Högskolan i Skövde

Neuroblastoma, a malignant paediatric tumour originating from neural crest cells, exhibits significant clinical and biological heterogeneity. MYCN amplification is a crucial genetic marker associated with aggressive disease and poor prognosis, making it essential to understand the dynamics and functional implications of MYCN overexpression for designing therapeutic therapies. This study developed and applied an XLone-MYCN PiggyBac inducible vector system to investigate MYCN overexpression in neuroblastoma cells. Stable integration of the MYCN gene was confirmed through antibiotic selection and PCR amplification of the MYCN insert in SK-N-AS cells. An inducible promoter allowed controlled MYCN expression upon doxycycline treatment, and successful integration was verified by PCR, gel electrophoresis, and Sanger sequencing. MYCN overexpression was induced in transfected cells, and its effects were analysed using immunostaining, Western blotting, and MTT assays. Immunostaining and Western blot results confirmed MYCN expression and nuclear localisation in doxycycline-treated cells. However, short-term MYCN overexpression did not lead to detectable changes in mitochondrial activity, suggesting that additional functional assays are needed to assess its impact on proliferation and metabolic reprogramming. This inducible system provides a valuable tool for studying the temporal dynamics of MYCN overexpression and its functional implications in neuroblastoma. Future studies should prioritise assays such as RNA sequencing, metabolic profiling, and apoptosis assays to further elucidate MYCN's role in proliferation, metabolic reprogramming, and apoptosis resistance. This system could contribute to identifying novel therapeutic targets for MYCN-driven neuroblastoma.