Högskolan i Skövde

Recent developments in mRNA vaccines have opened up new therapeutic opportunities for diseases that remain a global health concern. One of these is neuroblastoma, the most common solid cancer in childhood, with numerous high-risk cases every year. Neuroblastoma-associated transcript 1 (NBAT1) is a long noncoding RNA associated with tumour suppressor functions, and its low expression correlates with higher pathological grade. This study aimed to optimise a procedure for synthesising NBAT1 long non-coding RNA and further investigate if exogenous delivery would restore tumour suppressor function in the neuroblastoma cell line SH-SY5Y. NBAT1 and p53 upregulated modulator of apoptosis (PUMA) expression was profiled by real-time qPCR in SH-SY5Y and BE(2)-C cell lines. NBAT1 and PUMA sequences were cloned into an mRNA expression vector using cloning based on restriction enzymes, with insert identity confirmed by Sanger sequencing. In vitro transcription was performed to generate RNA, which was used to transfect SH-SY5Y cells. Overexpression following transfection was assessed by real-time qPCR, and an apoptosis assay was performed to evaluate downstream effects. NBAT1 and PUMA expression seemed to be higher in the SH-SY5Y cell line than in the BE(2)-C cell line, consistent with the genes’ relation to the p53 pathway and p53 mutation in the BE(2)-C cell line. NBAT1 insert identity was confirmed, though suboptimal plasmid preparation was noted. PUMA cloning was unsuccessful due to an off-target amplification. In vitro transcription reaction yielded RNA, but poor plasmid quality influenced RNA yield and transfection outcomes. Transfections of SH-SY5Y cells with NBAT1 constructs did not result in confirmed overexpression. The apoptosis assay suggested elevated apoptosis in transfected cells, though insufficient replicates did not allow for appropriate statistical analysis. The project represents a first step towards a therapeutic approach based on RNA targeting NBAT1 in neuroblastoma, providing a basis for future optimisation of the procedure setup.