E-GEOD-43622 - miR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.
Released on 1 April 2013, last updated on 2 June 2014
The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional level of Hypoxia Inducible Factor (HIF) dependent transcriptional regulation has emerged involving modulation of a specific set of miRNAs including miR-210. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. We therefore hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed Non-Small Cell Lung Cancer (NSCLC)-derived cell lines (A549 and H1975) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). MiR-210 expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. The cells were subjected to radiation levels ranging from 0 to 10Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of resistance than control cells in hypoxia. pmiR-210 cells under hypoxia showed a low mortality rate due to a decrease in apoptosis and an ability to grow even at 10Gy. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we show here that genomic double strand breaks (DSB) foci disappeared faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells (pmiR-210/HIF-1-) abolished radioresistance of cells showing that this mechanism was dependent upon HIF-1. In conclusion, miR-210 appears to be a major component in the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could even be used by tumor cells in conditions where hypoxia may not be present anymore and strongly suggests that strategies targeting miR-210 would enhance tumor radiosensitization. To identify the set of transcripts targeted by miR-210, we overexpressed has-miR-210 or a control miRNA (ce-miR-67) in human lung adenocarcinoma A549 cells by transduction using lentivirus. The resulting pmiR-67 and pmiR-210 that stably overexpress miR-210 were selected by puromycin. RNA samples were harvested at 2 different times following cell plating (24 hours or 48 hours). Experiments were performed in dye-swap.
transcription profiling by array
Kevin Lebrigand <email@example.com>, B Mari, K Lebrigand, P Barbry, S Grosso