Role of Non-Coding RNAs in TGF-β Signalling in Glioma

Brain tumours and Gliomas, in particular, are among the primary causes of cancer mortality worldwide. Glioma diagnosis and therapy have not significantly improved despite decades of efforts. Autocrine TGF-β signalling promotes glioma proliferation, invasion, epithelial-to-mesenchymal transition (EMT), and drug resistance. Non-coding RNAs such as miRNA, lncRNA, and circRNAs have emerged as critical transcriptional and post-transcriptional regulators of TGF-β pathway components in glioma. Here, we summarize the complex regulatory network among regulatory ncRNAs and TGF-β pathway during Glioma pathogenesis and discuss their role as potential therapeutic targets for Gliomas.


Introduction 1.Gliomas
Gliomas are a group of brain tumours clinically divided into four types from grade I to grade IV.Grade IV gliomas, known as Glioblastoma multiforme (GBM), are the most common form of adult brain cancer [1,2].The etiology of GBMs is complex and involves mutation or overexpression of multiple genes, and they have high intra-tumour heterogeneity [3,4].Based on the molecular characteristics of GBM, the World Health Organization (WHO) classifies it into three types: GBM isocitrate dehydrogenase (IDH) wild type, GBM IDH mutant, and GBM not otherwise specified (NOS) [5,6].Mutations in the IDH group of genes represent the most critical genetic alterations in GBM, which plays a vital role in therapeutic responses [7].GBMs likely originate from astrocytes; however, tracking the cell of origin in GBM is challenging due to their heterogeneity [8].Diffuse intrinsic pontine glioma (DIPG) is a form of pediatric malignancy that primarily grows in the pons with a dismal prognosis [9].DIPG shares resemblances with adult high-grade astrocytomas.However, this has been debated lately due to its distinct molecular alterations [9,10].Glioma stem cells (GSCs) are glioma-initiating cells that form a small subpopulation of GBM tumour cells and express stemness markers, such as CD133 [8].GSCs can differentiate into multiple tumour cell types, contributing to intratumour heterogeneity in GBM [4].GSCs contribute to tumour initiation, therapeutic resistance, and recurrence [8].
Current therapeutic strategies for GBM include maximum surgical resection and radioand chemotherapy with temozolomide (TMZ) [11,12].TMZ is an oral alkylating agent that alkylates DNA bases; it causes mismatch during DNA replication, leading to futile rounds of DNA repair, DNA double-strand breaks, and apoptosis [13].However, O6-methylguanine-DNA methyltransferase (MGMT) can resolve some TMZ-induced alterations and thus mediate the survival of GBM cells [14].MGMT inhibitors are considered beneficial for improving the action of TMZ in GBM patients [15].Localized application of pseudosubstrates or tumour-specific delivery of blocking peptides against MGMT increases TMZ efficiency [14].Overall, TMZ treatment extends the survival of GBM patients from 12.1 to 14.6 months [11], while tumour recurrence in GBM patients is inevitable.Resistance to radiation and chemotherapy in gliomas is also due to various other adaptive mechanisms, such as enhanced DNA repair capacity, cytoprotective autophagy, deregulated signalling pathways, intratumoral heterogeneity, phenotypic plasticity, and hypoxia [16].

Transforming Growth Factor-β (TGF-β)
TGF-β is a pleiotropic cytokine that regulates cell proliferation, differentiation, tissue homeostasis, motility, invasion, extracellular matrix production, angiogenesis, epithelial to mesenchyme transition (EMT), chemoresistance, and immune response in various cancers, including GBM [17,18].TGF-β also contributes to pathologies associated with virus and bacterial infections as an inflammatory cytokine [19].TGF-β superfamily consists of a large spectrum of ligands, including TGF-β1, TGF-β2, TGF-β3, activin, nodal, bone morphogenetic protein (BMP), growth and differentiation factors (GDF), and anti-mullerian hormone (AMH) [20].Seven types I and five types II transmembrane serine/threonineprotein kinase receptors exist for the TGF-β superfamily in the mammalian genome [21].TGF-β ligand-mediated signalling is initiated by the binding of TGF-β to the type II TGF-β receptor (TGF-βR II), which gets phosphorylated, alters its conformation, and phosphorylates the type I TGF-β receptor (TGF-βR1).The signal through TGFβR1 is transduced downstream either through SMAD proteins-in the canonical TGF-β pathway or through other effectors like MAPK, ERK, and JUN kinase-in the non-canonical TGF-β pathway [20].TGF-β target genes consist of evolutionarily conserved putative SMAD binding elements (SBEs) in their promoter regions.Nuclear translocation of the activated SMAD2/3 complex and binding of the translocated SMAD2/3 complex to the SBEs leads to the activation or repression of hundreds of TGF-β target genes [22].Non-canonical or non-SMAD pathways include various branches of MAP kinase pathways, Rho-like GTPase signalling pathways, and phosphatidylinositol-3-kinase/AKT pathways.For example, in the ERK-mediated signal transduction, the activated TGFβRI phosphorylates and activates the ShcA protein, forming the SHCA/GRB22/SOS complex, followed by the sequential activation of c-RAF, MEK, ERK [23].In normal cells and the early stages of cancer, TGF-β restrains cell proliferation whereas, in advanced stages of cancer, due to accumulation of mutations in the TGF-β pathway components or selective impairment of its tumour-suppressive function, it turns out to be oncogenic [24][25][26].TGF-β levels are elevated in glioma and are associated with increased histologic grade [27,28].TGF-β expression is also higher in the serum of GBM patients and correlates with poor survival [29].TGF-β promotes TMZ resistance by activating genes, such as connective tissue growth factor (CTGF), ZEB1, and SNAIL1 [30][31][32].TGF-β also promotes TMZ resistance in MGMT promoter hypomethylated GBM [33].Several anti-TGF-β antibodies, inhibitors, and antisense oligonucleotides (ASOs) against TGF-β pathway components have been evaluated in the pre-clinical and clinical trials for GBM, with limited success [18,[34][35][36].Systemic inhibition of TGF-β may not be ideal as it has both pro and anti-tumour activities and might also hamper the normal physiological functions of the TGF-β pathway.

Non-Coding RNAs (ncRNAs)
The human genome may be categorized into protein-coding genes (PCGs) and nonprotein-coding genes (NCGs).ncRNAs constitute >90% of the human genome.The non-coding category of the genome is highly heterogeneous, consisting of small non-coding RNAs < 200 bps in length and long ncRNAs (lncRNAs) > 200 bps in length.1.3.1. miRNAs miRNAs are small non-coding RNAs of 21-23 nucleotides in length that generally perform post-transcriptional gene silencing of their mRNA targets [37].They regulate gene expression by primarily interacting with the miRNA response elements (MREs) present on the 3 UTR of a transcript.However, interaction with 5 UTR, coding sequences, and gene promoters are also observed [38].They bind to the target gene and either degrade the transcript or, more often, limit the target gene's translation [39].The mRNA decay by miRNA occurs through the miRNA-induced silencing complex (miRISC) [40].Argonaute and GW182 are the core components of the RISC complex.GW182 interacts with PABP and recruits the PAN2-PAN3 and CCR4-NOT deadenylase complex, causing deadenylation, decapping, and mRNA decay [41].miRNAs also prevent protein synthesis by cap-dependent translational inhibition [42].It interferes with the assembly or activity of the translation initiation complex, eIF4F, via eIF4E-T and DDX6 [42].While these are the canonical miRISC-mediated silencing mechanisms, there are few non-canonical miRNAmediated gene silencing.For example, complete sequence complementarity between miRNA and target mRNA results in direct AGO2-mediated target cleavage [43].Also, the recruitment of Argonaute in the absence of GW182 inhibits translation without affecting mRNA stability [44].The differential association of Argonaute protein with other proteins mainly decides the outcome of mRNA decay or translation inhibition [40].Several miRNAs regulate multiple aspects of GBM pathogenesis and are potential diagnostic biomarkers and therapeutic targets for GBM [45].

LncRNAs
LncRNAs are transcripts longer than 200 bps with no ability to code for proteins [46][47][48][49][50][51][52][53].More than 100,000 lncRNAs in the human genome are listed in the NONCODE and other lncRNA databases [50,54].While most lncRNAs are generated from RNA polymerase II, which shares similarities with mRNAs, such as polyadenylation and 7-methylguanosine cap, some are generated from RNA pol I and RNA polymerase III [50].LncRNAs have a wide range of functions to modulate gene expression, chromatin architecture, transcription, RNA processing, splicing, editing, localization, stability, and protein translation [50].They modulate gene expression in cis and trans by interacting with DNA, RNAs, and proteins [55].To regulate gene expression, lncRNAs modulate (i) recruitment of a regulatory/transcription factor/epigenetic protein to a gene locus; (ii) inhibit the binding of a transcription factor to gene promoter by acting as a decoy; (iii) by acting as scaffold for protein complexes to either positively or negatively regulate gene expression; and (iv) large number of lncRNAs localized in the cytoplasm function as competing endogenous RNAs (ceRNAs) for miRNAs and stabilize the mRNA target of those miRNAs.LncRNAs regulate multiple aspects of GBM pathogenesis, such as proliferation, invasion, metastasis, and drug resistance [56].They have the potential to serve as potential diagnostic markers and therapeutic targets for GBM [57,58].

CircRNAs
Circular RNAs are covalently closed, single-stranded RNAs produced by a non-canonical back splicing of cellular non-coding RNAs and precursor messenger RNAs [54][55][56].CircRNAs are generated in the nucleus, but most are primarily present in the cytoplasm.Their synthesis is regulated by specific cis-acting elements and trans-acting factors [59][60][61][62][63].Many circRNAs act as non-coding RNAs and regulate gene expression by serving as decoys or competitors for microRNAs and proteins.In addition to sponging miRNAs and proteins, circRNAs also regulate the splicing of linear RNAs, regulate transcription, and form chromatin looping [60,64] A small percentage of circRNAs undergo cap-independent translation to encode functional peptides in response to specific cellular stresses [61].CircRNAs regulate proliferation, angiogenesis, cancer cell migration and invasion, and apoptosis in cancer [62].CircRNAs may act as diagnostic biomarkers and therapeutic targets in several cancer types, including GBM [62,63,65,66].
Several miRNAs, lncRNAs, and circRNAs have recently been identified, which modulate the TGF-β pathway to promote or repress GBM (Tables 1-3).Since TGF-β plays a role in tumour suppression at early stages of cancer development, its complete inactivation for cancer treatment is not ideal [24][25][26].NcRNAs regulated by the TGF-β pathway modulate numerous aspects of GBM pathogenesis.They may serve as attractive therapeutic targets downstream of TGF-β for countering the tumour-promoting effects of TGF-β.Here, we summarize the role of lncRNAs, miRNAs, and circRNAs in the TGF-β pathway in GBM pathogenesis (Figures 1-3).
Several miRNAs, lncRNAs, and circRNAs have recently been identified, which modulate the TGF-β pathway to promote or repress GBM (Tables 1-3).Since TGF-β plays a role in tumour suppression at early stages of cancer development, its complete inactivation for cancer treatment is not ideal [24][25][26].NcRNAs regulated by the TGF-β pathway modulate numerous aspects of GBM pathogenesis.They may serve as attractive therapeutic targets downstream of TGF-β for countering the tumour-promoting effects of TGF-β.Here, we summarize the role of lncRNAs, miRNAs, and circRNAs in the TGF-β pathway in GBM pathogenesis (Figures 1-3).miR-182 is over-expressed in GBM tissues and cells, whereas CYLD, a negative regulator of the NFκB pathway, is downregulated [78].The genomic location of miR-182, 7q32.1, is frequently amplified in gliomas [78].Interestingly, miR-182 is induced by TGF-β in U373MG and LN229 cells through the SMAD signalling pathway [78].miR-182 expression was also upregulated in Smad2/Smad4-overexpressing cells [78].ChIP assays confirmed the binding of SMAD2/3 to the promoter of miR-182 [78].These results suggest that TGF-β induced miR-182 expression in glioma cells through the SMAD signalling pathway.CYLD is a target of miR-182 [78].Up-regulation of miR-182 in U373MG and LN229 cells decreased the expression of CYLD.Also, miR-182 interacts with and degrades CYLD.Further, overexpression of miR-182 increased, while inhibition of miR-182 reduced the luciferase activity of NF-κB reporter and expression of NF-κB target genes.miR-182 overexpression significantly induced the phosphorylation of IKKβ, while miR-182 inhibitor reversed this effect.Importantly, in vitro kinase assay showed that endogenous IKK kinase activity was prolonged in miR-182 -transduced cells.These results indicate that miR-182 suppresses CYLD and enhances and sustains NF-κB activity in GBM [78].miR-182 up-regulation promotes anchorage-independent growth, colony formation, and invasion of GBM in vitro and in vivo [78].At the same time, miR-182 inhibition reversed these effects [78].Also, the tumour-promoting function of miR-182 overexpression was reversed with combined transfection of miR-182 mimics and IκBα dominant-negative mutant construct, indicating that miR-182 promotes GBM tumour through the NF-κB pathway [78].Further, miR-182 suppression inhibits NF-κB activity and malignant properties of patient-derived glioma cells (PDGCs) [78].The study also identified that TGF-β treatment in U373MG and LN229 cells significantly increased the NF-κB reporter activity, which was abolished upon miR-182 inhibition.This indicates that TGF-β-induced miR-182 is essential for sustained NF-κB activity in GBM [78].Overexpression of pSMAD2/3, miR-182, and several NF-κB target genes was observed in clinical GBM samples, which conferred poor survival of the patients [78].Also, the clinical samples showed a positive correlation between TGF-β, miR-182, and NF-κB target genes [78].Upon induction by TGF-β, miR-182 promotes GBM pathogenesis by activating and sustaining NF-κB activity by downregulating CYLD [78].

miR-15a
Guo et al., using a microarray screen, observed that miR-15a-5p is upregulated in glioma tissues [79].Bioinformatics analysis and luciferase reporter assay confirmed the direct interaction of miR-15a with SMAD7.Overexpression of miR-15a promoted migration and invasion of SHG139 cells [79].Whereas inhibition of miR-15a displayed the opposite effect, indicating the tumour-promoting ability of miR-15a [79].Anti-miRNA oligonucleotide (AMO)-mediated inhibition of miR-15a in SHG139 cells reduced migration and downregulated mesenchymal markers-Vimentin and N-cadherin.However, the combined knockdown of miR-15a and SMAD7 reversed these effects [79].Further, miR-15a inhibition attenuated GBM tumour growth in vivo.Hence, this study indicates the oncogenic function of miR-15a in GBM by inhibiting SMAD7 activity [79].

miR-193b
miR-193b levels are upregulated in glioma cell lines and GBM tumour samples [80].miR-193b depletion reduces the proliferation of U87 and U251 cells [80].Cell cycle analysis upon miR-193b knockdown revealed G0/G1 arrest in U87 and U251 cells [80].Bioinformatics analysis and luciferase reporter assays demonstrated that SMAD3 is the primary target of miR-193b [80].Inhibition of miR-193b significantly increased the protein levels of SMAD3 in U87 and U251 cells [80].Further, to know whether miR-193b regulates GBM proliferation through the TGF-β pathway, one of the primary targets of the TGF-β pathway, p21 levels were evaluated upon miR-193b inhibition.Inhibition of miR-193b and the subsequent up-regulation of SMAD3 in GBM cells displayed a significant accumulation of p21 [80].Also, the down-regulation of miR-193b decreased the proliferation of U87 and U251 cells [80].Hence, the study demonstrates that miR-193b is an oncogene that promotes cell growth by directly targeting SMAD3 and restricting the tumour-suppressive effects of SMAD3 through p21 down-regulation in GBM [80].

miR-148a
Quaking (QKI), an essential negative regulator of the TGF-β signalling, is downregulated, and miR-148a is upregulated in GBM tissues and cell lines [82].Low levels of QKI and overexpression of miR-148a confer poor prognosis in GBM patients [82].Microribonucleoprotein (miRNP) IP and luciferase reporter assay confirmed the interaction between miR-148a and QKI [82].Overexpression of miR-148a reduced the protein levels of QKI, while inhibition of miR-148a reduced this effect [82].Moreover, SKP1, a member of the E3 ubiquitin ligase complex that degrades SMAD3, is also an essential target of miR-148a [82].Consequently, miR-148a overexpression enhanced the SMAD luciferase reporter activity and phosphorylation of SMAD2/3 [82].GSEA and TCGA database analysis revealed that miR-148a positively correlates with the gene signatures of TGF-β signalling [82].Overexpression of miR-148a is associated with poor prognosis in GBM patients, indicating that miR-148a is an oncogenic miRNA [82].High levels of miR-148a are positively correlated with p-SMAD3 expression and the DNA binding activity of NF-κB [82].Also, the promoter of miR-148a contains multiple NF-κB binding domains.Consequently, NF-κB activation increased the expression of miR-148a in GBM cells [82].Further, in vitro experiments in LN18 and U-138MG cells and in vivo experiments demonstrated that miR-148a promotes the invasion, migration, and aggressiveness of GBM [82].These results illustrate that the hyperactive NF-κB signalling in GBM promotes the expression of miR-148a oncogene, which further promotes the aggressive phenotypes of GBM by downregulating the expression of QKI and SKP1 and activating the TGF-β signalling [82].Thus, miR-148a establishes an essential link between NF-κB and TGF-β signalling in promoting GBM pathogenesis [82].

miR-503
Analysis of the gene expression omnibus data of glioblastoma samples revealed that miR-503 is overexpressed in GBM tissue compared to normal tissue [86].Also, TGF-β treatment significantly increased the expression of miR-503 in T98G cells through SMAD signalling [86].miR-503 overexpression decreased the proliferation, migration, and colony formation ability and restricted apoptosis in GBM [86].Dual luciferase reporter assay indicated that PDCD4 is the primary target of miR-503 [86].Overexpression of miR-503 dramatically reduced the mRNA and protein levels of PDCD4 in GBM cells [86].Further, the combination of miR-503 inhibition with varying doses of TMZ showed a synergistic decrease in proliferation and increased apoptosis of A172 and U251 cells, indicated by enhanced cleaved PARP [86].These results suggest that miR-503 is an oncogenic miRNA induced by TGF-β in GBM [86].Upon induction by TGF-β, miR-503 enhances the proliferation, invasion, migration, and drug resistance in GBM by targeting PDCD4 [86].

Tumour Suppressor miRNAs Involved in the TGF-β Pathway in Gliomas 2.2.1. miR-127-3p
RNAseq analysis of GBM and normal brain tissues indicated that miR-127 is downregulated in GBM tissues compared to normal brain tissues [87].miR-127-3p gene is located in chromosome 14 between two lincRNAs (ENSG00000214548 and ENSG00000258399).
DNA methylation and histone acetylase inhibition resulted in the down-regulation of miR-127-3p in GBM tissues [87].Additionally, overexpression of miR-127-3p in LN229 and T98G cells reduced the proliferation and caused cell cycle arrest compared to control cells.However, the overexpression of miR-127-3p did not significantly affect the apoptosis of glioma cells.In vivo nude mice model with miR-127-3p overexpressing LN229 cells displayed reduced colony formation and tumour volume compared to the control group.Hence, miR-127-3p functions as a tumour suppressor in GBM [87].Bioinformatics analysis and luciferase reporter assays revealed that SKI, RGMA, ZWINT, SERPINB9, and SFRP1 are the primary targets of miR-127-3p.SKI is an essential negative regulator of the TGF-β pathway [87].It binds to SMAD proteins, blocking the ability of the SMAD complexes to activate TGF-β signalling in GBM.Overexpression of miR-127-3p or knockdown of SKI promotes TGFBR1 expression, phosphorylation of SMAD3, and induces cell cycle arrest of LN229 cells [87].Hence, miR-127-3p is an essential tumour suppressor miRNA that attenuates GBM proliferation by inhibiting the TGF-β signalling [87].

miR-564
miR-564 is downregulated in glioma cells and tumour tissues compared to normal human astrocytes and normal brain tissues, respectively [88].miR-564 mimics decreased the proliferation and invasion of U87 cells [88].Bioinformatics analysis and luciferase reporter assay indicated that TGF-β1 is the primary target of miR-564 [88].miR-564 overexpression markedly decreased the mRNA and protein levels of TGF-β1 [88].In addition, miR-564 also reduced the expression of SMAD4 protein and phosphorylated SMAD2 protein levels [88].Moreover, protein levels of EGFR and MMP9 were also significantly reduced upon miR-564 overexpression in U87 cells [88].EGFR and MMP9 are upregulated in GBM tissues compared to normal brain tissues [88].Also, a significant negative correlation was observed between miR-564, EGFR, and MMP9 [88].Cell proliferation and invasion assays indicated that the increase in proliferation and invasion by TGF-β treatment was attenuated by miR-564 overexpression [88].Further, the U87-engrafted in vivo GBM tumour model indicated a reduction in tumour growth upon miR-564 overexpression [88].Also, the mRNA and protein expression of TGF-β1 was lower in miR-564treated tumours than in scrambletreated tumours [88].Hence, miR-564 is a tumour suppressor miRNA, which restricts proliferation and invasion in GBM by targeting TGF-β1, and its downstream targets EGFR and MMP9 [88].

LncRNAs Involved in the TGF-β Pathway in GBM
3.1.Oncogenic lncRNAs Involved in the TGF-β Pathway in Gliomas 3.1.1.LncRNA-ATB LncRNA-ATB levels are higher in glioma tissues and U251, A172 cell lines than in normal brain tissues [89].Increased expression of lncRNA-ATB correlated with poor survival of GBM patients [89].Further, loss of function studies depicted a reduced proliferation, migration, and invasion of U251 and A172 cells [89].The study also indicated a negative correlation between the expression of lncRNA-ATB and miR-200a in GBM tissues.miR-200a is downregulated in GBM tissues and cell lines, and the knockdown of lncRNA-ATB significantly increased miR-200a expression in U251 and A172 cells [89].Luciferase reporter assay and Ago2 pulldown assays validated that lncRNA-ATB and TGF-β2 are direct targets of miR-200a.Also, miR-200a inhibition results in up-regulation of TGF-β2 [89].LncRNA-ATB knockdown mediated a reduction in cell proliferation, colony formation, and invasion of U251 and A172 cells, which is attenuated upon miR-200a inhibition.Additionally, the knockdown of lncRNA-ATB significantly reduced the levels of TGF-β2 expression, which was rescued by miR-200a inhibition.Glioma samples show a positive correlation between lncRNA-ATB and TGF-β2 and a negative correlation between miR-200a and TGF-β2.The reduction in mRNA and protein levels of TGF-β2 upon lncRNA-ATB depletion was further downregulated upon miR-200a overexpression.In contrast, TGF-β2 expression was rescued with the combination of lncRNA-ATB knockdown and miR-200a inhibition [89].Studies in nude mice models upon lncRNA-ATB depletion demonstrated a reduction in tumour volume, tumour weight, and reduced proliferation index indicated by Ki67 staining, supporting the oncogenic role of lncRNA-ATB in GBM.These results suggest that lncRNA-ATB could competitively bind miR-200a to stabilize TGF-β2 and promote TGF-β2-mediated GBM pathogenesis [89].
Another study by Tang et al. reported that lncRNA-ATB is upregulated by TGF-β1 treatment in LN18 and U251 cells [90].The up-regulation of lncRNA-ATB by TGF-β treatment was abrogated upon treatment with TGF-β inhibitor, SB505124, indicating the SMAD2/3 mediated regulation of lncRNA-ATB expression [90].LncRNA-ATB overexpression combined with TGF-β1 treatment increases the invasion of U251 and LN18 cells.Also, lncRNA-ATB overexpression and TGF-β1 treatment promote the phosphorylation of p65, the nuclear translocation of p65, and the phosphorylation of p38.These results indicate the activation of the NF-κB and p38/MAPK pathways by TGF-β regulated lncRNA-ATB [90].The increase in invasion in LN18 and U251 cells upon lncRNA-ATB overexpression and TGF-β treatment was significantly abolished upon treatment with NF-κB and p38/MAPK pathway inhibitors.These results suggest that the SMAD2/3 transcription factor induces lncRNA-ATB expression in GBM, and it promotes TGF-β-mediated GBM invasion through the NF-κB and p38/MAPK pathways.

LINC00115
RNA-sequencing of TGF-β treated GSCs revealed up-regulation of LINC00115 [93].Moreover, LINC00115 expression is higher in GBM tumour samples than in normal tissues and correlates with poor patient prognosis [93].LINC00115 knockdown inhibits GSC proliferation and neurosphere formation in vitro and also inhibits tumour formation in the xenograft model [93].LINC00115 physically associates with miR-200b and miR-200c [93].Also, LINC00115 depletion reduced the expression of ZEB1 and ZNF596 and reduced invasion in GSCs [93].Reporter assays indicate that ZEB1 and ZNF596 are targets of miR-200b and miR-200c.Down-regulation of ZEB1 and GBM invasion upon LINC00115 knockdown was reversed upon miR-200b overexpression, indicating that LINC00115 competitively binds to miR-200b to promote ZEB1-mediated GBM invasion [93].LINC00115 and its target ZNG596 are co-expressed in clinical glioma samples.Concomitantly, exogenous expression of ZNF596 in LINC00115-depleted GSCs reversed the inhibition of cell proliferation caused by LINC00115 depletion, indicating that ZNF596 is the downstream effector of LINC00115-driven GBM tumourigenicity [93].Also, LINC00115 binds to miR-200 to promote the expression of ZNF596 [93].CRISPR-mediated knockout of ZNF596 indicated that EZH2 is a direct target of ZNF596.ZNF296 is a transcription factor promoting the expression of EZH2 [93].LINC00115 depletion results in loss of EZH2 expression, which is reversed upon ZNF596 overexpression [93].LINC00115 further activates STAT3 downstream of EZH2 through ZNF596, indicating that LINC00115 activates EZH2/STAT3 signalling through ZNF596, thereby promoting GSC self-renewal and tumourigenicity [93].LINC00115 aids GSC's self-renewal by acting as a ceRNA for transcription factors ZEB1 and ZNF596 by sponging miR-200 [93].It also promotes GSC's tumourigenicity through the ZNF596/EZH2/STAT3 signal axis [93].

LncRNA RPSAP52
Wang et al. identified that lncRNA RPSAP52 is overexpressed in GBM tumour samples [97].High expression of RPSAP52 is associated with poor survival in GBM patients [97].Wang et al. also observed a positive correlation between RPSAP52 and TGF-β1expression in GBM samples [97].Further, overexpression of RPSAP52 increased TGF-β1 protein expression, and knockdown of RPSAP52 exhibited the opposite effect [97].Overexpression of RPSAP52 and TGF-β1 individually increased the percentage of CD133+ cells.Further, the overexpression of TGF-β1 rescued the reduction in the percentage of CD133+ cells observed upon RPSAP52 silencing [97].

LncRNA Plasmacytoma Variant Translocation-1 (PVT1)
Li et al. reported overexpression of lncRNA PVT1 and down-regulation of p53 in higher grades of glioma compared to normal brain cells [94].Also, the expression of PVT1 increased with increasing grades of glioma [94].Kaplan-Meier survival analysis revealed that high PVT1 levels are associated with poor survival in GBM patients.Clinical GBM samples show high expression of PVT1, TGF-β, and pSMAD2/3 levels and low p53 levels [94].Further, the knockdown of p53 decreased PVT1 levels in U373 cells, while overexpression of p53 showed a reverse effect [94].RIP assay revealed the direct interaction between PVT1 and p53 [94].Bioinformatics analysis using lncATLAS provided evidence of the interaction of p53 with the PVT1 promoter.Also, the dual luciferase reporter assay indicated that p53 binds to the promoter of PVT1 to attenuate the expression of PVT1 [94].Loss-of-function studies demonstrated that the knockdown of PVT1 reduced proliferation, viability, migration, and invasion, induced cell cycle arrest at S and G2/M phases, and promoted apoptosis in GBM [94].However, the knockdown of p53 showed the opposite effects [94].Expression of mesenchymal markers, N-cadherin, MMP-9, and MMP-2 was reduced, and E-cadherin was upregulated upon lncRNA PVT1 depletion.At the same time, the knockdown of p53 had a reverse effect.Furthermore, the study demonstrated that lncRNA PVT1 overexpression increased the transcription activity of the TGF-β, as depicted in the dual luciferase reporter assay, and increased the pSMAD2/3 levels [94].Knockdown of lncRNA PVT1 decreased the transcription activity of TGF-β, while p53 knockdown displayed the opposite effects [94].However, the combined knockdown of PVT1 and p53 counteracted the suppressive effects of p53 on TGF-β activity, indicating that p53 attenuated the TGF-β/SMAD pathway in GBM by targeting PVT1 [94].In vivo nude mice model demonstrated that the knockdown of PVT1 and p53 individually suppressed the tumour growth [94].In contrast, the combined knockdown of PVT1 and p53 counteracted the tumour-suppressive effects of p53 in GBM [94].The levels of TGF-β and pSMAD2/3 were determined from in vivo nude tumour tissues transfected with shRNA against lncRNA PVT1 or p53 [94].P53 knockdown increased PVT1 levels, TGF-β, and pSMAD2/3 levels [94].At the same time, PVT1 knockdown displayed the opposite effects [94].Also, the action of lncRNA PVT1 depletion on TGF-β activity was counteracted by p53 knockdown [94].This study demonstrates that p53 potentially contributes to downregulating the oncogenic lncRNA PVT1, thereby suppressing the activation of TGF-β and TGF-β mediated GBM progression by modulating the lncRNA PVT1-TGF-β axis [94].
3.1.9.LncRNA-MUF Using a genome-wide microarray screen, we identified that lncRNA-MUF is induced upon TGF-β treatment in T98G cells [95].Also, lncRNA-MUF induction upon TGF-β treatment is observed across other GBM cell lines-LN229, U87-MG, and LN18.Moreover, levels of lncRNA-MUF are elevated in GBM tumour samples, and its expression is associated with poor survival and prognosis [95].LncRNA-MUF induction by TGF-β is completely abolished upon treatment with TGFBR1 inhibitor SB505124 in glioma cells.In addition, the ChIP qPCR assay demonstrates the enrichment of SMAD2/3 antibody in the promoter of lncRNA-MUF upon TGF-β stimulation [95].Loss-of-function assays using siRNA against lncRNA-MUF revealed that lncRNA-MUF promotes proliferation, migration, and invasion in GBM [95].In addition, we show that loss of lncRNA-MUF sensitizes glioma cells to TMZ-induced cell death [95].Knockdown of lncRNA-MUF downregulated its cis oncogene, CAPRIN2, and various trans genes from the TGF-β ontology group (VIMENTIN, CTGF, c-MYC, and SNAIL1) [95].Western blotting analysis of mesenchymal markers revealed the down-regulation of N-cadherin, VIMENTIN, and SNAIL1 upon lncRNA-MUF knockdown in T98G and U87-MG cells [95].Bioinformatics analysis and dual luciferase reporter assay demonstrated the direct interaction between lncRNA-MUF and miR-34a, and overexpression of miR-34a reduces lncRNA-MUF expression.miR-34a has a potential tumour-suppressor role in glioma by targeting several oncogenes, particularly SNAIL1, and it is downregulated in GBM tissues compared to normal tissues [95].SNAIL1 is a crucial transcription factor that promotes tumour cell invasion and EMT and is upregulated in GBM.We observed a positive correlation between MUF and SNAIL1 expression in GBM tumour samples [95].Down-regulation of Snail and reduction in invasion upon lncRNA-MUF knockdown was rescued upon miR-34a inhibition in T98G and U87-MG cells.These experiments indicate that TGF-β-induced lncRNA-MUF sponges miR-34a to promote SNAIL1-induced invasion in GBM [95].Our study suggests that TGF-β induced lncRNA-MUF promotes GBM invasion through the miR-34a/Snail axis [95].

LINC01711
We have shown that TGF-β induces LINC01711 expression in glioma cells and that the levels of LINC01711 are elevated in GBM tumour samples, and its expression is associated with poor patients' survival and prognosis [96].Like lncRNA MUF, LINC01711 is also induced by the SMAD2/3 transcription factors downstream of TGF-β signalling.Down-regulation of LINC01711 reduces proliferation, migration, and invasion and induces apoptosis in GBM [96].LINC01711 knockdown results in downregulating ZEB1, a crucial transcription factor that promotes tumour cell invasion and EMT [96].LINC01711 also interacts with miR-34a, and ZEB1 is a target of miR-34a [96].Reduction of ZEB1 expression due to LINC01711 knockdown was rescued upon miR-34a inhibition [96].Further, the invasion assay revealed that miR-34a inhibition could reverse the reduction in invasion caused by LINC01711 knockdown in T98G and U87-MG cells [96].We also observed that ZEB1 overexpression could partially reverse the LINC01711 knockdown-mediated reduction in invasion in GBM [96].Further, we tested if LINC01711 could promote TMZ resistance in GBM.LINC01711 depletion significantly reduced proliferation and increased caspase 3/7 activity in T98G and LN229 cells, suggesting that LINC01711 promotes resistance to TMZ in GBM.Given the role of ZEB1 in TMZ resistance and that LINC01711 depletion results in ZEB1 inhibition, we evaluated if LINC01711 knockdown-mediated sensitization of GBM cells to TMZ-induced apoptosis is associated with a reduction in ZEB1 levels.ZEB1 protein levels were significantly downregulated during TMZ treatment in LINC01711-depleted cells compared to cells treated with TMZ alone [96].We found that in addition to TMZmediated apoptosis, LINC01711 knockdown could induce cisplatin-mediated apoptosis in GBM.Hence, upon induction by TGF-β, LINC01711 promotes GBM proliferation, migration, invasion, and drug resistance by modulating the LINC01711/miR-34a/ZEBl signalling axis [96].
3.2.Tumour Suppressor lncRNAs Involved in the TGF-β Pathway in Gliomas 3.2.1.LncRNA TCONS_00020456 Tang et al., using a microarray screen, identified 1759 upregulated and 1932 downregulated lncRNAs in U251 cells [99].Among these differentially expressed lncRNAs, they characterized the most downregulated lncRNA-TCONS_00020456 role in GBM pathogenesis [99].The expression of TCONS_00020456 decreased with increasing glioma grades, and the low expression of TCONS_00020456 indicated poor survival of GBM patients [99].Further, the siRNA-mediated knockdown of TCONS_00020456 significantly promoted the invasion and migration of U251 and U87 cells [99].While overexpression of TCONS_00020456 significantly inhibited invasion and migration in GBM [99].Bioinformatic analysis suggests that various mRNAs with oncogenic function negatively correlated with TCONS_00020456 expression [99].Among them, SMAD2 and PKCα were the top hits [99].Further, western blotting analysis revealed that the knockdown of TCONS_00020456 increased the expression of SMAD2, PKCα, N-cadherin, vimentin, and down-regulation of E-cadherin.Also, the phosphorylation of JNK and ERK was elevated upon TCONS_0002045 knockdown [99].The overexpression of TCONS_0002045 reversed these effects [99].These results indicate that TCONS_0002045 abrogates GBM invasion and migration by targeting SMAD2 and PKCα pathways [99].In vivo, analysis of TCONS_0002045 in nude mice model revealed a decrease in tumour size and weight in the TCONS_0002045 overexpression group compared to the TCONS_002045 knockdown group [99].In addition, the immunohistochemical staining of tumour tissues from the nude mice indicated increased expression of SMAD2 and PKCα in the TCONS_002045 knockdown group compared to the TCONS_0002045 overexpression group [99].Computational analysis using the miRDB database revealed several miRNAs targeting TCONS_0002045, SMAD2, and PKCα [99].Among these miRNAs, miR-1279 was identified as the common miRNA target between the three.LncRNA TCONS_0002045 abrogates GBM migration and invasion by targeting SMAD2 and PKCα [99].However, the exact mechanism of down-regulation of SMAD2 and PKCα by TCONS_0002045 and the role of miR-1279 needs further investigation.

Discussion
The TGF-β signalling pathway is an attractive therapeutic target for GBM.However, the development of therapeutics targeting the TGF-β pathway has been hindered mainly by its critical regulatory roles in normal physiology and due to its ability to function as both tumour promoter and inhibitor in a context-dependent manner [104].Hence, there is a need to specifically target tumour-promoting functions of TGF-β.Aberrant TGF-β signalling in GBM alters regulatory ncRNA expression and vice versa to promote GBM pathogenesis.NcRNAs can modulate the TGF-β pathway in GBM in the following ways (i) they act as downstream effectors of the TGF-β pathway, (ii) they can regulate components of the TGF-β pathway, and (iii) they can form a positive or negative feedback loop with the TGF-β pathway.
Many ncRNAs are in clinical trials for potential biomarkers and therapeutic targets for cancers and other diseases.LncRNA MFI2-AS1 is in clinical trials for use as a diagnostic biomarker for kidney cancer [105], and lncRNAs UCA1 and WRAP53 are in clinical trials for use as diagnostic biomarkers for hepatocellular carcinoma [106].Results from safety trials of RNA-targeted therapies using ASO against lncRNAs are also promising [107].Andes-1537, a short single-stranded phosphorothioate-deoxyoligonucleotide against antisense non-coding mitochondrial RNA (ASncmtRNA), was evaluated in phase I clinical trial by subcutaneous administration in patients with solid tumours.The results of this study displayed low toxicity of the oligonucleotide, with significant anti-tumour activity in pancreatic and cholangiocarcinoma patients [107].A phase I trial for a liposomal mimic of miR-34a (MRX34) was carried out in patients with renal-cell carcinoma, hepatocellular carcinoma, melanoma, lung cancer, and gastrointestinal stromal tumours [108,109].The MRX34 treatment in patients pretreated with dexamethasone displayed significant dosedependent modulation of miR-34a target genes and manageable toxicity [109].However, studies on ncRNAs as biomarkers and therapeutic agents in GBM are still in the pre-clinical testing stage.The major challenge associated with delivering RNA-targeted therapies to the brain is to cross the blood-brain barrier.ASO-mediated therapies against ncRNAs are best delivered when conjugated with nanoparticulate formulations [110].ASO-loaded glucosylated-polyion complex micelles have shown promise to effectively deliver ASOs across the blood-brain barrier through the intravenous route [111].
Furthermore, the non-canonical TGF-β downstream targets, such as NF-κB and PI3K/AKT/mTOR pathways, promote GBM pathogenesis.Specific lncRNAs and miR-NAs establish a link between TGF-β and these non-canonical signalling pathways.For example, lncRNA-ATB, induced by TGF-β, promotes GBM invasion through the NF-κB and P38/MAPK pathways [90].Also, TGF-β-induced miR-182 suppresses CYLD and promotes sustained activation of NF-κB in GBM [78].Similarly, the hyperactive NF-κB signalling in GBM promotes the expression of miR-148a oncogene.miR-148a, upon induction, promotes the GBM pathogenesis by activating the TGF-β signalling by promoting the expression of pSMAD3 and downregulating the negative regulators (QKI and SKI) of the TGF-β signalling [82].It needs to be tested if targeting such ncRNAs, which modulate crosstalk between multiple oncogenic pathways, can achieve better therapeutic efficacy for GBMs.
Most studies on the TGF-β modulating lncRNAs and circRNAs in GBM have focused on their ability to function as ceRNAs to sponge miRNAs.However, lncRNAs and circRNAs also function by interacting with proteins [49,59].Further studies are needed to identify RNA binding proteins interacting with these lncRNAs and circRNAs to promote GBM pathogenesis and fully understand their potential as therapeutic targets.Dysregulation of several ncRNAs, such as: miR-129-2, lncRNAs AF086127, AF086217, AF086391, AF119852, AK021535, AK022370, AL050068, BC012548, and BC041658 occurs in DIPG [112].However, studies are required to understand the relationship between ncRNAs and the TGF-β pathway in DIPG.
In summary, tumour-promoting ncRNAs involved in the TGF-β pathway have the potential to serve as attractive biomarkers and therapeutic targets for GBM.

Figure 1 .
Figure 1.Role miRNAs and their targets in regulating the TGF-β pathway in GBM.TGF-β (yellow circled) promotes the expression of oncogenic miRNAs (red boxed), which can control post-transcriptional gene expression of its targets (brown circled) to promote TGF-β-mediated GBM pathogenesis.Few tumour suppressor miRNAs (green boxed) target the TGF-β pathway's components and downregulate the TGF-β signalling, thereby attenuating GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.

Figure 1 .
Figure 1.Role miRNAs and their targets in regulating the TGF-β pathway in GBM.TGF-β (yellow circled) promotes the expression of oncogenic miRNAs (red boxed), which can control posttranscriptional gene expression of its targets (brown circled) to promote TGF-β-mediated GBM pathogenesis.Few tumour suppressor miRNAs (green boxed) target the TGF-β pathway's components and downregulate the TGF-β signalling, thereby attenuating GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.

Figure 2 .
Figure 2. Role of LncRNAs involved in TGF-β pathway in regulating GBM pathogenesis.TGF-β (yellow circled) promotes the expression of oncogenic lncRNAs (red boxed), which can control transcriptional/post-transcriptional gene expression of its targets (brown circled) by interacting with miRNAs or proteins to promote TGF-β-mediated GBM pathogenesis.Few oncogenic lncRNAs are not induced by TGF-β but promote the TGF-β signalling-mediated GBM pathogenesis.Tumour suppressor lncRNAs (green boxed) target the components of the TGF-β pathway and downregulate the TGF-β signalling, thereby attenuating GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.

Figure 3 .
Figure 3. Role of circular RNAs in regulating the TGF-β pathway in GBM.TGF-β (yellow circled) promotes the expression of oncogenic circRNA (red boxed), which controls the gene expression of its targets (brown circled) to promote TGF-β-mediated GBM pathogenesis.Tumour suppressor circRNA (green boxed) targets the TGF-β pathway's components, downregulates the TGF-β signalling, and attenuates GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.

Figure 2 . 26 Figure 2 .
Figure 2. Role of LncRNAs involved in TGF-β pathway in regulating GBM pathogenesis.TGF-β (yellow circled) promotes the expression of oncogenic lncRNAs (red boxed), which can control transcriptional/post-transcriptional gene expression of its targets (brown circled) by interacting with miRNAs or proteins to promote TGF-β-mediated GBM pathogenesis.Few oncogenic lncRNAs are not induced by TGF-β but promote the TGF-β signalling-mediated GBM pathogenesis.Tumour suppressor lncRNAs (green boxed) target the components of the TGF-β pathway and downregulate the TGF-β signalling, thereby attenuating GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.

Figure 3 .
Figure 3. Role of circular RNAs in regulating the TGF-β pathway in GBM.TGF-β (yellow circled) promotes the expression of oncogenic circRNA (red boxed), which controls the gene expression of its targets (brown circled) to promote TGF-β-mediated GBM pathogenesis.Tumour suppressor circRNA (green boxed) targets the TGF-β pathway's components, downregulates the TGF-β signalling, and attenuates GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.

Figure 3 .
Figure 3. Role of circular RNAs in regulating the TGF-β pathway in GBM.TGF-β (yellow circled) promotes the expression of oncogenic circRNA (red boxed), which controls the gene expression of its targets (brown circled) to promote TGF-β-mediated GBM pathogenesis.Tumour suppressor circRNA (green boxed) targets the TGF-β pathway's components, downregulates the TGF-β signalling, and attenuates GBM progression.The red arrows indicate inhibitory function, and the green arrows indicate a stimulatory role.
LncRNA-UCA1 is induced by TGF-β.It Acts as a molecular sponge for miR-1 and miR-203a to promote Slug expression and Slug-mediated GBM cell stemness

Type of Regulation Function Mechanism of Action in GBM Type of Model Cell Lines Biomarkers/ Therapeutic Target Reference Oncogenic Circular RNA
2. miRNAsInvolved in the TGF-β Pathway in GBM 2.1.Oncogenic miRNAs Involved in the TGF-β Pathway in Gliomas 2.1.1.miR-182