Neuroblastoma is the most common extra-cranial solid tumor in childhood. Tumors with amplification of the MYCN proto-oncogene are characterized by aggressive biology and poor survival of the patients. To improve future treatment options, it is of fundamental interest to understand MYCN’s role in tumorigenesis and determine factors regulating MYCN expression. In this thesis focuses on the interactions between MYCN and miRNAs, a group of endogenous small regulatory RNA molecules that can act as both tumor suppressors and oncogenes. In paper I, we knocked-down MYCN in a neuroblastoma cell lines with MYCN amplification by short hairpin RNA (shRNA) (method described in the appendix paper) and performed a miRNA expression profiling study to elucidate miRNAs that are correlated to MYCN expression. This approach is different from other studies used to investigate the role of N-myc on miRNAs, as MYCN knockdown in addition induces significant neuronal differentiation of the cells. We observed both up- and down-regulation of miRNAs. MiRNAs with positive correlation to MYCN included members of the oncogenic mir-17-92 cluster. One of the most prominently up-regulated miRNAs upon MYCN knockdown was mir-21. However, we were not able to establish a functional role for this miRNA during differentiation. Mir-92a and mir-92b were both positively correlated to MYCN expression. In paper III, we demonstrated that both miRNAs target the tumor suppressor DKK3 in neuroblastoma and repress secretion of the DKK3 protein. Finally, we demonstrated in paper II that the interaction between MYCN and miRNAs is mutual, as the MYCN mRNA itself is targeted by several miRNAs. Some of these miRNAs showed anti-proliferative properties. Re-establishment of these miRNAs in MYCN-amplified neuroblastoma may prove to be of therapeutic value.

The papers and appendix in this thesis are not available in Munin due to publishers' restrictions: 1. Buechner J, Henriksen JR, Haug BH, Tomte E, Flaegstad T and Einvik C: 'Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells', Differentiation (2011) 81(1):25-34. Available at http://dx.doi.org/10.1016/j.diff.2010.09.184 2. Buechner J, Tømte E, Haug BH, Henriksen JR, Løkke C, Flægstad T and Einvik C: 'Tumoursuppressor microRNAs let-7 and mir-101 target the proto-oncogene MYCN and inhibit cell proliferation in MYCN-amplified neuroblastoma', British Journal of Cancer (2011) 105, 296 – 303. Available at http://dx.doi.org/10.1038/bjc.2011.220 3. Haug BH, Henriksen JR, Buechner J, Geerts D, Tomte E, Kogner P, Martinsson T, Flaegstad T, Sveinbjornsson B and Einvik C: 'MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma', Carcinogenesis (2011) 32(7):1005-1012. Available at http://dx.doi.org/10.1093/carcin/bgr073. The accepted manuscript version of this article is freely available in Munin at http://hdl.handle.net/10037/3550 4. Henriksen JR, Buechner J, Lokke C, Flaegstad T and Einvik C: 'Inhibition of gene function in mammalian cells using short-hairpin RNA (shRNA)', Methods in Molecular Biology (2011) 703:189-204. Available at http://dx.doi.org/10.1007/978-1-59745-248-9_13