Hypoxic signature of microRNAs in glioblastoma: insights from small RNA deep sequencing

BMC Genomics

Ritu Kulshreshtha³  Chitra Sarkar¹  Vivek Dwivedi³  Prerana Jha¹  Priyatama Pandey²  Rahul Agrawal³ 

[1] Department of Pathology, All India Institute of Medical Sciences, 110029 New Delhi, India;School of Computational and Integrative sciences, Jawaharlal Nehru University, 110067 New Delhi, India;Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, 110016 New Delhi, India

关键词: miR-210;    A172;    U251MG;    U87MG;    Deep sequencing;    MicroRNA;    Glioblastoma;   

Others  :  1216264
DOI  :  10.1186/1471-2164-15-686

 received in 2014-03-28, accepted in 2014-08-12,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Agrawal et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Brown JM, Wilson WR: Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer 2004, 4:437-447.
• [2]Melillo G: Inhibiting hypoxia-inducible factor 1 for cancer therapy. Mol Cancer Res 2006, 4:601-605.
• [3]Louis DN: Molecular pathology of malignant gliomas. Annu Rev Pathol 2006, 1:97-117.
• [4]Rich JN, Bigner DD: Development of novel targeted therapies in the treatment of malignant glioma. Nat Rev Drug Discov 2004, 3:430-446.
• [5]Brat DJ, Mapstone TB: Malignant glioma physiology: cellular response to hypoxia and its role in tumor progression. Ann Intern Med 2003, 138:659-668.
• [6]Jensen RL: Brain tumor hypoxia: tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic target. J Neurooncol 2009, 92:317-335.
• [7]Said HM, Polat B, Staab A, Hagemann C, Stein S, Flentje M, Theobald M, Katzer A, Vordermark D: Rapid detection of the hypoxia-regulated CA-IX and NDRG1 gene expression in different glioblastoma cells in vitro. Oncol Rep 2008, 20:413-419.
• [8]Said HM, Hagemann C, Staab A, Stojic J, Kühnel S, Vince GH, Flentje M, Roosen K, Vordermark D: Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1α in human glioma in vitro and in vivo. Radiother Oncol 2007, 83:398-405.
• [9]Seidel S, Garvalov BK, Wirta V, von Stechow L, Schänzer A, Meletis K, Wolter M, Sommerlad D, Henze A-T, Nistér M, Reifenberger G, Lundeberg J, Frisén J, Acker T: A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. Brain 2010, 133:983-995.
• [10]Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer 2006, 6:857-866.
• [11]Croce CM: Oncogenes and cancer. N Engl J Med 2008, 358:502-511.
• [12]Lawler S, Chiocca EA: Emerging functions of microRNAs in glioblastoma. J Neurooncol 2009, 92:297-306.
• [13]Ciafrè SA, Galardi S, Mangiola A, Ferracin M, Liu CG, Sabatino G, Negrini M, Maira G, Croce CM, Farace MG: Extensive modulation of a set of microRNAs in primary glioblastoma. Biochem Biophys Res Commun 2005, 334:1351-1358.
• [14]Krichevsky AM, Gabriely G: miR-21: a small multi-faceted RNA. J Cell Mol Med 2009, 13:39-53.
• [15]Gillies JK, Lorimer IAJ: Regulation of p27Kip1 by miRNA 221/222 in glioblastoma. Cell Cycle 2007, 6:2005-2009.
• [16]Xia H, Qi Y, Ng SS, Chen X, Li D, Chen S, Ge R, Jiang S, Li G, Chen Y, He ML, Kung HF, Lai L, Lin MC: microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs. Brain Res 2009, 1269:158-165.
• [17]Sasayama T, Nishihara M, Kondoh T, Hosoda K, Kohmura E: MicroRNA-10b is overexpressed in malignant glioma and associated with tumor invasive factors, uPAR and RhoC. Int J Cancer 2009, 125:1407-1413.
• [18]Moore LM, Kivinen V, Liu Y, Annala M, Cogdell D, Liu X, Liu C-G, Sawaya R, Yli-Harja O, Shmulevich I, Fuller GN, Zhang W, Nykter M: Transcriptome and small RNA deep sequencing reveals deregulation of miRNA biogenesis in human glioma. J Pathol 2013, 229:449-459.
• [19]Kulshreshtha R, Ferracin M, Wojcik SE, Garzon R, Alder H, Agosto-Perez FJ, Davuluri R, Liu C-G, Croce CM, Negrini M, Calin GA, Ivan M: A microRNA signature of hypoxia. Mol Cell Biol 2007, 27:1859-1867.
• [20]Hu H, Gatti RA: MicroRNAs: New players in the DNA damage response. J Mol Cell Biol 2011, 3:151-158.
• [21]Ivan M, Harris AL, Martelli F, Kulshreshtha R: Hypoxia response and microRNAs: no longer two separate worlds. J Cell Mol Med 2008, 12:1426-1431.
• [22]Fasanaro P, Greco S, Lorenzi M, Pescatori M, Brioschi M, Kulshreshtha R, Banfi C, Stubbs A, Calin GA, Ivan M, Capogrossi MC, Martelli F: An integrated approach for experimental target identification of hypoxia-induced miR-210. J Biol Chem 2009, 284:35134-35143.
• [23]Shen G, Li X, Jia Y, Piazza GA, Xi Y: Hypoxia-regulated microRNAs in human cancer. Acta Pharmacol Sin 2013, 34:336-341.
• [24]Hara S, Hamada J, Kobayashi C, Kondo Y, Imura N: Expression and characterization of hypoxia-inducible factor (HIF)-3alpha in human kidney: suppression of HIF-mediated gene expression by HIF-3alpha. Biochem Biophys Res Commun 2001, 287:808-813.
• [25]Mittelbronn M: The current WHO classification of tumours of the central nervous system: histopathology and additional diagnostic methods. Curr Med Imaging Rev 2010, 6:200-219.
• [26]Crosby ME, Kulshreshtha R, Ivan M, Glazer PM: MicroRNA regulation of DNA repair gene expression in hypoxic stress. Cancer Res 2009, 69:1221-1229.
• [27]Vaz C, Ahmad HM, Sharma P, Gupta R, Kumar L, Kulshreshtha R, Bhattacharya A: Analysis of microRNA transcriptome by deep sequencing of small RNA libraries of peripheral blood. BMC Genomics 2010, 11:288.
• [28]Tyagi S, Vaz C, Gupta V, Bhatia R, Maheshwari S, Srinivasan A, Bhattacharya A: CID-miRNA: a web server for prediction of novel miRNA precursors in human genome. Biochem Biophys Res Commun 2008, 372:831-834.
• [29]Jiang P, Wu H, Wang W, Ma W, Sun X, Lu Z: MiPred: classification of real and pseudo microRNA precursors using random forest prediction model with combined features. Nucleic Acids Res 2007, 35:W339-W344.
• [30]Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T: MiRecords: an integrated resource for microRNA-target interactions. Nucleic Acids Res 2009, 37:D105-D110.
• [31]Messeguer X, Escudero R, Farré D, Núñez O, Martínez J, Albà MM: PROMO: detection of known transcription regulatory elements using species-tailored searches. Bioinformatics 2002, 18:333-334.
• [32]Farré D, Roset R, Huerta M, Adsuara JE, Roselló L, Albà MM, Messeguer X: Identification of patterns in biological sequences at the ALGGEN server: PROMO and MALGEN. Nucleic Acids Res 2003, 31:3651-3653.
• [33]Henriksen M, Johnsen KB, Andersen HH, Pilgaard L, Duroux M: MicroRNA expression signatures determine prognosis and survival in glioblastoma multiforme—a systematic overview. Mol Neurobiol 2014, 10:420-435.
• [34]Moller HG, Rasmussen AP, Andersen HH, Johnsen KB, Henriksen M, Duroux M: A systematic review of MicroRNA in glioblastoma multiforme: micro-modulators in the mesenchymal mode of migration and invasion. Mol Neurobiol 2013, 47:131-144.
• [35]Wang GL, Jiang BH, Rue EA, Semenza GL: Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci U S A 1995, 92:5510-5514.
• [36]Bruning U, Cerone L, Neufeld Z, Fitzpatrick SF, Cheong A, Scholz CC, Simpson DA, Leonard MO, Tambuwala MM, Cummins EP, Taylor CT: MicroRNA-155 promotes resolution of hypoxia-inducible factor 1{alpha} activity during prolonged hypoxia. Mol Cell Biol 2011, 31:4087-4096.
• [37]Huang X, Le Q-T, Giaccia AJ: MiR-210–micromanager of the hypoxia pathway. Trends Mol Med 2010, 16:230-237.
• [38]Evans SM, Judy KD, Dunphy I, Jenkins WT, Hwang W-T, Nelson PT, Lustig RA, Jenkins K, Magarelli DP, Hahn SM, Collins RA, Grady MS, Koch CJ: Hypoxia is important in the biology and aggression of human glial brain tumors. Clin Cancer Res 2004, 10:8177-8184.
• [39]Murat A, Migliavacca E, Hussain SF, Heimberger AB, Desbaillets I, Hamou MF, Rüegg C, Stupp R, Delorenzi M, Hegi ME: Modulation of angiogenic and inflammatory response in glioblastoma by hypoxia. PLoS One 2009, 4:e5947.
• [40]Cheng Q, Yi B, Wang A, Jiang X: Exploring and exploiting the fundamental role of microRNAs in tumor pathogenesis. Onco Targets Ther 2013, 6:1675-1684.
• [41]Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL: The Vienna RNA websuite. Nucleic Acids Res 2008, 36:W70-W74.
• [42]Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringnér M, Mörgelin M, Bourseau-Guilmain E, Bengzon J, Belting M: Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci U S A 2013, 110:7312-7317.
• [43]Kelly TJ, Souza AL, Clish CB, Puigserver P: A hypoxia-induced positive feedback loop promotes hypoxia-inducible factor 1alpha stability through miR-210 suppression of glycerol-3-phosphate dehydrogenase 1-like. Mol Cell Biol 2011, 31:2696-2706.
• [44]Makino Y, Cao R, Svensson K, Bertilsson G, Asman M, Tanaka H, Cao Y, Berkenstam A, Poellinger L: Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 2001, 414:550-554.
• [45]Taguchi A, Yanagisawa K, Tanaka M, Cao K, Matsuyama Y, Goto H, Takahashi T: Identification of hypoxia-inducible factor-1 alpha as a novel target for miR-17-92 microRNA cluster. Cancer Res 2008, 68:5540-5545.
• [46]Cha S-T, Chen P-S, Johansson G, Chu C-Y, Wang M-Y, Jeng Y-M, Yu S-L, Chen J-S, Chang K-J, Jee S-H, Tan C-T, Lin M-T, Kuo M-L: MicroRNA-519c suppresses hypoxia-inducible factor-1alpha expression and tumor angiogenesis. Cancer Res 2010, 70:2675-2685.
• [47]Yang M-H, Wu M-Z, Chiou S-H, Chen P-M, Chang S-Y, Liu C-J, Teng S-C, Wu K-J: Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol 2008, 10:295-305.
• [48]Krishnan J, Suter M, Windak R, Krebs T, Felley A, Montessuit C, Tokarska-Schlattner M, Aasum E, Bogdanova A, Perriard E, Perriard JC, Larsen T, Pedrazzini T, Krek W: Activation of a HIF1α-PPARγ Axis Underlies the Integration of Glycolytic and Lipid Anabolic Pathways in Pathologic Cardiac Hypertrophy. Cell Metab 2009, 9:512-524.
• [49]Zhang F-L, Shen G-M, Liu X-L, Wang F, Zhao Y-Z, Zhang J-W: Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions. J Cell Mol Med 2012, 16:1889-1899.
• [50]Pulkkinen K, Malm T, Turunen M, Koistinaho J, Ylä-Herttuala S: Hypoxia induces microRNA miR-210 in vitro and in vivo. Ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210. FEBS Lett 2008, 582:2397-2401.
• [51]Cloonan N, Wani S, Xu Q, Gu J, Lea K, Heater S, Barbacioru C, Steptoe AL, Martin HC, Nourbakhsh E, Krishnan K, Gardiner B, Wang X, Nones K, Steen JA, Matigian NA, Wood DL, Kassahn KS, Waddell N, Shepherd J, Lee C, Ichikawa J, McKernan K, Bramlett K, Kuersten S, Grimmond SM: MicroRNAs and their isomiRs function cooperatively to target common biological pathways. Genome Biol 2011, 12:R126.
• [52]Chan SY, Zhang YY, Hemann C, Mahoney CE, Zweier JL, Loscalzo J: MicroRNA-210 controls mitochondrial metabolism during hypoxia by repressing the Iron-Sulfur Cluster Assembly Proteins ISCU1/2. Cell Metab 2009, 10:273-284.
• [53]Zhang Z, Sun H, Dai H, Walsh RM, Imakura M, Schelter J, Burchard J, Dai X, Chang AN, Diaz RL, Marszalek JR, Bartz SR, Carleton M, Cleary MA, Linsley PS, Grandori C: MicroRNA miR-210 modulates cellular response to hypoxia through the MYC antagonist MNT. Cell Cycle 2009, 8:2756-2768.
• [54]Fasanaro P, D’Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, Capogrossi MC, Martelli F: MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 2008, 283:15878-15883.
• [55]Lages E, Guttin A, El Atifi M, Ramus C, Ipas H, Dupré I, Rolland D, Salon C, Godfraind C, de Fraipont F, Dhobb M, Pelletier L, Wion D, Gay E, Berger F, Issartel JP: MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes. PLoS One 2011, 6:e20600.
• [56]Chen L, Han L, Zhang K, Shi Z, Zhang J, Zhang A, Wang Y, Song Y, Li Y, Jiang T, Pu P, Jiang C, Kang C: VHL regulates the effects of miR-23b on glioma survival and invasion via suppression of HIF-1α/VEGF and β-catenin/Tcf-4 signaling. Neuro Oncol 2012, 14:1026-1036.
• [57]Jiang J, Sun X, Wang W, Jin X, Bo X, Li Z, Bian A, Jiu J, Wang X, Liu D, Hui X, Wang Y, Wang A, Ding L: Tumor microRNA-335 expression is associated with poor prognosis in human glioma. Med Oncol 2012, 29:3472-3477.
• [58]Ujifuku K, Mitsutake N, Takakura S, Matsuse M, Saenko V, Suzuki K, Hayashi K, Matsuo T, Kamada K, Nagata I, Yamashita S: miR-195, miR-455-3p and miR-10a( *) are implicated in acquired temozolomide resistance in glioblastoma multiforme cells. Cancer Lett 2010, 296:241-248.
• [59]Srinivasan S, Patric IRP, Somasundaram K: A Ten-microRNA expression signature predicts survival in Glioblastoma. PLoS One 2011, 6:e17438.
• [60]Tanaka H, Sasayama T, Tanaka K, Nakamizo S, Nishihara M, Mizukawa K, Kohta M, Koyama J, Miyake S, Taniguchi M, Hosoda K, Kohmura E: MicroRNA-183 upregulates HIF-1α by targeting isocitrate dehydrogenase 2 (IDH2) in glioma cells. J Neurooncol 2013, 111:273-283.
• [61]Cortez MA, Nicoloso MS, Shimizu M, Rossi S, Gopisetty G, Molina JR, Carlotti C, Tirapelli D, Neder L, Brassesco MS, Scrideli CA, Tone LG, Georgescu M-M, Zhang W, Puduvalli V, Calin GA: miR-29b and miR-125a regulate podoplanin and suppress invasion in glioblastoma. Genes Chromosomes Cancer 2010, 49:981-990.