miR-210 Regulates ISCU Figure 4. MiR-210 affects cell survival and is inversely correlated to ISCU in hypoxic tumor xenografts. A, apoptosis (Annexin V+ cells) was measured in MCF7 (left) and HCT116 (right) cells treated with miR-210 inhibitor or mimic, after 48 hours exposure to 0.1% oxygen or normoxia. Mean 6 s.e.m. is representative of 3 independent experiments (* p,0.05, ** p,0.01, *** p,0.001). B, after transfecting MCF7 cells with scrambled LNA or mir-210 LNA and exposed to 0.1% oxygen or normoxia (48 hrs), the cells were re-plated (100 and 500 cells/well). After a 12-day incubation, colonies were fixed, stained, and the surviving fractions were calculated based upon the plating efficiency. C, surviving fractions were calculated as in (B) in HeLa cells, which were transfected with anti-ctrl or anti-210 (Applied Biosystems/Ambion, Austin, TX, USA) and exposed to 0.01% oxygen or normoxia. In all colony assays, mean 6 s.e.m. is representative of 2 independent experiments carried out in triplicate employing 2 different plating densities. (** p,0.01, *** p,0.001). D, RT-PCR for ISCU and miR-210 in U87 xenografts treated with anti-angiogenic therapy (bevacizumab). Relative expression of ISCU normalised to b-actin, miR-210 normalised to three small nucleolar controls, RNU43, RNU44 and RNU48. Mean expression 6 s.e.m. in 4 animals/group is shown (** p,0.01, ***p,0.001). doi:10.1371/journal.pone.0010345.g004 control. Immunohistochemistry of these tumours demonstrated Avastin-induced necrosis, expression of HIF1a and the HIF target genes CA9 and VEGF (data not shown). We analysed the mRNA from the tumours and found marked upregulation of miR-210 and reciprocal downregulation of ISCU mRNA (Figure 4D). The only data available for comparison of miR-210 with ISCU RNA in human tumour samples is from our breast and head and neck series. There was a highly significant inverse relationship of miR-210 to ISCU expression in 216 patients with breast cancer (rho = 20.39, p,0.001) (Figure 5A, left). There was a highly significant inverse correlation of ISCU with more aggressive high grade tumours (p = 0.008) and a poor relapse free survival (Figure 5A, right). In a multivariate analysis, ISCU remained significant (p = 0.028), along with nodes, grade and age (Supporting Table S2). In 2 other series of breast cancer (Rotterdam [26], 286 cases: Uppsala [27]; 235 cases) low ISCU was a poor prognostic factor in multivariate analysis (Supporting Tables S3 and S4) (p = 0.038 and 0.015 respectively). In the Uppsala series [27] we could assess the precursor miR-210 using the Affymetrix probes (Affymetrix U133b, 230710_at) and this also showed an inverse relationship of the precursor to ISCU and poor survival (Figure 5B). In our head and neck cancer series [25], there was an inverse relationship of these variables (Figure 5C, left). In a series with published follow up, Chung et al [28], there was a strong association of suppressed ISCU with poor outcome (Figure 5C, right). Analysis of expression in normal tissues versus tumour tissues in 9 tumour data sets using Oncomine showed in all studies suppression compared to normal tissues (Supporting Figure S4). PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e10345