hsa-miR-210 Is Induced by Hypoxia and Is an Independent Prognostic Factor in Breast Cancer

Abstract

Purpose: MicroRNA (miRNA) expression alterations have been described in cancer. Many cancers are characterized by areas of hypoxia, enhanced hypoxia-inducible factor (HIF) levels, and increased expression of hypoxically regulated genes, all of which correlate with patient outcome. We examined hypoxia-induced miRNA expression changes to identify markers of survival in breast cancer. Experimental Design: We used microarrays to analyze miRNA expression changes induced by hypoxia in MCF7 breast cancer cell lines and validated results by quantitative-PCR (Q-PCR). Small interfering RNA against HIF-1a and HIF-2a, and RCC4 cells transfected with the von Hippel-Lindau (VHL) protein were used to investigate HIF dependency of miRNA expression. miRNA Q-PCR assays were done on 219 early breast cancer samples with long-term follow-up. Correlation of expression with clinical variables was done using Pearson and Spearman's rank tests, univariate, and Cox multivariate analysis. Results: hsa-miR-210 induction was the most significant change under hypoxia by microarray analysis (3.4-fold, P < 0.001). hsa-miR-210 expression changes were validated by Q-PCR and detected in other cancer cell lines. Using small interfering RNAs and RCC4 cells transfected with VHL, we showed that the regulation by hypoxia of hsa-miR-210 was mediated by the HIF-1a/VHL transcriptional system but not HIF-2a. hsa-miR-210 expression levels in breast cancer samples correlated directly with a hypoxia score based on the expression of 99 genes. hsa-miR-210 expression levels showed an inverse correlation with disease-free and overall survival, significant in both univariate and multivariate analyses. Conclusions: We show that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1aâ ndVHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor.Hypoxia in cancer appears as a consequence of the growth of a malignant tumor but can also act to promote tumor development. Hypoxic conditions in solid malignancies may confer resistance to conventional therapies and are associated with a poorer prognosis (1 -3). The exposure of cells to hypoxia leads to the coordinated regulation of many genes. The protein products of these genes have a wide variety of critical roles in processes such as energy metabolism, angiogenesis, growth, and apoptosis. Studies of the mechanisms underlying the regulation of such genes have implicated a central role for the transcription factor hypoxia-inducible factor (HIF), which exists as a heterodimer of an a and a h subunit (4). In the presence of oxygen, HIF-a molecules undergo prolyl hydroxylation which is catalyzed by three homologous 2-oxoglutarate -dependent dioxygenases, PHD1, PHD2, and PHD3. The von HippelLindau (VHL) protein recognizes and binds to two specific hydroxyprolyl residues in HIF-1a and HIF-2a, and facilitates ubiquitination leading to rapid proteasomal degradation. Further oxygen-regulated control of HIF-a is achieved by another dioxygenase (FIH-1), which catalyze...

hsa‐miR‐210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer

“…17 If hsa-miR-10b had a role in metastasis we would expect a positive correlation with factors such as the presence or number of tumorinvolved lymph nodes at the time of diagnosis, but as in breast cancer, 32 we found no significant correlation. Similarly, hsa-miR-21 was not associated with invasiveness.…”

“…cDNA was synthesized from 5 ng of total RNA using TaqMan microRNA-specific primers and the TaqMan MicroRNA Reverse Transcription Kit (both from Applied Biosystems). Real-time PCR was done in triplicate as described in Camps et al 17 Fold changes in microRNA expression between treatments and controls were determined by the 2-DDCt method, 28 normalizing the results using the small nRNAs RNU43, RNU44, and RNU48 and control tissue.…”

“…This transcript also shows HIF-1alpha-dependent hypoxic regulation, in keeping with the coordinated transcription of intronic microRNAs and their host genes that has been seen for many intronic microRNAs. 17 We examined the correlation between this transcript, which can be detected on mRNA microarrays, and the mature form detected by real-time PCR. There were 2 probes to AK123483 in the Affymetrix U133 array, 236480_at and 230710_at, and these strongly correlated with the mature form of hsa-miR-210 (236480_at Spearman rho ¼ 0.618, P < .001; 230710_at Spearman rho ¼ 0.646, P < .001; 1 representative probe shown in Fig.…”

“…Validated targets of hsa-miR-210 include the receptor tyrosine kinase ligand ephrin-A3, 14 the transcription factor E2F3, 15 and the DNA repair enzyme RAD52. 16 However, the effect of hsa-miR-210 may depend on the cancer type; whereas high level of expression is an independent prognostic factor in breast cancer, 17 it is frequently deleted in epithelial ovarian cancer. 15 Thus, its role needs to be investigated in individual types of cancer.…”

A positive feedback loop promotes HIF‐1α stability through miR‐210‐mediated suppression of RUNX3 in paraquat‐induced EMT

“…Based on these results, miR‐210 may play a role in the development of PQ‐induced pulmonary fibrosis. A direct regulatory role of HIF‐1α in the transcription of miR‐210 has recently been reported 20, 30, 31. The silencing of HIF‐1α expression also reduced miR‐210 expression.…”