Abstract
The lncRNA HOTAIR has been implicated in several human cancers. Here, we evaluated the molecular alterations and upstream regulatory mechanisms of HOTAIR in glioma, the most common primary brain tumors, and its clinical relevance. HOTAIR gene expression, methylation, copy-number and prognostic value were investigated in human gliomas integrating data from online datasets and our cohorts. High levels of HOTAIR were associated with higher grades of glioma, particularly IDH wild-type cases. Mechanistically, HOTAIR was overexpressed in a gene dosage-independent manner, while DNA methylation levels of particular CpGs in HOTAIR locus were associated with HOTAIR expression levels in GBM clinical specimens and cell lines. Concordantly, the demethylating agent 5-Aza-2′-deoxycytidine affected HOTAIR transcriptional levels in a cell line-dependent manner. Importantly, HOTAIR was frequently co-expressed with HOXA9 in high-grade gliomas from TCGA, Oncomine, and our Portuguese and French datasets. Integrated in silico analyses, chromatin immunoprecipitation, and qPCR data showed that HOXA9 binds directly to the promoter of HOTAIR. Clinically, GBM patients with high HOTAIR expression had a significantly reduced overall survival, independently of other prognostic variables. In summary, this work reveals HOXA9 as a novel direct regulator of HOTAIR, and establishes HOTAIR as an independent prognostic marker, providing new therapeutic opportunities to treat this highly aggressive cancer.
Highlights
Gliomas encompass the majority of primary central nervous system tumors, and have been historically classified based on their histopathological features, according to four grades of malignancy (World Health Organization – WHO – grade I-IV)
HOX Transcript Antisense Intergenic RNA (HOTAIR) can act as modular scaffold, with different binding sites for harboring demethylase (LSD1, that mediates the enzymatic demethylation of histone H3 dimethyl lysine 4, H3K4me2) and methylase (PRC2 and the histone H3 lysine 27 – H3K27 – methylase EZH2) complexes, resulting in the transcriptional inhibition of the HOXD locus on chromosome 2 by epigenetic control of the chromatin structure [10, 12]
We performed anti-HOXA9 chromatin-immunoprecipitation (ChIP) on U251 GBM cell line, U87MG-HOXA9 (HOXA9 overexpression models) and their HOXA9-negative counterparts. In both U251 and U87MG-HOXA9, ChIPqPCR revealed significantly increased HOXA9 occupancy of the promoter region of HOTAIR in comparison to their respective controls (Figure 2D and 2E; p < 0.0002 for U251; p = 0.0148 for U87MG-HOXA9), confirming that HOXA9 directly interacts with the promoter region of HOTAIR. These results indicate that HOXA9 is a direct activator of HOTAIR expression in GBM cells, supporting the strong co-expression of these genes observed in glioma cell lines
Summary
Gliomas encompass the majority of primary central nervous system tumors, and have been historically classified based on their histopathological features, according to four grades of malignancy (World Health Organization – WHO – grade I-IV). Recent studies have explored the complexity of the transcriptome and reported the existence of a large number of transcripts that do not code for a protein, which have the critical ability to regulate gene expression [7]. Among others, these transcripts include long non-coding RNAs (lncRNA) [8, 9] that are defined as spliced, polyadenylated and >200 nucleotides long RNAs that modify the genome in a highly tissuespecific manner [9, 10]. HOTAIR can act as modular scaffold, with different binding sites for harboring demethylase (LSD1, that mediates the enzymatic demethylation of histone H3 dimethyl lysine 4, H3K4me2) and methylase (PRC2 and the histone H3 lysine 27 – H3K27 – methylase EZH2) complexes, resulting in the transcriptional inhibition of the HOXD locus on chromosome 2 by epigenetic control of the chromatin structure [10, 12]
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