirely unknown. Knockdown of the miR processing enzymes Dicer and Drosha reduces miR expression. Therefore, we first investigated the general role of Dicer and Drosha for endothelial cell function. HUVEC were transfected with siRNA against Dicer and Drosha to inhibit miR biogenesis. Knockdown of Dicer and Drosha significantly reduced sprouting of EC (Dicer: 12±3%, Drosha: 58±11% of scrambled siRNA transfected cells, n=3, p<0.05) as measured in a three-dimensional spheroid assay. Migration of HUVEC using a modified Boyden chamber assay was slightly reduced in Dicer siRNA-transfected cells (73±3% control, n=4), whereas Drosha siRNA had no effect (95±8% of control, n=4). Dicer knockdown significantly reduced viability after 24 h (89±6% of scrambled, n=3) and 48 h (68±0.8% of scrambled, n=3, p<0.05) as measured by MTT assay. In contrast, Drosha knockdown did not significantly affect EC viability after 48 h (115±0.6% scrambled, n=3). Having shown that Dicer and Drosha specifically regulates EC sprouting, we attempted to identify specific miR involved in EC function. A screening analysis of 160 human miR using real-time-PCR revealed that miR-92–1 was highly expressed in HUVEC. In silico analysis predicted targets for miR-92–1 involved in endothelial cell biology include Wnt 5a, FGF-5, Hand1, Hand2, SOCS5 and SIRT1. Taken together, knockdown of miR-regulating enzymes Dicer and Drosha specifically impairs EC sprouting suggesting an important function of miR in EC biology. miR-92–1 appears to be an attractive target for mediating functional effects in EC.