P152 Long non-coding RNA HOTAIR affects DNA methylation patterns in gastrointestinal stromal tumours (GIST)

Aberrant alterations of DNA methylation patterns have been recognized as early and common events in oncogenesis emphasizing high therapeutic and preventive potential. Concurrent epigenetic silencing of multiple tumour suppressor genes has been reported in different tumours suggesting the existence of a directed program, whereby groups of genes are regulated by promoter methylation. The molecular bases of such a program remain largely unclear. Certain long non-coding RNAs (lncRNAs) have been recently identified that are responsible for target specificity of the histone modification complexes. It remains incompletely understood, however, if lncRNAs may play a role in patterning DNA methylation. In this study, we asked by using gastrointestinal stromal tumours as a model if an epigenetic related HOX Antisense Intergenic RNA, or HOTAIR, is involved in establishment of DNA methylation patterns. To this end, we first showed high up-regulation of HOTAIR in patient samples of high risk compared to low and intermediate risk groups (n=66, p=6.7×10−6), what is supported by the earlier reports on common carcinomas. Highest levels of HOTAIR endogenous expression were next detected also in cell lines GIST T1, GIST48b and GIST882. Stable knockdown of HOTAIR was achieved in GIST T1 and GIST48b cells by RNAi using lentiviral transduction. As expected epigenetic alterations due to HOTAIR knockdown could develop with a delay, genome-wide DNA methylation profiling was performed at passage 12 after the transduction on the Infinium HumanMethylation450 BeadChip platform in triplicates. DNA methylation data were analysed by an R package RnBeads. A total of 218 CpG sites got hypomethylated upon HOTAIR knockdown in GIST T1 and GIST48b cells (Δβ>0.3, FDR