Abstract 4911: miR-200c is Up-regulated by Oxidative Stress and Induces Endothelial Cell Apoptosis and Senescence
Background. microRNAs (miR) are small non-coding RNAs that inhibit translation of messenger RNAs (mRNA) into proteins and play a key role in the modulation of cell function. The objective of this work was to examine the effect of oxidative stress on human umbilical vein endothelial cells (HUVEC) miR expression.
Methods and results. HUVEC were exposed to 200 μM H2O2 for 8 to 24 hours; out of 254 miRNAs examined only 2 increased > 8-fold, miR-200c (28.30 ± 8.8 fold vs control; p < 0.01) and the co-transcribed miR-141(26.72 ± 10.23 fold vs control; p < 0.02); peak expression was achieved after 16 hours exposure to oxidative stress. All other members of the miR-8 gene family to which miR-200c and miR-141 belong, i.e. miR429, miR200a and miR200b were induced by H2O2, albeit to a lower level: i.e. 4 – 8 fold increase. This response to oxidative stress was not limited to HUVEC since a similar increase in miR-200c was also found in C2C12 cells growing or differentiated in myotubes exposed to H2O2. Further, oxidative stress induced with 0.25 mM BCNU also up-regulated miR-200c (3.18 ± 0.96 fold; p < 0.02) 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 (3.36 ± 1.05 fold vs control; p < 0.01) and induced cellular senescence as indicated by positive beta-galactosidase (β-gal)staining (scramble miR:12.66% ± 2.27 β-gal positive cells; miR-200c: 87.36%± 4.85 β-gal positive cells); all these effects on cell proliferation, apoptosis and senescence are known to be caused by oxidative stress. ZEB1 is a well-known target of miR-200c and lack of ZEB1 has been linked to an enhanced cellular senescence. It was found that under our experimental conditions in HUVEC exposed to H2O2 both ZEB1 mRNA and protein decreased and the over-expression of miR-200c in HUVEC down-regulates ZEB1 mRNA and protein.
Conclusions. Oxidative stress markedly increases miR-200c and its family members and down-regulates ZEB1, which is a known miR-200c target. Further, the over-expression of miR-200c induces cell growth arrest, enhances apoptosis and induces cell senescence. Thus, miR-200c may play a key role in mediating the effect of oxidative stress on cell function and survival.