miR-210 is robustly induced by hypoxia in many cell lines, via HIF1a [14]. We recently analysed its expression in a series of 216 breast cancer patients and showed miR-210 expression was correlated with many HIF1a targets at mRNA level (as measured by a hypoxia metagene) and was strongly associated with poor patient survival. Derived only from sequence-based algorithms, some of the previously validated targets of miR-210 include Ephrin A3 [15], E2F3 [16], RAD52 [17], CASP8AP2 [18], and MNT [19]. We combined publically available algorithms, with our gene array datasets, to predict potential miR targets of importance in cancer cells. We found that the mitochondrial iron sulfur cluster homologue ISCU was the highest predicted target for miR-210. Recently, ISCU has been identified as a miR-210 target also in normal pulmonary endothelial cells [20], where it contributes to the Pasteur effect and controls the level of ROS production in hypoxia, suggesting its potential adaptive role to hypoxia in the context of pulmonary endothelium. Iron sulfur clusters [Fe-S] are present in the active sites of many enzymes and proteins, critical for their activity and capable of conferring regulation by redox status [21]. These clusters are assembled in mitochondria [22] by a complex series of chaperones and enzymes including ISCU, then exported to the cytoplasm, where they are assembled into the relevant protein [23]. Amongst the Fe-S cluster proteins involved are several that comprise key components of complex I, II and III in the mitochondria, and components of the Krebs cycle such as succinate dehydrogenase and aconitase. The cytoplasmic form of the latter regulates iron metabolism via its function as a translational regulator-IRP1 [24]. In this report we show the major biological effects of miR-210 targeting ISCU, all of which are likely to contribute to important phenotypes in cancer. By downregulating ISCU, miR-210 decreases Krebs cycle enzyme activity and mitochondrial function, provides a major mechanism for the increased free radical generation in hypoxia, increases cell survival under hypoxia, induces a switch to glycolysis in normoxia and hypoxia (Warburg and Pasteur effects) and upregulation of the iron uptake required for cell growth. Importantly, analysis of over 900 patients with different tumour types showed that suppression of ISCU is strongly correlated with a worse prognosis. This study thus reveals a new pathway activated in hypoxic tumours, mediated by miR- 210 affecting mitochondrial enzyme activity and free radical generation and highlights the importance of mitochondrial metabolism in hypoxia biology [3]. Results Selection of ISCU as a potential target We compared miR-210 expression in our published series of breast cancer [14] with our hypoxia metagene of clustered mRNAs [25] and combined assessment with target prediction algorithms showed ISCU was the highest predicted target, and three known target genes also sit in highly-ranked positions were selected by this approach (Supporting Material and Methods S1, Supporting Table S1). MiR-210 and ISCU mRNA undergo reciprocal regulation In agreement with previous publications [13,14], miR-210 was robustly induced in MCF7 and HCT116 by hypoxia (1% or 0.1% oxygen) at 24 and 48 hrs, with maximal induction observed at 48 hours in 0.1% oxygen (Figure 1A and Supporting Figure S1A). ISCU mRNA quantified by RTPCR was inversely correlated to miR-210 and decreased most significantly when cells were exposed to more severe hypoxia for longer periods (Figure 1B and Supporting Figure S1B). Similar to the regulation of miR-210 miR-210 Regulates ISCU under hypoxia, ISCU suppression at the transcript level was dependent on HIF1a and not HIF2a (Supporting Figures S1C and S1D). Furthermore, the phenomenon of reciprocal regulation of miR-210 and ISCU by hypoxia was also found in many other cell lines (Supporting Figures S1E and S1F). Mimic-210 suppressed ISCU in normoxia and anti-210 reversed the suppression in hypoxia We recapitulated the observed hypoxic induction of miR-210 by transfecting MCF7 and HCT116 with mimic-210 in normoxia. Mimic-210 suppressed ISCU mRNA levels by approximately 60% (Figure 1C and Supporting Figure S2A). We then antagonised miR-210 induced under hypoxic conditions with anti-210. Inhibition of endogenous miR-210 significantly rescued the suppression of ISCU mRNA, although this was not complete (Figure 1C and Supporting Figure S2A). ISCU has two main alternatively spliced isoforms. ISCU1 has an alternative N-terminal sequence and is localised to the cytosol, while ISCU2 is associated with the mitochondria. To confirm the specificity of the antibody used in these studies, MCF7 cells were treated with siRNAs against ISCU. The band detected in the control cells was completely suppressed upon transfection with siISCU1 and siISCU3 (Supporting Figure S2B). The two isoforms of ISCU could not be distinguished by Western blotting of whole cell lysates because of the minor difference in molecular weight. However, the immunoreactive band was detected in both the cytosolic and mitochondrial fractions (Supporting Figure S2C). This band is referred to as ISCU protein and its molecular weight is compatible with the results of Tong et. al. [23]. Under hypoxia, MCF7 and HCT116 cells showed a reduction in ISCU protein, and this was replicated with mimic-210 in normoxia (Figure 1D and Supporting Figure S2D). Moreover, anti-210 partially reversed the hypoxic suppression of ISCU protein (Figure 1D and Supporting Figure S2D). Hypoxia strongly suppressed a luciferase reporter containing the 39UTR of ISCU that included the putative miR-210 target site and this suppression could be partially rescued by transient transfection of anti-210 (Supporting Figure S2E). In addition, mimic-210 substantially suppressed the luciferase activity compared to the control in normoxic MCF7 cells (Supporting Figure S2E). Finally, while siRNA against ISCU led to a downregulation of both endogenous ISCU and an exogenous tagged ISCU lacking the 39UTR, mimic- 210 mediated downregulation was only observed on the endogenous ISCU protein (Supporting Figure S2F). Taken together, these observations demonstrate that miR-210 downreg- ulates ISCU mRNA and protein levels by targeting its 39UTR. Effects of ISCU downregulation on Fe-S proteins An expected effect of downregulation of ISCU would be a reduction of Fe-S delivery to target proteins. Therefore we analysed the impact of ISCU repression on two enzymes that require Fe-S for their activities, aconitase and mitochondrial complex I. siRNA against ISCU significantly reduced aconitase activity in MCF7 and HCT116 cells compared to control cells in normoxia (Figure 2A). A similar decrease in aconitase activity was evident in both cell lines upon transfection of mimic-210 (Figure 2A). However there was no change in aconitase protein levels as assessed by Western blot (Supporting Figure S3A). Aconitase depleted of Fe-S acts as the translational regulator- IRP1, to increase uptake of iron. We found the mimic-210 increased iron uptake in HCT116 cells (Supporting Figure S3B). There was also clear inhibition of mitochondrial complex I activity induced by mimic-210, in the two cell lines (Figure 2B). In both cell lines there was down regulation of activity by hypoxia, to a PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10345