Brain tumours account for the most frequent type of solid tumours among children. Despite advances in surgery and chemotherapy, brain tumours are still the main cause of cancer deaths in children. Furthermore, little is known about DNA methylation changes in paediatric astrocytoma. Recent investigations suggest that many tumours are initiated not only by genetic abnormalities, but also caused by epigenetic changes. DNA methylation is a key epigenetic mechanism that controls the regulation of gene expression. Interestingly, unlike DNA mutations, epigenetic abnormalities are reversible. The reversibility of epigenetic abnormalities upon pharmacological unmasking has prompted interest in developing epigenetic therapy with the crucial goal of restoring the expression of aberrantly silenced genes. The focus of this study was to utilise a combination of different microarray strategies to develop an integrative candidate gene approach to identify several novel frequently methylated genes in a cohort of paediatric HGA (High grade glioma) samples. In addition, to investigate the potential of therapeutic efficacy of a DNA methyltransferase inhibitor, 5-Aza-dC in paediatric HGA. There were 147 genes commonly identified to be potentially methylated in IN699 cells using the two different array strategies integration; re-expression array and Illumina Infinium Human Methylation 450k array. Furthermore, using two complementary microarray strategies including methylation 450k array and expression array, this work identified 55 genes that were both methylated and under-expressed in these HGA cultures. Following validation with CoBRA and RT-PCR coupled with the response of hypermethylated promoters to the demethylating agent 5-Aza-dC, six novel genes (CXCL14, PRR5L, ELTD1, ITGA2, KRT8 and NTM) that are frequently silenced in paediatric astrocytoma were identified. This study suggests that re-expression of ii CXCL14 inhibited the colony formation and cell growth and reduces the migration rate significantly in IN699 short term culture and likely have functional significance in the development of paediatric HGA and an excellent candidate gene for further analysis. In parallel, the efficacy of 5-Aza-dC treatment was examined in paediatric HGA aiming to introduce this epigenetic therapy as a potential mechanism in management of this tumours. This study demonstrated that, relatively low dose of 5-Aza-dC sharply reduced the colony formation and inhibited proliferation and not through the apoptotic effect. It is likely that this reduction in proliferation without cell death is due to using relatively low doses that do not acutely kill cells, thus, allow the sustained alterations in both gene expression patterns and appearance of a new phenotype to emerge. Taken together, this work contributes to a more detailed understanding of the effect of epigenetic silencing on paediatric HGA. This investigation also demonstrated the use of epigenetic drug, 5-aza-dC to reverse the gene silencing for the potential treatment of paediatric HGA.

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation-induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation-associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5-aza-2′-deoxycitidine (5-Aza-dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation. was partially reversible. TSA was more efficient than 5-Aza-dC in inducing TMS1 expression, and the combination of 5-Aza-dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation-associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5-Aza-dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5-Aza-dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way.