Emerging Role of Long Non-Coding RNAs in the Pathobiology of Glioblastoma

Glioblastoma is the utmost aggressive diffuse kind of glioma which is originated from astrocytes, neural stem cells or progenitors. This malignant tumor has a poor survival rate. A number of genetic aberrations and somatic mutations have been associated with this kind of cancer. In recent times, the impact of long non-coding RNAs (lncRNAs) in glioblastoma has been underscored by several investigations. Up-regulation of a number of oncogenic lncRNAs such as H19, MALAT1, SNHGs, MIAT, UCA, HIF1A-AS2 and XIST in addition to down-regulation of other tumor suppressor lncRNAs namely GAS5, RNCR3 and NBAT1 indicate the role of these lncRNAs in the pathogenesis of glioblastoma. Several in vitro and a number of in vivo studies have demonstrated the contribution of these transcripts in the regulation of cell proliferation and apoptosis, cell survival, invasion and metastasis of glioblastoma cells. Moreover, some lncRNAs such as SBF2-AS1 are involved in conferring resistance to temozolomide. Finally, few circularRNAs have been identified that influence the evolution of glioblastoma. In this paper, we discuss the impacts of lncRNAs in the pathogenesis of glioblastoma, their applications as markers and their implications in the therapeutic responses in this kind of cancer.


INTRODUCTION
Being considered as grade IV glioma tumors, glioblastomas are the utmost aggressive diffuse kind of glioma originating from the astrocytes, neural stem cells or progenitors (1). This type of brain tumor includes about half of all glioma tumors and less than 20% of all primary brain tumors (2). Although being a rare tumor, the poor prognosis and low survival rate of glioblastoma have made it an important public health problem (3). It is more frequent in men compared with females, in Western countries compared with developing world and in some ethnicities such as Asians, Latinos and Whites (3). The etiology of this kind of tumor is largely unclarified, as no causal carcinogen has been linked with it. High dose ionizing radiation is the solitary environmental element that is highly associated with risk of glioblastoma (4). A number of genetic aberrations such as activation of growth factor cascade through amplification and mutations in receptor tyrosine kinase genes, induction of the PI3K proteins and loss of the p53 and Rb tumor suppressor genes have been identified in glioblastoma (5). Genome-wide and direct sequencing techniques have also detected recurrent disease-causing mutations in glioblastoma samples in a number of genes such as IDH1 (6) and TERT promoter (7). Moreover, contemporary studies have conveyed anomalous expression of long non-coding RNAs (lncRNAs) in glioblastoma samples indicating the impact of these transcripts in the pathobiology of this kind of cancer (8). These transcripts are larger than 200 nucleotides and regulate expression of numerous genes at transcriptional, post-transcriptional, and epigenetic phases (9). In the current paper, we discuss the impact of lncRNAs in the pathobiology of glioblastoma and their effects on the regulation of cell proliferation and apoptosis, cell survival, invasion and metastatic aptitude of glioblastoma cells.

ONCOGENIC LNCRNAS IN GLIOBLASTOMA
Several oncogenic lncRNAs have been up-regulated in glioblastoma samples. For instance, MIR22HG is an oncogenic lncRNA which has been shown to be highly dysregulated in glioblastoma via assessment of accessible datasets. This lncRNA hosts miR-22-3p and miR-22-5p. Further studies have unraveled over-expression of the MIR22HG/miR-22 route in glioblastoma and glioma stem-like cells. Over-expression of MIR22HG in glioblastoma samples has been related with poor patients' outcome. Knock down of this lncRNA has led to inactivation of the Wnt/b-catenin route via modulating miR-22-3p and miR-22-5p expressions. Functionally, MIR22HG silencing has diminished cell proliferation, invasion and tumor growth in xenograft models. The mentioned miRNAs have been shown to target SFRP2 and PCDH15. Taken together, MIR22HG has been acknowledged as an important activator of the Wnt/bcatenin signaling pathway, and its silencing has been proposed as a therapeutic modality in this kind of cancer (10). The small nucleolar RNA host gene 5 (SNHG5) is another up-regulated lncRNA in glioblastoma which enhances cell proliferation and suppresses cell apoptosis in these cells. Expression of this lncRNA is activated by the Yin Yang 1 (YY1) transcription factor. This lncRNA exerts its oncogenic role via stimulation of the p38/MAPK axis (11). SNHG9 has also been demonstrated to be over-expressed in glioblastoma samples in association with poor survival of patients. SNHG9 has a role in suppression of miR-199a-5p expression and enhancement of Wnt2 expression in glioblastoma cells. This lncRNA has been revealed to enhance aerobic glycolysis and cell proliferation (12). Expression of SAMMSON has been increased in the plasma of patients with glioblastoma but not in those with diffuse neurosarcoidosis, a disorder that shares MRI signs with glioblastoma. This lncRNA has been displayed to suppress expression of miR-622 in glioblastoma cells and subsequently enhance cell (13). MIAT is another up-regulated lncRNA in glioblastoma. Bountali et al. have knocked down this lncRNA in glioblastoma cell lines and analyzed RNA profile of these cells via RNA sequencing method.
They reported differential expression of several genes including those participating in cancer-associated functions, namely cell growth and viability, apoptotic features, reactive oxygen species creation and migration. Functionally, MIAT silencing abolishes long-term viability and migration and enhances apoptosis in these cells (14). A genome-wide expression profiling in glioblastoma cells has identified MALAT1 as one of the most remarkably over-expressed genes following treatment with temozolomide (TMZ). Expression of this lncRNA has been coregulated by p50 and p53 through kBand p53-binding sites which are located in coding sequence of this lncRNA. MALAT1 silencing has increased sensitivity of patient-originated glioblastoma cells to TMZ and improved the effects of this drug in xenograft mice models (15). UCA1 is another oncogenic lncRNA which enhances cell proliferation and migration, while suppressing cell apoptosis. Figure 1 depicts the molecular mechanisms through which UCA1 participates in the pathogenesis of glioblastoma. Table 1 reviews the function of oncogenic lncRNAs in glioblastoma.

TUMOR SUPPRESSOR LNCRNAS IN GLIOBLASTOMA
Expression of GAS5 has been decreased in glioblastoma and its levels have been negatively correlated with miR-34a levels (61). In addition, expression of AC016405.3 has been decreased in glioblastoma tissues in association with numerous aggressive characteristics of this type of cancer. Up-regulation of this lncRNA inhibits proliferation and metastatic ability of glioblastoma cells. The oncogenic miRNA, miR-19a-5p has been identified as a downstream miRNA of AC016405.3. AC016405.3 has been shown to be targeted by miR-19a-5p. Functionally, AC016405.3 inhibits cell proliferation and metastasis via regulation of TET2 by serving as a sponge for miR-19a-5p (62). LINC00657 is another tumor suppressor lncRNA whose expression has been decreased in glioblastoma sections compared with neighboring normal section. Upregulation of this lncRNA has suppressed cell proliferation, colony formation, invasiveness and migratory potential of glioma cells through activating cell apoptosis. LINC00657 has been acknowledged as a direct target of miR-190a-3p, a miRNA that negatively regulates PTEN expression. The tumor suppressive role of LINC00657 has also been verified in xenograft models (63). The lncRNA AC003092.1 has been shown to be down-regulated in TMZ resistance cells compared with their original cells. Moreover, down-regulation of this lncRNA has been correlated with resistance to TMZ, higher possibility of tumor relapse, and poor patients' outcome. Cell line studies has shown improvement of TMZ sensitivity following up-regulation of AC003092.1. The effect of this lncRNA in the modulation of TMZ sensitivity is exerted via regulation of TFPI-2-associated cell apoptosis through sponging miR-195 (64). RNCR3 is another down-regulated lncRNA in glioblastoma. Over-expression of this lncRNA significantly suppresses cell survival and proliferation of glioblastoma cells, while enhancing cell apoptosis and activity caspase-3/7. Besides, up-regulation of this lncRNA enhances expression of Krüppel-like factor 16 (KLF16) via suppressing miR-185-5p (65). Table 2 gives an outline of studies which assessed function of tumor suppressor lncRNAs in glioblastoma.

DIAGNOSTIC AND PROGNOSTIC VALUE OF LNCRNAS IN GLIOBLASTOMA
Expression levels of lncRNAs can distinguish patients with glioblastoma from cancer-free individuals. Moreover, these transcripts can possibly differentiate different brain tumors. For instance, plasma levels of SAMMSON can differentiate glioblastoma from both diffuse neurosarcoidosis and healthy controls (13). Among lncRNAs whose diagnostic power has been assessed in glioblastoma, HOTAIR has exhibited the most promising results.
Tan et al. have demonstrated significant higher levels of this lncRNA in sera of glioblastoma patients compared with controls. The area under the receiver operating characteristic (ROC) curve was 0.913 indicating the ideal feature of HOTAIR for this purpose. Moreover, they reported significant correlation between its levels and high tumor grade. Notably, there was significant correlation between tumor and serum levels of this lncRNA. Finally, exosomes extracted from the serum samples have been shown to contain this lncRNA, further emphasizing the application of this lncRNA in the prognostic and diagnostic processes in glioblastoma (49). In addition, Kaplan-Meier analysis has indicated the correlation between expression levels of several lncRNAs such as SNHG9, TRG-AS1, AGAP2-AS1, lnc-TALC, SBF2-AS1, SNHG20, AC016405.3, LINC-ROR, HOXB-AS1, H19, LINC00152, RAMP2-AS1 and GAS5 and patients' prognosis in the terms of overall survival, disease-free survival and progression free survival. Table 3 gives a summary of studies which assessed such aspect of lncRNAs in glioblastoma.  (16). In addition, UCA1 decreases miR-627-5p levels. As miR-627-5p inhibits NR2C2 expression, down-regulation of miR-627-5p by UCA1 enhances expression of NR2C2. NR2C2 binds with the promoter region of UCA1 and increase its expression through a positive feedback loop. Moreover, NR2C2 enhances expression of SPOCK1 increasing cell proliferation, migration and invasiveness of tumor cells (17).  -AHIF was up-regulated in GBM cells after radiotherapy and affects GBM cell clonogenic formation, DNA repair and apoptosis.
Higher levels of AGAP2-AS1 correlated with lower OS. - 79 GBM patients ---OS in patients with TMZ therapy and low expression of lnc-TALC was increased, whereas high expression of lnc-TALC and therapy with TMZ reduced OS.
TMZ chemotherapy was correlated with the OS of patients with low lnc-TALC expression.
77 with high levels of SBF2-AS1 and 77 with low levels of SBF2-AS1 ---OS decreases in patients with high levels of SBF2-AS1. - 45 patients with low levels of SNHG20 and 33 patients with high levels of SNHG20 ---High levels of SNHG20 was correlated with lower rate of OS. - Two groups of 32 patients with high and low levels of AC016405.3 ---Low expression of AC016405.3 was correlated with a shorter survival rate, a larger size of tumor, a higher grade, and more common distant metastasis.
-   (72). Table 4 summarizes the expression and function of circRNAs in glioblastoma.

DISCUSSION
Both candidate gene and high throughput expression studies have reported anomalous expression of several lncRNAs in glioblastoma samples indicating the oncogenic roles for some lncRNAs and tumor suppressor roles for a number of other lncRNAs. Yet, the function of the former group of lncRNAs has been more assessed in this kind of cancer. Like other cancers, the role of lncRNAs in the pathogenesis of glioblastoma can be exerted through their effects on the expression of miRNAs. Accordingly, several lncRNA/miRNA/ mRNA axes have been identified in this context among them are SNHG9/miR-199a-5p/Wnt2, MIR155HG/miR-185/ANXA2, TRG-AS1/miR-877-5p/SUZ12, LINC01579/miR-139-5p/EIF4G2, AC016405.3/miR-19a-5p/TET2, AC003092.1/miR-195/TFPI-2, LINC00657/miR-190a-3p/PTEN, RNCR3/miR-185-5p/KLF16, and MALAT1/miR-203/thymidylate synthase axes. Thus, comprehensive assessment of these three types of transcripts would facilitate identification of the molecular pathways underlying the pathogenesis of this type of cancer. Moreover, a number of recent studies revealed the role of circRNAs in regulation of expression of miRNAs, thus adding an extra level of complexity in gene regulation networks. An example of the circRNA/miRNA/ mRNA functional axis in glioblastoma is represented by circ_0001946/miR-671-5p/CDR1. Association between lncRNA expression levels and resistance to TMZ has been assessed in several studies. Notably, expressions of oncogenic lncRNAs lnc-TALC, LncSBF2-AS1, MALAT1, TP73-AS1, and H19 as well as expression of tumor suppressor lncRNAs AC003092.1, TUSC7, and RP11-838N2.4 have been shown to alter this phenotype in glioblastoma cells. Therefore, a panel of these lncRNAs might be applied to predict response of pateints to this chemotherapeutic agent and establish a personalized strategy for these patients.
Finally, several oncogenic and tumor suppressor lncRNAs have been identified as modulators of glioblastoma patients' survival indicating the appropriateness of these transcripts as prognostic biomarkers. The diagnostic power of lncRNAs SAMMSON, HOTAIR, MALAT1, H19, and LINR-ROR has been assessed in serum or tissue samples of pateints with glioblastoma revealing the best results for the first two mentioned lncRNAs based on the high values of the area under the reciver operating characteristic curves. Considering the unavialbility of tissue samples for the purpose of early diagnosis and ambiguity of imaging techniques in early stages of the disease, assessment of expression of lncRNAs in serum samples provides a non-invasive method for early detection of this kind of malignant tumor.
In brief, dysregulation of several lncRNAs has been deteceted in glioblastoma cells leading to abnormal regualtion of cancerassociated pathways and cellular processes namely apoptosis, proliferation and survival. These transcripts provide promising tools for early detection of glioblastoma and prediction of patients' prognosis and response to therapeutic choices particularly TMZ. However, a limitation of in vitro studies in this regard is that most of them has been executed using traditional serum-grown cell lines such as U87 or U251. Furhther functional in vitro and in vivo investigations are required to verify the obtained data.