Myocardial Infraction Associated Transcript (MIAT) is a subnuclear lncRNA that interferes with alternative splicing and is associated with increased risk of various heart conditions and nervous system tumours. The current study aims to elucidate the role of MIAT in cell survival, apoptosis and migration in neuroblastoma and glioblastoma multiforme. To this end, MIAT was silenced by MIAT-specific siRNAs in neuroblastoma and glioblastoma cell lines, and RNA sequencing together with a series of functional assays were performed. The RNA sequencing has revealed that the expression of an outstanding number of genes is altered, including genes involved in cancer-related processes, such as cell growth and survival, apoptosis, reactive oxygen species (ROS) production and migration. Furthermore, the functional studies have confirmed the RNA sequencing leads, with our key findings suggesting that MIAT knockdown eliminates long-term survival and migration and increases basal apoptosis in neuroblastoma and glioblastoma cell lines. Taken together with the recent demonstration of the involvement of MIAT in glioblastoma, our observations suggest that MIAT could possess tumour-promoting properties, thereby acting as an oncogene, and has the potential to be used as a reliable biomarker for neuroblastoma and glioblastoma and be employed for prognostic, predictive and, potentially, therapeutic purposes for these cancers.

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Missense somatic mutations affecting histone H3.1 and H3.3 proteins are now accepted as the hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) as well as a subset of adult glioblastoma multiforme (GBM). Different mutations give rise to one of three amino acid substitutions at two critical positions within the histone tails, K27M, G34R/V. Several studies have highlighted gene expression and epigenetic changes associated with histone H3 mutations; however their precise roles in tumourigenesis remain incompletely understood. Determining how such amino acid substitutions in a protein affect its properties can be challenging because of difficulties in detecting and tracking mutant proteins within cells and tissues. Here we describe a strategy for the generation of antibodies to discriminate G34R and G34V mutant histone H3 proteins from their wild-type counterparts. Antibodies were validated by western blotting and immunocytochemistry, using recombinant H3.3 proteins and paediatric GBM cell lines. The H3-G34R antibody demonstrated a high degree of selectivity towards its target sequence. Accordingly, immunostaining on a cohort of 22 formalin-fixed paraffin embedded tumours with a previously known H3.3 G34R mutation status, detected successfully the corresponding mutant protein in 11/11 G34R cases. Since there was a high concordance between genotype and immunohistochemical analysis of G34R mutant tumour samples, we analysed a series of tissue microarrays (TMAs) to assess the specificity of the antibody in a range of paediatric brain tumours, and noted immunoreactivity in 2/634 cases. Importantly, we describe the generation and validation of highly specific antibodies for G34 mutations. Overall our work adds to an extremely valuable portfolio of antibodies, not only for histopathologic detection of tumour-associated mutant histone sequences, but also facilitating the study of spatial/anatomical aspects of tumour formation and the identification of downstream targets and pathways in malignant glioma progression.

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