Abstract 14400: miR-200c is Up-Regulated by Oxidative Stress and Induces Endothelial Cell Apoptosis and Senescence via ZEB1 Inhibition
Background: microRNAs (miRNAs) are small non coding RNAs that function as negative gene regulators. Our aim was to examine the effect of reactive oxygen species (ROS) on miRNAs expression in Human umbilical vein endothelial cells (HUVEC) in vitro, in mouse skeletal muscle following acute hindlimb ischemia, and to establish modulated miRNAs functional role.
Methods and Results: HUVEC were exposed to 200 μM hydrogen peroxide (H2O2) for 8 to 24 hours; miRNAs profiling showed that out of 250 miRNAs examined only 2 increased > 8-fold, miR-200c (29.3 ± 1.1 fold vs control; p<0.001) and the co-transcribed miR-141 (23.3 ± 0.5 fold vs control; p<0.001). All other members of the miR-200 gene family to which miR-200c and miR-141 belong, miR-200a, -200b and -429 were also induced by H2O2, albeit to a lower level: i.e. 4–8 fold. Moreover, miR-200c up-regulation was not endothelium-restricted, since it occurred also in C2C12 muscle cells, growing or differentiated in myotubes, exposed to H2O2. Further, oxidative stress induced with 0.25 mM BCNU also up-regulated miR-200c (3.2 ± 1 fold; p<0.05) and this response was prevented by pre-incubation with the free radical scavenger NAC (10 mM). Over-expression of miR-200c in HUVEC induced cell growth arrest, enhanced apoptosis (3.4 ± 1.1 fold vs control; p<0.01) and induced cellular senescence as indicated by positive β-galactosidase (β-gal) staining (scramble miR: 12.7 ± 2.3% β-gal-positive cells; miR-200c: 87.4 ± 4.9% β-gal-positive cells; p<0.001); all these effects are known to be caused by oxidative stress and were partially rescued by miR-200c inhibition. miR-200c target ZEB1 mRNA and protein were down-modulated by H2O2 and by miR-200c over-expression. ZEB1 knockdown recapitulated miR-200c-induced responses. Finally, oxidative stress due to acute hindlimb ischemia, significantly enhanced miR-200c in mice skeletal muscle (5.1 ± 0.4 fold vs control; p<0.01). In contrast, in p66ShcA −/− mice, which display lower levels of oxidative stress after ischemia, up-regulation of miR-200c was markedly inhibited (2.6 ± 0.8 fold vs control; p<0.05; p<0.05 vs WT).
Conclusions: Oxidative stress induces miR-200c and its family members; the ensuing down-modulation of ZEB1 plays a key role in ROS-induced apoptosis and senescence.
- © 2010 by American Heart Association, Inc.