Targeted MYCN suppression and its effect on miRNA in neuroblastoma

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Thesis introduction (PDF)
Jørn R. Henriksen, Cecilie Løkke, Martin Hammerø, Dirk Geerts, Rogier Versteeg, Trond Flægstad and Christer Einvik: 'Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines', Nucleic Acids Research, 2007, Vol. 35, No. 9 (reprinted with permission) (PDF)
Jørn R Henriksen, Bjørn Helge Haug, Jochen Buechner, Ellen Tømte, Cecilie Løkke, Trond Flaegstad and Christer Einvik: Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma', BMC Developmental Biology 2011, 11:1 (reprinted with permission) (PDF)
Jochen Buechner, Joern R. Henriksen, Bjørn Helge Haug, Ellen Tømte, Trond Flaegstad and Christer Einvik: 'Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells'. (Accepted manuscript, reprinted with permission.) Published version: Differentiation, Volume 81, Issue 1, January 2011, Pages 25-34 (Elsevier), available at http://dx.doi.org/10.1016/j.diff.2010.09.184 (PDF)
Henriksen JR, Büchner J, Løkke C, Flægstad T and Einvik C.: 'Inhibition of gene function in mammalian cells using short-hairpin RNA (shRNA)' (Accepted manuscript, reprinted with permission.) Published version: Methods in Molecular Biology, 2011, Volume 703, Part 1, 189-204 (Springer), available at http://dx.doi.org/10.1007/978-1-59745-248-9_13 (PDF)
Bjørn Helge Haug, Jørn Remi Henriksen, Jochen Buechner, Per Kogner,Tommy Martinsson, Trond Flægstad, Baldur Sveinbjørnsson and Christer Einvik: 'MYCN-regulated miRNAs inhibit secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma' (manuscript, reprinted with permission). Later published in Carcinogenesis (2011) 32 (7): 1005-1012, and available at http://dx.doi.org/10.1093/carcin/bgr073 (PDF)
Entire thesis in one comprehensive file (PDF)
Date
2010-12-03
Type
Doctoral thesis
Doktorgradsavhandling

Author
Henriksen, Jørn Remi
Abstract
Neuroblastoma is the most common extra-cranial tumour disease in children, and accounts for 15% of all childhood cancer deaths. MYCN is a transcription factor and a proto-oncogene that most often initiates transcription of target genes involved in increased proliferation and inhibition of differentiation. MYCN amplified neuroblastoma represents the most aggressive form of this disease. Silencing of MYCN is an attractive approach for understanding the role of this gene in development and disease. MYCN is involved in development, proliferation and differentiation. In order to explore its functions in detail, it is a great advantage to be able to silence its expressions in all cells present for an indefinite period of time, and at any suitable time-point. In paper I we describe a novel design of an inducible H1 promoter capable of delivering shRNA in a conditional manner. This versatile system allows inducible expression of any desired shRNA. In paper II this promoter is used to control the conditional expression of anti-MYCN shRNAs from cassettes stably integrated in two MYCN-amplified cell lines. Here MYCN is silenced when the inducer doxycycline is added to the media, resulting in increased differentiation and reduced proliferation of the MYCN amplified cell lines. MiRNAs are small non-coding RNA shown to regulate the expression of a vast amount of proteins in humans. In paper III we investigate the effect of MYCN suppression on the global miRNA expression in MYCN-amplified neuroblastoma. We find that reduced MYCN expression leads to both increased and reduced expression of several miRNAs. The function of mir-21 is investigated further, and no effect on neither proliferation nor differentiation is found. Other miRNAs demonstrated to be regulated by MYCN, is mir-92a and mir-92b. In paper IV we demonstrate how these miRNAs suppresses the expression of DKK3, a tumour suppressor gene often found to be down-regulated in MYCN-amplified neuroblastoma.
Publisher
Universitetet i Tromsø
University of Tromsø
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