Long non‐coding RNAs in brain tumours: Focus on recent epigenetic findings in glioma

Abstract Glioma biology is a major focus in tumour research, primarily due to the aggressiveness and high mortality rate of its most aggressive form, glioblastoma. Progress in understanding the molecular mechanisms behind poor prognosis of glioblastoma, regardless of treatment approaches, has changed the classification of brain tumours after nearly 100 years of relying on anatomopathological criteria. Expanding knowledge in genetic, epigenetic and translational medicine is also beginning to contribute to further elucidating molecular dysregulation in glioma. Long non‐coding RNAs (lncRNAs) and their main representatives, large intergenic non‐coding RNAs (lincRNAs), have recently been under scrutiny in glioma research, revealing novel mechanisms of pathogenesis and reinforcing others. Among those confirmed was the reactivation of events significant for foetal brain development and neuronal commitment. Novel mechanisms of tumour suppression and activation of stem‐like behaviour in tumour cells have also been examined. Interestingly, these processes involve lncRNAs that are present both during normal brain development and in brain malignancies and their reactivation might be explained by epigenetic mechanisms, which we discuss in detail in the present review. In addition, the review discusses the lncRNAs‐induced changes, as well as epigenetic changes that are consequential for tumour formation, affecting, in turn, the expression of various types of lncRNAs.

provide more precise diagnosis and treatment. 6 However, the quest for potential therapeutic targets remains unfulfilled; 7,8 thus, new strategies are emerging to explore the molecular basis of brain tumour development for clinical exploitation.
An expanding research area in epigenetic regulation of tumorigenesis includes the field of non-coding RNAs. Among non-coding species, lncRNAs are being intensively investigated, and enticing progress has been made in recent years, revealing their roles in chromatin remodelling, transcription, posttranscriptional processing and intracellular trafficking. 9,10 lincRNAs consist of separate transcript units that are located between, but do not overlap with, protein-coding genes (PCGs) and represent the largest subclass of functional characterized lncRNAs. 11 lincRNAs have highly tissuespecific expression and are primarily involved in epigenetic regulation of PCG expression as well as in normal development processes, including embryogenesis, stem cell pluripotency and differentiation. 12,13 Aberrant expression of lncRNAs is associated with several types of cancer, including glioma. Normal expression of lncRNAs is affected by functional mutations or epigenetic alterations, transforming them into cancer-associated transcripts present at every step of tumour development. Several lincRNAs have been associated with oncogenic mechanisms in gliomas, such as interference with glial cell differentiation and maintenance of stemness in glial cancer stem cells, detailed later in the present review. The differential expression patterns of lncRNAs between tumour and normal tissues, along with expression discrepancies in tumours with different clinical features, suggest that lncRNAs could act as diagnostic and prognostic biomarkers and pharmaceutical targets in gliomas.
This review focuses on lncRNA-related mechanisms found to be activated in glioma molecular biology, some of which are significant to the developmental biology of the central nervous system, becoming aberrantly reactivated in adulthood. Several lncRNAs involved in embryogenesis that are found to be reactivated in brain tumour are discussed in the section dedicated to lncRNA functions in glioma biology. We will also review the two sides of lncRNA epigenetic regulation in glioma, as both targets and drivers. Finally, arguments in favour of using lncRNAs as diagnostic tools and therapeutic targets in glioma will be presented.

FUNCTIONS OF LNCRNAS
Completion of human genome sequencing and de novo mammalian transcriptome characterization has revealed tens of thousands of lncRNA sequences emanating from uncharacterized genomic regions. 14,15 LncRNA transcripts are lengthy (>200 nucleotides), often containing multiple functional domains capable of interacting with DNA, proteins or RNAs (mRNAs and microRNAs). The versatility of lncRNAs to fold into a variety of secondary and tertiary structures explains the diversity of their interacting partners and the complexity of their molecular functions.
Four types of molecular mechanism for lncRNAs have been distinguished, whereby lncRNAs act as signals, decoys, guides or scaffolds. 16,17 Signal lncRNAs regulate transcriptional activity or signalling initiation. Decoy lncRNAs bind and titrate away gene regulatory elements (proteins, mRNAs, microRNAs). In the nucleus, lncRNAs can bind transcription factors (TFs) or chromatin modifiers, whereas in the cytoplasm, they function as a sponge to attract proteins and miRNA/RISC complexes away from their targets. In Table 1, we summarized some of significant lncRNA-miRNA interactions with impact on glioma biology and pathology, where lncRNA is acting as competing endogenous to sponge miRNAs. Their interactions are discussed in detail in the next sections. Guide lncRNAs direct their molecular target (proteins) either in cis on neighbouring genes or in trans to distantly located genes. Scaffold lncRNAs bind and assemble multiple effector partners (proteins, RNAs) into complexes, controlling their formation and localization. 18,19 Based on these characterized mechanisms, lncRNAs can initiate regulatory networks with high complexity at epigenetic, transcriptional and posttranscriptional levels required for cellular functions.
Though lncRNAs exhibit poor sequence conservation and are less expressed than are PCGs, they present the highest specificity with respect to cell type, subcellular compartment, developmental stage and in response to environmental stimuli. 11,20,21 LncRNAs play regulatory and structural roles in diverse cellular processes, including embryogenesis, stem cell pluripotency, differentiation and senescence. Interestingly, compared to that in other organs, the highest number of expressed lncRNAs has been found in the brain, and their number surpasses that of the brain PCG transcripts. 22,23 The brain's lncRNAs have the highest tissue specificity and are the most evolutionarily conserved, with similar spatiotemporal expression patterns across multiple species. [23][24][25] In neural development, lncRNAs have important roles in regulating stem cell maintenance and differentiation programmes, including cell fate specification 26 and neural lineage commitment. 27,28 Under a complex programme of differentiation, partially controlled by lncRNAs, diverse types of neuroprogenitors develop into different neuronal and glial cell subtypes. 29 Moreover, differentially expressed lncRNAs across various stages of differentiation indicate that they can amplify and consolidate the molecular differences between cell types that are required to control cell identity and lineage commitment. 30 Some of these embryonically active programmes are reactivated during adulthood, primarily during oncogenic transformation, which will be discussed in this review.

IN GLIOMA PATHOL OGY
Systemic high-throughput studies, including lncRNA microarray and RNA sequencing on hundreds of classified glioma samples and normal brain tissues, have demonstrated that lncRNAs, in addition to their involvement in normal biological processes, represent key players during tumorigenesis. 31 Zhang et al 32 identified 129 lncRNAs differentially expressed between glioma and normal brain tissues. Two lncRNAs, colorectal neoplasia differentially expressed (CRNDE) and HOX antisense intergenic RNA myeloid 1 (HOTAIRM1), showed the highest expression in glioma (grade I-IV) compared with that in normal tissue. Interestingly, both lncRNAs are also involved in brain development and neuronal differentiation, 31,33 with HOTAIRM1 highly expressed in foetal brain. 34 Comparing recurrent glioma samples with primary tumours, CRNDE and HOTAIRM1 were significantly up-regulated among thousands of differentially expressed lncRNAs. The same study demonstrated that many dysregulated lncRNA-mRNA pairs from the recurrent group were closely related to cancer or neural differentiation. 35 Several studies have correlated lncRNA expression profiles with different histological subtypes and malignancy grades in gliomas. 32,36,37 Global gene expression analyses identified 27 lncRNAs that are differentially expressed between astrocytomas and oligodendrogliomas. 32 For GBM, six lncRNAs were found to be significantly associated with patient prognosis but were independent of patient age or MGMT promoter methylation status. 38 One transcript, KIAA0495, was abundantly expressed in GBM tissues and associated with reduced survival. Myocardial infarction-associated transcript (MIAT/Gomafu), a neuron-specific component of the nuclear matrix 39 involved in neurogenesis and neural stem cells differentiation, 40 was identified as a significantly down-regulated lncRNA in glioma.
Gliomas can be classified into three different groups based on their dysregulated pattern of lncRNA expression and may be further associated with mutational status, molecular subtypes and clinical outcome. 41 In addition, an individual lncRNA can indicate stages in tumour progression and might be useful as an independent biomarker for diagnosis and prognosis. For example, the HOX transcript antisense RNA (HOTAIR) exhibits reduced expression in low-grade gliomas (LGG) compared with that in GBM. Furthermore, differentiated levels of expression between GBM subtypes were revealed, with higher levels of HOTAIR in classical and mesenchymal subtypes than those in proneural, a neural subtype. 37 Recently, hundreds of lncRNAs differentially expressed between glioma samples (grades I-IV) and normal brain tissues were identified and were subsequently associated with glioma pathology. 42 Among them, CRNDE was found to exhibit a 40-fold higher expression in GBM than that in normal tissues. Additionally, TUNA (TCL1 upstream neural differentiation-associated RNA) was shown to be severely down-regulated in all glial tumours by 45-fold in GBMs and

14-fold in
LGGs. TUNA performs a regulatory function in pluripotency and neural differentiation of ESCs, acting as a scaffold for RNA-binding proteins. TUNA also regulates the expression of several key neurogenic genes, including SOX2, and its depletion causes down-regulation of SOX2 and subsequent loss of neurogenesis. 43 Therefore, TUNA represents another example of an lncRNA involved in both neurogenesis and brain tumour progression.  in response to cell cycle, p53 and STAT3 pathway activation. 44 A novel four-lncRNA signature that accurately predicts survival in GBM patients was recently reported, and results of a functional analysis suggest that co-expressed genes tend to cluster within nine immunerelated processes and four biological pathways. 45 While dysregulation of lncRNAs has been correlated with glioma pathology and alteration of diverse signalling pathways, their functional significance in cancer is only beginning to be explored. Importantly, identification of key lncRNAs involved in neurogenesis and normal brain development (e.g CRNDE, HORAIRM1 and TUNA) as the most dysregulated lncRNAs in glioma suggests aberrant reactivation as a mechanism that promotes oncogenesis ( Figure 1).

| FUNCTIONS OF LNCRNAS IN GLIOMA BIOLOGY
The hallmark of brain tumours includes rapid cell proliferation, resistance to apoptosis, invasion of surrounding brain tissue, elevated levels of angiogenesis and the existence of therapy-resistant GSCs.
The functional role of lncRNAs is beginning to be validated by systematic experimental analysis of lncRNA activity and effects in normal and transformed cells, including both in vitro and in vivo models.

| ONCOGENIC LNCRNAS
CRNDE, one of the most highly expressed lncRNAs during neuronal differentiation, in induced pluripotent stem cells (iPSCs) and within gliomas 32,35,50 exerts its oncogenic function by promoting glioma cell growth, invasion and migration through different signalling pathways. 51 Acting as a decoy, lncRNAs support glioma progression by reducing miR-136-5p expression, directly impacting PI3K/AKT/mTOR signalling pathways. 52 Knockdown of CRNDE combined with overexpression of miR-384 restrained glioma tumour growth and increased survival in a nude mouse model. CRNDE promotes GSC malignancy by negatively regulating miR-186. 31 Interestingly, the expression of CRNDE in stem cells is regulated by c-Myc, a multipotent TF that stimulates gene amplification and is overexpressed in many cancers, including glioma. 50 SOX2OT is involved in neurogenesis and glioma development. 53 Silencing SOX2OT inhibits proliferation, migration and invasion of NEAT1 is essential for nuclear paraspeckle formation and is overexpressed in most solid tumours. 56 The expression of NEAT1 is closely correlated with higher WHO grade and recurrence in gliomas. 31 Oncogenic NEAT1 depletion through the dual-CRISPR/Cas9 system inhibited GBM cell growth and invasion both in vitro and in vivo.  Nuclear MALAT1/NEAT2 is one of the most highly conserved lncRNAs among mammals and is significantly up-regulated in primary tissues and serum samples from GBM patients who exhibit resistance to TMZ-based treatment. 45 Furthermore, MALAT1 is more  Overexpression of TUG1 in glioma cells leads to the alteration of apoptosis-related mediator proteins. 46 In addition, TUG1 is a downstream effector of the p53-regulatory network, promoting proliferation and invasion of glioma cells and inhibiting apoptosis. In a xenograft GBM model, TUG1 was shown to enhance tumourinduced angiogenesis and VEGF expression through inhibition of miR-299. 64 More importantly, TUG1 is an inducer of GSC differentiation via the Notch pathway, whose increased activity promotes brain tumour growth. 65

| TUMOUR-SUPPRESSOR LNCRNAS
The cellular mechanism of tumour-suppressor lncRNAs consists of inhibiting uncontrolled cellular growth, migration and invasion or inducing apoptosis. MEG3 is highly expressed in normal brain tissue and is down-regulated in gliomas, inhibiting cell proliferation/ DNA synthesis by stimulating expression of p53 protein and modulating its binding to target gene promoters. 32,47 Functional studies revealed that restored expression of MEG3 inhibited GBM cell proliferation and induced apoptosis as well as autophagy. 61 In MEG3 knockout mice, expression of genes involved in the VEGF angiogenic pathway and vascularization in the brain is affected. 74 The ADAMTS9-AS2 antisense transcript of ADAMTS9 gene was shown to be significantly down-regulated in glioma tissues and was negatively correlated with tumour grade and prognosis. The Tumorigenesis results from a complex interplay of both genetic alterations and epigenetic changes affecting various cellular processes. In primary GBM, 80% of studied cases exhibit severe global hypomethylation of DNA. 6 The CpG island methylator phenotype is frequently found in secondary and recurrent GBM tumours and was correlated with high rates of IDH1 and TP53 mutations, early age of diagnosis and better prognosis. 4,6 In GBM, genome-wide sequencing has identified an enormous number of mutations in epigenetic regulatory genes, including histone deacetylase (HDAC) 2 and 9, histone demethylases and methyltransferases. 84  lator, was also demonstrated. 90 The activity of tumour suppressor ADAMTS9-AS2 is also controlled by DNA methylation in glioma.
Additionally, the expression of its transcript was positively correlated with ADAMTS9 and DNMT3A expressions. 76 A recent study showed that the lncRNA LOC285758 expression is regulated by DNA methylation and differs within glioma subtypes, with overexpression and higher promoter hypomethylation in GBM. 91 Histone modifications play an essential role in the regulation of lncRNA expression. LncRNA promoters exhibit specific histone marks, including methylated H3K4, H3K27, H3K36, and acetylated H3K9 and H3K27, suggesting that they undergo epigenetic regulation similar to that of PCG. 21    however, epigenetic pathways can be important therapeutic targets.
Until now, a major unsolved issue with epigenetic therapy for cancer was target specificity. Through their peculiar features, such as tissue and genomic location specificity, and their modular structure interacting with several proteins involved in epigenetic mechanisms, lncRNAs might yield novel approaches for specific epigenome-targeted therapies for gliomas or novel biomarkers for diagnostic, prognostic and monitoring purposes.

ACKNOWLEDG EMENT
The authors would like to thank Dr.

CONFLI CTS OF INTEREST
The authors confirm that there are no conflicts of interest.