miR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.. Homo sapiens

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. Overall design: 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.

缺氧细胞对放疗与化疗的耐药性，是当前癌症治疗领域的核心难题之一。近期研究揭示，缺氧诱导因子（Hypoxia Inducible Factor, HIF）依赖的转录调控存在额外调控层级，其涉及对包括miR-210在内的特定microRNA（miRNA）子集的调控。本团队此前研究证实，HIF-1对miR-210的诱导作用，可通过正反馈环路实现HIF-1自身的稳定。基于此，我们提出假说：在常氧条件下，miR-210通过稳定HIF-1，可使癌细胞获得辐射抗性。本研究构建了稳定过表达miR-210（命名为pmiR-210）或对照miRNA（命名为pmiR-Ctl）的非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）源细胞系，分别为A549与H1975细胞。过表达miR-210的细胞中，HIF-1稳定性显著提升，同时伴随线粒体功能缺陷与糖酵解表型的出现。我们分别在常氧与缺氧条件下，对上述细胞施加0至10Gy梯度剂量的电离辐射。常氧条件下过表达miR-210的细胞，其辐射抗性水平与缺氧条件下的对照细胞相当。缺氧条件下的pmiR-210细胞死亡率较低，这源于细胞凋亡水平的降低，且即便在10Gy辐射剂量下仍具备增殖能力。本研究证实，该辐射抗性与p53状态及细胞周期进程无关。此外，本研究发现，pmiR-210细胞中基因组双链断裂（double strand breaks, DSB）焦点的清除速度快于pmiR-Ctl细胞，提示miR-210的表达可促进DSB的高效修复。最后，我们对pmiR-210细胞进行HIF-1敲除（构建pmiR-210/HIF-1⁻细胞系）后，其辐射抗性完全消失，证实该调控机制依赖于HIF-1。综上，miR-210是缺氧癌细胞获得辐射抗性的核心调控因子之一。鉴于多数miRNA稳定性较强，即便在缺氧条件不复存在的场景中，肿瘤细胞仍可借助miR-210的这一特性；该结果强烈提示，靶向miR-210的干预策略可有效增强肿瘤的辐射敏感性。总体实验设计：为鉴定miR-210的靶标转录本群体，我们通过慢病毒转导，在人肺腺癌A549细胞中过表达has-miR-210或对照miRNA（ce-miR-67）。随后利用嘌呤霉素筛选获得稳定过表达miR-210的pmiR-210细胞，以及稳定过表达对照的pmiR-67细胞。在细胞铺板后的2个不同时间点（24小时与48小时）收集RNA样本。实验采用染料交换（dye-swap）设计进行。