Agrawal Rahul, Pandey Priyatama, Jha Prerana, Dwivedi Vivek, Sarkar Chitra, Kulshreshtha Ritu
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, 110016, New Delhi, India.
BMC Genomics. 2014 Aug 17;15(1):686. doi: 10.1186/1471-2164-15-686.
Hypoxia is a critical aspect of the glioma microenvironment and has been associated with poor prognosis and resistance to various therapies. However, the mechanisms responsible for hypoxic survival of glioma cells remain unclear. Recent studies strongly suggest that microRNAs act as critical mediators of the hypoxic response. We thus hypothesized their prominent role in hypoxia resistance in glioblastoma (GBM) and aimed to identify those.
With this study, we present the first detailed analysis of small RNA transcriptome of cell line U87MG, a grade IV glioma cell line, and its alteration under hypoxic condition. Based on deep sequencing and microarray data, we identify a set of hypoxia regulated microRNAs, with the miR-210-3p and its isomiRs showing highest induction in GBM cell lines U87MG and U251MG. We show miR-210-3p, miR-1275, miR-376c-3p, miR-23b-3p, miR-193a-3p and miR-145-5p to be up-regulated, while miR-92b-3p, miR-20a-5p, miR-10b-5p, miR-181a-2-3p and miR-185-5p are down-regulated by hypoxia. Interestingly, certain hypoxia-induced miRNAs are also known to be over-expressed in GBM tumors, suggesting that hypoxia may be one of the factors involved in establishing the miRNA signature of GBM. Transcription factor binding sites for Hypoxia inducible factor 1 A (HIF1A) were identified in the promoter region (5 kb upstream) of 30 hypoxia-induced miRNAs. HIF-1A over-expression and silencing studies show regulation of specific miRNAs, including miR-210-3p, to be HIF1A dependent. On the other hand, miR-210-3p leads to an increase in transcriptional activity of HIF and its target genes vascular endothelial growth factor (VEGF) and carbonic anhydrase 9 (CA9). MiR-210-3p levels were found to be high in GBM patient samples and showed good correlation with the known hypoxia markers CA9 and VEGF. We show that miR-210-3p promotes hypoxic survival and chemoresistance in GBM cells and targets a negative regulator of hypoxic response, HIF3A. Additionally, a total of 139 novel miRNAs were discovered by the analysis of deep sequencing data and three of these were found to be differentially expressed under hypoxia.
Overall, our study reveals a novel miRNA signature of hypoxia in GBM and suggests miR-210-3p to be an oncogenic player and a novel potential intrinsic marker of hypoxia in glioblastoma.
缺氧是胶质瘤微环境的一个关键方面，与预后不良和对各种治疗的抗性有关。然而，胶质瘤细胞缺氧存活的机制仍不清楚。最近的研究强烈表明，微小RNA作为缺氧反应的关键介质。因此，我们推测它们在胶质母细胞瘤（GBM）的缺氧抗性中起重要作用，并旨在识别这些微小RNA。
在本研究中，我们首次对IV级胶质瘤细胞系U87MG的小RNA转录组及其在缺氧条件下的变化进行了详细分析。基于深度测序和微阵列数据，我们鉴定出一组缺氧调节的微小RNA，其中miR-210-3p及其异源异构体在GBM细胞系U87MG和U251MG中诱导程度最高。我们发现miR-210-3p、miR-1275、miR-376c-3p、miR-23b-3p、miR-193a-3p和miR-145-5p上调，而miR-92b-3p、miR-20a-5p、miR-10b-5p、miR-181a-2-3p和miR-185-5p在缺氧条件下下调。有趣的是，某些缺氧诱导的微小RNA在GBM肿瘤中也已知过度表达，这表明缺氧可能是参与建立GBM微小RNA特征的因素之一。在30个缺氧诱导的微小RNA的启动子区域（上游5 kb）鉴定出缺氧诱导因子1A（HIF1A）的转录因子结合位点。HIF-1A过表达和沉默研究表明，包括miR-210-3p在内的特定微小RNA的调节是HIF1A依赖性的。另一方面，miR-210-3p导致HIF及其靶基因血管内皮生长因子（VEGF）和碳酸酐酶9（CA9）的转录活性增加。发现GBM患者样本中miR-210-3p水平较高，并且与已知的缺氧标志物CA9和VEGF显示出良好的相关性。我们表明，miR-210-3p促进GBM细胞的缺氧存活和化疗抗性，并靶向缺氧反应负调节因子HIF3A。此外，通过深度测序数据分析发现了总共139个新的微小RNA，其中三个在缺氧条件下差异表达。
总体而言，我们的研究揭示了GBM中缺氧的新微小RNA特征，并表明miR-210-3p是胶质母细胞瘤中致癌的参与者和缺氧的新型潜在内在标志物。
