miR-210 Regulates ISCU Figure 1. MiR-210 regulates ISCU expression in MCF7 cells. A, miR-210 increases under hypoxia, with the most robust induction seen at 48 hrs with 0.1% oxygen. B, under the same conditions, the strongest downregulation of ISCU mRNA is observed at 48 hrs with 0.1% oxygen. The expression levels of miR-210 and ISCU mRNA under hypoxia are relative to their normoxic expression levels at 24 hrs (N). Mean 6 s.e.m. of three independent experiments is shown (* p,0.05, ** p,0.01, *** p,0.001). C, transfection of anti-210 partially rescues the hypoxic suppression of ISCU mRNA and mimic-210 overexpression decreases ISCU mRNA in normoxia at 48 hrs. Expression of ISCU mRNA is relative to the anti-ctrl in normoxia (N). Mean 6 s.e.m. of three independent experiments is shown (** p,0.01, *** p,0.001). D, (top panel) ISCU protein is downregulated in MCF7 lysates at 0.1% oxygen compared to normoxic lysates (N) when the anti-ctrl is transfected for 48 hrs. This decrease in protein level under hypoxic conditions is partially rescued when the anti-210 is transfected. Under normoxic conditions, mimic-210 suppresses ISCU protein levels compared to mimic-ctrl transfected cells. D, (bottom panel), quantification of the rescue of ISCU protein level upon transfection of anti-210. Transfection of anti-210 rescues the ISCU protein levels in MCF7 by approximately 40%. Mean 6 S.E.M of two independent experiments is shown. (* p,0.05). doi:10.1371/journal.pone.0010345.g001 similar extent to that induced by mimic-210 or by ISCU depletion. Moreover, transfection of anti-210 in MCF7 cells was able to significantly increase aconitase activity in hypoxia (Figure 2C). As the decrease in aconitase and complex I activity reduces the Krebs cycle and mitochondrial function we investigated whether there was a shift to glycolysis and lactate production. Our results showed a highly significant reduction of pyruvate and increase in lactate in normoxia with the mimic-210, with an increase in lactate pyruvate ratio. There was also a decrease in lactate production with the anti-210 in hypoxia (Figure 2D). Effects of ISCU downregulation on free radical production The loss of Fe-S from complex I is likely to affect the transport of electrons in the electron transport chain and impact on free radical production. We therefore measured the production of superoxide in MCF7 and HCT116 cells. In normoxia, both cell lines showed a marked increase in free radical production upon transfection of mimic-210 compared to mimic-control (Figure 3A). In hypoxia we noted a highly significant increase in superoxide production, which was nearly completely reversed by anti-210 (Figure 3B). Additionally, transfection of the ISCU2 construct nearly completely reversed the free radical induced by miR-210 (Figure 3C). This demonstrates a major new mechanism for regulation of ROS in hypoxia, and that it is not a passive effect of reduced oxygen availability. Effects of miR-210 on apoptosis and survival in normoxia and hypoxia Previous studies in yeast have shown the lethal consequences of the ISCU homologs deletion [23]. This notion led us to investigate the effects of miR-210 on apoptosis in normoxia and hypoxia. There was a striking difference in the effects of mir-210 in these opposing conditions. In normoxia, mimic-210 substantially increased apoptosis as measured by annexin V staining, but in hypoxia, antagonism of miR-210 increased apoptosis (Figure 4A). To evaluate effects of miR-210 on cell proliferation under hypoxic conditions, a clonogenic assay was used with a locked PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10345