title: Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.forename: Rahul
authors.initials: R
authors.lastname: Agrawal
authors.forename: Priyatama
authors.initials: P
authors.lastname: Pandey
authors.forename: Prerana
authors.initials: P
authors.lastname: Jha
authors.forename: Vivek
authors.initials: V
authors.lastname: Dwivedi
authors.forename: Chitra
authors.initials: C
authors.lastname: Sarkar
authors.affiliation: Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, 110016, New Delhi, India. drritukulshreshtha@gmail.com.
authors.forename: Ritu
authors.initials: R
authors.lastname: Kulshreshtha
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000502:physiology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020218:Response Elements; D017423:Sequence Analysis, RNA; D015533:Transcriptional Activation; D059467:Transcriptome; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D035683:MicroRNAs; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases