Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

De-repression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures

Subjects

Abstract

All-trans retinoic acid is a potent promoter of cellular differentiation processes, which is used in cancer therapy. Glioblastoma spheroid cultures are enriched in tumor-initiating cells, and provide a model to test new treatment options in vitro. We investigated the molecular mechanisms of response to exposure to differentiation-promoting conditions in such cultures. Microarray analyses of five independent cultures showed that after induction of differentiation, inhibitors of transforming growth factor-β/bone morphogenetic protein, Wnt/β-catenin and IGF signaling were upregulated, whereas expression of several microRNAs decreased, particularly that of the miR-17-92 cluster. In primary astrocytic gliomas (n=82), expression of several members of miR-17-92 was significantly higher relative to those of normal brain (n=8) and significantly increased with tumor grade progression (P<0.05). A high-level amplification of the miR-17-92 locus was detected in one glioblastoma specimen. Transfection of inhibitors of miR-17-92 induced increased apoptosis and decreased cell proliferation in glioblastoma spheroids. Mir-17-92 inhibition was also associated with increased messenger RNA (mRNA) and/or protein expression of CDKN1A, E2F1, PTEN and CTGF. The CTGF gene was shown to be a target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays. Our results suggest that miR-17-92 and its target CTGF mediate effects of differentiation-promoting treatment on glioblastoma cells through multiple regulatory pathways.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  • Aguado T, Carracedo A, Julien B, Velasco G, Milman G, Mechoulam R et al. (2007). Cannabinoids induce glioma stem-like cell differentiation and inhibit gliomagenesis. J Biol Chem 282: 6854–6862.

    Article  CAS  PubMed  Google Scholar 

  • Chan JA, Krichevsky AM, Kosik KS . (2005). MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 65: 6029–6033.

    Article  CAS  PubMed  Google Scholar 

  • Chen HC, Chen GH, Chen YH, Liao WL, Liu CY, Chang KP et al. (2009). MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma. Br J Cancer 100: 1002–1011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Conti A, Aguennouz M, La Torre D, Tomasello C, Cardali S, Angileri FF et al. (2009). MiR-21 and 221 upregulation and miR-181b downregulation in human grade II-IV astrocytic tumors. J Neurooncol 93: 325–332.

    Article  CAS  PubMed  Google Scholar 

  • Corsten MF, Miranda R, Kasmieh R, Krichevsky AM, Weissleder R, Shah K . (2007). MicroRNA-21 knockdown disrupts glioma growth in vivo and displays synergistic cytotoxicity with neural precursor cell delivered S-TRAIL in human gliomas. Cancer Res 67: 8994–9000.

    Article  CAS  PubMed  Google Scholar 

  • Das A, Banik NL, Ray SK . (2008). Retinoids induced astrocytic differentiation with down regulation of telomerase activity and enhanced sensitivity to taxol for apoptosis in human glioblastoma T98G and U87MG cells. J Neurooncol 87: 9–22.

    Article  CAS  PubMed  Google Scholar 

  • D'Ercole J, Ye P . (2008). Expanding the mind: insulin-like growth factor I and brain development. Endocrinology 149: 5958–5962.

    Article  CAS  Google Scholar 

  • Dews M, Homayouni A, Yu D, Murphy D, Sevignani C, Wentzel E et al. (2006). Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster. Nat Genet 38: 1060–1065.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dictus C, Tronnier V, Unterberg A, Herold-Mende C . (2007). Comparative analysis of in vitro conditions for rat adult neural progenitor cells. J Neurosci Methods 161: 250–258.

    Article  CAS  PubMed  Google Scholar 

  • Ernst A, Hofmann S, Ahmadi R, Becker N, Korshunov A, Engel F et al. (2009). Genomic and expression profiling of glioblastoma stem cell-like spheroid cultures identifies novel tumor-relevant genes associated with survival. Clin Cancer Res 15: 6541–6550.

    Article  CAS  PubMed  Google Scholar 

  • Ferrara N . (2002). Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin Oncol 29: 10–14.

    Article  CAS  PubMed  Google Scholar 

  • Fontana L, Fiori ME, Albini S, Cifaldi L, Giovinazzi S, Forloni M et al. (2008). Antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through CDKN1A and BCL2L11. PLoS ONE 3: e2236.

    Article  PubMed  PubMed Central  Google Scholar 

  • Foshay KM, Gallicano GI. . (2009). MiR-17 family miRNAs are expressed during early mammalian development and regulate stem cell differentiation. Dev Biol 326: 431–443.

    Article  CAS  PubMed  Google Scholar 

  • Gal H, Pandi G, Kanner AA, Ram Z, Lithwick-Yanai G, Amariglio N et al. (2008). MIR-451 and Imatinib mesylate inhibit tumor growth of Glioblastoma stem cells. Biochem Biophys Res Commun 376: 86–90.

    Article  CAS  PubMed  Google Scholar 

  • Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S et al. (2004). Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64: 7011–7021.

    Article  CAS  PubMed  Google Scholar 

  • Gangaraju VK, Lin H . (2009). MicroRNAs: key regulators of stem cells. Nat Rev Mol Cell Biol 10: 116–125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gillies JK, Lorimer IA . (2007). Regulation of p27Kip1 by miRNA 221/222 in glioblastoma. Cell Cycle 6: 2005–2009.

    Article  CAS  PubMed  Google Scholar 

  • Godlewski J, Nowicki MO, Bronisz A, Williams S, Otsuki A, Nuovo G et al. (2008). Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res 68: 9125–9130.

    Article  CAS  PubMed  Google Scholar 

  • Inoki I, Shiomi T, Hashimoto G, Enomoto H, Nakamura H, Makino K et al. (2002). Connective tissue growth factor binds vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis. FASEB J 16: 219–221.

    Article  CAS  PubMed  Google Scholar 

  • Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada K . (2009). MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes. Blood 113: 396–402.

    Article  CAS  PubMed  Google Scholar 

  • Karmakar S, Banik NL, Patel SJ, Ray SK . (2007). Combination of all-trans retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice. Apoptosis 12: 2077–2087.

    Article  CAS  PubMed  Google Scholar 

  • Kefas B, Godlewski J, Comeau L, Li Y, Abounader R, Hawkinson M et al. (2008). MicroRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Res 68: 3566–3572.

    Article  CAS  PubMed  Google Scholar 

  • Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB . (2003). Prediction of mammalian microRNA targets. Cell 115: 787–798.

    Article  CAS  PubMed  Google Scholar 

  • Lichter P, Tang CJ, Call K, Hermanson G, Evans GA, Housman D et al. (1990). High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247: 64–69.

    Article  CAS  PubMed  Google Scholar 

  • Lu J, Zhang F, Zhao D, Hong L, Min J, Zhang L et al. (2008). ATRA-inhibited proliferation in glioma cells is associated with subcellular redistribution of beta-catenin via up-regulation of Axin. J Neurooncol 87: 271–277.

    Article  CAS  PubMed  Google Scholar 

  • Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL . (2007). Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol 310: 442–453.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Malaterre J, Ramsay RG, Mantamadiotis T . (2007). Wnt–Frizzled signalling and the many paths to neural development and adult brain homeostasis. Front Biosci 12: 492–506.

    Article  CAS  PubMed  Google Scholar 

  • Malzkorn B, Wolter M, Liesenberg F, Grzendowski M, Stühler K, Meyer H et al. (2009). Identification and functional characterization of microRNAs involved in the malignant progression of gliomas. Brain Pathol (in press).

  • Medina R, Zaidi SK, Liu CG, Stein JL, van Wijnen AJ, Croce CM et al. (2008). MicroRNAs 221 and 222 bypass quiescence and compromise cell survival. Cancer Res 68: 2773–2780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mendell JT . (2008). MiRiad roles for the miR-17-92 cluster in development and disease. Cell 133: 217–222.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mendrzyk F, Radlwimmer B, Joos S, Kokocinski F, Benner A, Stange DE et al. (2005). Genomic and protein expression profiling identifies CDK6 as novel independent prognostic marker in medulloblastoma. J Clin Oncol 23: 8853–8862.

    Article  CAS  PubMed  Google Scholar 

  • Monzo M, Navarro A, Bandres E, Artells R, Moreno I, Gel B et al. (2008). Overlapping expression of microRNAs in human embryonic colon and colorectal cancer. Cell Res 18: 823–833.

    Article  CAS  PubMed  Google Scholar 

  • Navarro A, Marrades RM, Viñolas N, Quera A, Agustí C, Huerta A et al. (2009). MicroRNAs expressed during lung cancer development are expressed in human pseudoglandular lung embryogenesis. Oncology 76: 162–169.

    Article  CAS  PubMed  Google Scholar 

  • Northcott PA, Fernandez-L A, Hagan JP, Ellison DW, Grajkowska W, Gillespie Y et al. (2009). The miR-17/92 polycistron is up-regulated in sonic hedgehog-driven medulloblastomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors. Cancer Res 69: 3249–3255.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Obayashi S, Tabunoki H, Kim SU, Satoh J . (2009). Gene expression profiling of human neural progenitor cells following the serum-induced astrocyte differentiation. Cell Mol Neurobiol 29: 423–438.

    Article  CAS  PubMed  Google Scholar 

  • O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT . (2005). c-Myc-regulated microRNAs modulate E2F1 expression. Nature 435: 839–843.

    Article  CAS  PubMed  Google Scholar 

  • Ohgaki H, Kleihues P . (2005). Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol 64: 479–489.

    Article  CAS  PubMed  Google Scholar 

  • Ohgawara T, Kubota S, Kawaki H, Kondo S, Eguchi T, Kurio N et al. (2009). Regulation of chondrocytic phenotype by micro RNA 18a: involvement of Ccn2/Ctgf as a major target gene. FEBS Lett 583: 1006–1010.

    Article  CAS  PubMed  Google Scholar 

  • Ohno R, Asou N, Ohnishi K . (2003). Treatment of acute promyelocytic leukemia: strategy toward further increase of cure rate. Leukemia 17: 1454–1463.

    Article  CAS  PubMed  Google Scholar 

  • Papagiannakopoulos T, Shapiro A, Kosik KS . (2008). MicroRNA-21 targets a network of key tumor-suppressive pathways in glioblastoma cells. Cancer Res 68: 8164–8172.

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW . (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfister S, Remke M, Toedt G, Werft W, Benner A, Mendrzyk F et al. (2007). Supratentorial primitive neuroectodermal tumors of the central nervous system frequently harbor deletions of the CDKN2A locus and other genomic aberrations distinct from medulloblastomas. Genes Chromosomes Cancer 46: 839–851.

    Article  CAS  PubMed  Google Scholar 

  • Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G et al. (2006). Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444: 761–765.

    Article  CAS  PubMed  Google Scholar 

  • Schulte JH, Horn S, Otto T, Samans B, Heukamp LC, Eilers UC et al. (2008). MYCN regulates oncogenic MicroRNAs in neuroblastoma. Int J Cancer 122: 699–704.

    Article  CAS  PubMed  Google Scholar 

  • Shi L, Cheng Z, Zhang J, Li R, Zhao P, Fu Z et al. (2008). Hsa-mir-181a and hsa-mir-181b function as tumor suppressors in human glioma cells. Brain Res 1236: 185–193.

    Article  CAS  PubMed  Google Scholar 

  • Silber J, Lim DA, Petritsch C, Persson AI, Maunakea AK, Yu M et al. (2008). MiR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med 6: 14.

    Article  PubMed  PubMed Central  Google Scholar 

  • Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al. (2004). Identification of human brain tumour-initiating cells. Nature 432: 396–401.

    Article  CAS  PubMed  Google Scholar 

  • Solinas-Toldo S, Lampel S, Stilgenbauer S, Nickolenko J, Benner A, Döhner H et al. (1997). Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20: 399–407.

    Article  CAS  PubMed  Google Scholar 

  • Suárez Y, Fernández-Hernando C, Yu J, Gerber SA, Harrison KD, Pober JS et al. (2008). Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis. Proc Natl Acad Sci USA 105: 14082–14087.

    Article  PubMed  PubMed Central  Google Scholar 

  • Uziel T, Karginov FV, Xie S, Parker JS, Wang YD, Gajjar A et al. (2009). The miR-1792 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma. Proc Natl Acad Sci USA 106: 2812–2817.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E et al. (2005). MicroRNA expression in zebrafish embryonic development. Science 309: 310–311.

    Article  CAS  PubMed  Google Scholar 

  • Xia H, Qi Y, Ng SS, Chen X, Li D, Chen S et al. (2009). MicroRNA-146b inhibits glioma cell migration and invasion by targeting MMPs. Brain Res 1269: 158–165.

    Article  CAS  PubMed  Google Scholar 

  • Yu J, Wang F, Yang GH, Wang FL, Ma YN, Du ZW et al. (2006). Human microRNA clusters: genomic organization and expression profile in leukemia cell lines. Biochem Biophys Res Commun 349: 59–68.

    Article  CAS  PubMed  Google Scholar 

  • Yuan X, Curtin J, Xiong Y, Liu G, Waschsmann-Hogiu S, Farkas DL et al. (2004). Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23: 9392–9400.

    Article  CAS  PubMed  Google Scholar 

  • Zeng Y, Yang Z, Xu JG, Yang MS, Zeng ZX, You C . (2009). Differentially expressed genes from the glioblastoma cell line SHG-44 treated with all-trans retinoic acid in vitro. J Clin Neurosci 16: 285–294.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F Engel, M Zapatka, S Anders and W Huber for bioinformatic support. We also thank S Hofmann for support with array comparative genomic hybridization, A Korshunov for support with specimen analysis and B Malzkorn for sharing data. This study was supported by Grants 01GS0883, 01GS0884 and 01GS0886 of the German Bundesministerium für Bildung und Forschung, by Grant MRTN-CT-2006-035733 from the European Union FP6 Marie Curie Research Training Network; by a Grant from the Tumorzentrum Heidelberg-Mannheim; by the Sibylle Assmus Foundation; and by the Verein zur Förderung der Krebsforschung e.V.

Data Deposition: Gene expression data are available at Gene Expression Omnibus accession no. GSE17227 (http://www.ncbi.nlm.nih.gov/geo/).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to B Radlwimmer.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ernst, A., Campos, B., Meier, J. et al. De-repression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures. Oncogene 29, 3411–3422 (2010). https://doi.org/10.1038/onc.2010.83

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.83

Keywords

This article is cited by

Search

Quick links