The Role of Noncoding RNA in MGMT‐Positive Glioblastoma: A Narrative Review

ABSTRACT

Glioblastoma (GBM) is the most aggressive primary malignant tumor of the central nervous system and is characterized by rapid progression, frequent recurrence, and poor survival outcomes. The current standard of care, comprising maximal safe surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ), has improved clinical management; however, the therapeutic benefit remains limited by both intrinsic and acquired resistance to TMZ. O6‐methylguanine‐DNA methyltransferase (MGMT), a DNA repair protein that removes TMZ‐induced O6‐methylguanine adducts, is a major determinant of TMZ responsiveness. In particular, methylation of the MGMT promoter is associated with reduced MGMT expression and improved sensitivity to TMZ, although this molecular marker alone does not fully explain interpatient variability in treatment response, which is further modulated by the immunosuppressive tumor microenvironment. Increasing evidence indicates that non‐coding RNAs (ncRNAs), including microRNAs (miRNAs), long non‐coding RNAs (lncRNAs), and circular RNAs (circRNAs), participate in the regulatory network controlling MGMT expression. These ncRNAs may regulate MGMT directly at the posttranscriptional level or indirectly through competing endogenous RNA networks, epigenetic mechanisms, and signaling pathways associated with DNA repair and chemoresistance. Consequently, dysregulated ncRNA expression may contribute to TMZ resistance and GBM progression. Moreover, ncRNAs have potential clinical value as biomarkers for molecular stratification and prognostic assessment, as well as therapeutic targets for restoring TMZ sensitivity. This narrative review summarizes current evidence regarding ncRNA‐mediated regulation of MGMT in GBM and discusses the potential implications of targeting these regulatory mechanisms to overcome TMZ resistance.

Abbreviations

3′UTR

3′ untranslated region

AKT

protein kinase B

ATM

ataxia telangiectasia mutated

BCNU

carmustine

ceRNA

competing endogenous RNA

circRNA

circular RNA

CNS

central nervous system

COAD

colon adenocarcinoma

CXCL14

C‐X‐C motif chemokine ligand 14

DSB

double‐strand breaks

EZH2

enhancer of zeste homolog 2

FOXO3

forkhead box protein O3

GBM

glioblastoma

GSC

glioma stem cells

HLA‐G

human leukocyte antigen G

HOTAIR

HOX transcript antisense RNA

IL‐6

interleukin‐6

lncRNA

long non‐coding RNA

MDSCs

myeloid‐derived suppressor cells

MGMT

O⁶‐methylguanine‐DNA methyltransferase

miRNA

microRNA

MMP‐2/‐9

matrix metalloproteinase‐2/9

ncRNA

non‐coding RNA

NK

natural killer

NSCLC

non‐small cell lung cancer

O⁶‐meG

O⁶‐methylguanine

PRADX

PRC2‐ and DDX5‐associated lncRNA

PRC2

polycomb repressive complex 2

RBP

RNA‐binding protein

RNase R

ribonuclease R

ROS

reactive oxygen species

SANP

self‐assembled nanoparticle

Sp1

specificity protein 1

TME

tumor microenvironment

TMZ

temozolomide

UCA1

urothelial carcinoma‐associated 1

XIST

X‐inactive specific transcript

1. Introduction

Glioblastoma (GBM) is the most prevalent and aggressive primary malignant brain tumor in adult patients [1]. According to epidemiological data, GBM accounts for 51.5% of all primary malignant brain and other central nervous system tumors, with an average annual age‐adjusted incidence rate of 3.27 per 100,000 population [2]. Despite extensive research and multimodal therapy over the past few decades, GBM remains one of the tumors with the poorest prognosis, exhibiting a median survival of only 9 months and a 5‐year relative survival rate of 7.1% [2]. Temozolomide (TMZ) serves as the primary chemotherapeutic agent for the treatment of GBM. The mechanism of action involves inducing DNA alkylation. This process primarily occurs at the N⁷ position of guanine, the N³position of adenine or guanine, and the O⁶ position of guanine. In the DNA damage induced by TMZ, N⁷‐methylguanine accounts for approximately 80%–85%, and N⁶‐methyladenine or N⁶‐methylguanine accounts for approximately 8%–20%, while O⁶‐methylguanine (O⁶‐meG) constitutes approximately 8% [3, 4]. The primary toxic mechanism of TMZ treatment stems from O⁶‐meG‐induced DNA mismatch. This process consequently induces inhibition of DNA synthesis and generates double‐strand breaks (DSB) [3]. Severe DNA damage can further activate the ataxia telangiectasia mutated serine/threonine kinase/checkpoint kinase 2 signaling pathway, inducing p53‐associated G2/M cell cycle arrest [4] and ultimately leading to tumor cell apoptosis and necrosis [5].

O⁶‐methylguanine‐DNA methyltransferase (MGMT) is an enzyme that repairs DNA alkylation damage. This repair process involves the transfer of an alkyl group—most commonly a methyl group, but also larger alkyl groups such as ethyl, benzyl, or chloroethyl groups—from the O⁶ position of guanine to its own cysteine residue, thereby repairing the damaged guanine nucleotide in a suicide reaction that inactivates the enzyme [6]. Studies have shown that high MGMT mRNA and protein expression is closely associated with resistance to TMZ therapy in cancer [7]. The primary mechanism regulating the MGMT gene lies in the methylation status of its promoter region [8], which can suppress the transcription of the MGMT gene [9], thereby reducing the synthesis of MGMT protein. Clinical trials have clearly demonstrated that patients with MGMT gene promoter methylation exhibit significantly prolonged overall survival following treatment with TMZ combined with radiotherapy [10]. However, some studies have revealed that even among patients with GBM without MGMT promoter methylation, there are still individuals who exhibit poor response to TMZ treatment [11, 12]. Therefore, investigating the regulation of MGMT expression is of great significance in addressing drug resistance in patients with GBM. For the purposes of this review, the term “MGMT‐positive glioblastoma” refers to tumors with detectable MGMT protein expression—typically associated with an unmethylated MGMT promoter—that retain functional DNA repair capacity and consequently exhibit reduced sensitivity to TMZ.

Although MGMT promoter methylation is an established predictive biomarker of response to TMZ, it represents only one component of the complex biological determinants of treatment response in GBM. The immunosuppressive tumor microenvironment (TME) also plays a critical role in therapeutic resistance and disease progression [13]. GBM cells can express human leukocyte antigen G (HLA‐G), a nonclassical major histocompatibility complex Class I molecule that engages inhibitory receptors such as ILT2/ILT4 and KIR2DL4 on natural killer (NK) cells, thereby suppressing NK‐cell activation and cytotoxic activity. In parallel, myeloid‐derived suppressor cells (MDSCs) accumulate in the GBM TME and impair NK‐cell function through multiple mechanisms, including arginase‐1‐mediated L‐arginine depletion and the production of reactive oxygen species (ROS), thereby favoring immune evasion. These TME‐driven mechanisms may influence not only the efficacy of immunotherapeutic approaches but also the response to conventional chemoradiotherapy, and may contribute to inter‐patient heterogeneity that is not captured by MGMT status alone. Therefore, integrating established molecular biomarkers such as MGMT promoter methylation with TME features—including HLA‐G‐mediated immune suppression and MDSC‐associated NK‐cell dysfunction—could provide a more comprehensive understanding of therapeutic resistance and help identify patients who may benefit from rational combination or immune‐modulating strategies. Notably, ncRNAs are key mediators of tumor–immune crosstalk and can regulate both immune evasion and MGMT expression; thus, the interplay between TME‐driven immune suppression and ncRNA‐mediated MGMT regulation deserves further investigation.

Non‐coding RNAs (ncRNAs) comprise a diverse group of functional RNA transcripts that lack protein‐coding capacity, among which microRNAs (miRNAs), long non‐coding RNAs (lncRNAs), and circular RNAs (circRNAs) are the most extensively characterized categories [14, 15]. Accumulating evidence indicates that ncRNAs are important regulators of cellular homeostasis and malignant transformation. In particular, miRNAs and lncRNAs, which represent major components of the non‐coding transcriptome, have been implicated in both transcriptional and posttranscriptional control of gene expression [16]. Through these regulatory mechanisms, they influence multiple stages of tumorigenesis, including tumor initiation, disease progression, and the development of therapeutic resistance [17, 18, 19, 20]. In glioblastoma (GBM), increasing attention has therefore been directed toward the regulatory interactions between miRNAs, lncRNAs, and MGMT expression during tumor progression [21, 22, 23, 24]. In addition, exosomes constitute important intercellular communication vehicles that transport ncRNAs between cells, facilitating the dissemination of regulatory signals within the tumor microenvironment and potentially promoting chemoresistance in GBM [25]. Changes in miRNA abundance may, for example, promote the degradation of MGMT transcripts and consequently suppress MGMT expression [26, 27]. Conversely, lncRNAs can modulate miRNA‐mediated regulation through direct or indirect interactions with miRNAs, thereby altering their ability to regulate target mRNAs [28].

With the advancement of several cutting‐edge technologies, such as next‐generation high‐throughput sequencing, gene silencing, and gene editing, increasing evidence has demonstrated that not only miRNAs and lncRNAs but also circRNAs possess regulatory functions and are associated with various diseases [29, 30, 31, 32]. CircRNAs can interact with protein complexes, RNA molecules, and DNA molecules to exert their functions and regulate various physiological and pathological processes [33, 34, 35].

2. Literature Search Strategy

A comprehensive literature search was conducted using PubMed, Web of Science, and Embase databases for peer‐reviewed articles published in English up to January 2026. Search terms included combinations of keywords: (“Glioblastoma” OR “GBM” OR “glioma”) AND (“O6‐methylguanine‐DNA methyltransferase” OR “MGMT”) AND (“non‐coding RNA” OR “microRNA” OR “miRNA” OR “long non‐coding RNA” OR “lncRNA” OR “circular RNA” OR “circRNA”) AND (“temozolomide” OR “chemoresistance” OR “chemosensitivity”). Original research articles providing experimental evidence (in vitro, in vivo, or clinical data) demonstrating a direct or indirect regulatory link between a specific non‐coding RNA and MGMT expression in GBM were prioritized, while relevant review articles were also included for background context and reference cross‐checking. Conference abstracts, case reports, and non‐English publications were excluded.

3. miRNAs and MGMT

miRNAs are small non‐coding RNAs composed of 19–22 nucleotides that function by sequence‐specifically suppressing mRNA expression or promoting mRNA degradation [26, 36]. miRNAs play a pivotal role in regulating cellular homeostasis by modulating diverse cellular processes, including cell proliferation, migration, cell cycle progression, and apoptosis, thereby exerting a decisive influence on the maintenance of cellular functions [37, 38]. Accumulating evidence has linked aberrant microRNA (miRNA) expression to the pathogenesis of diverse human diseases, including malignancies, neurodegenerative disorders, and cardiovascular diseases. In GBM, dysregulated miRNA profiles have been associated with tumor initiation, progression, and maintenance, highlighting their potential value as predictive biomarkers [39]. Depending on their target genes and biological context, miRNAs may exert either oncogenic or tumor‐suppressive effects; thus, they can function as oncomiRs or tumor‐suppressive miRNAs in GBM [26]. Mechanistically, miRNAs exert their regulatory effects through posttranscriptional control: they sequence‐specifically bind to complementary sites within the 3′ untranslated region (3′UTR) of target mRNAs, thereby inducing either transcript degradation or translational repression.

3.1. miRNA in GBM

Accumulating evidence indicates that aberrant miRNA expression is a widespread feature of human malignancies and is closely associated with tumor initiation and progression. In GBM, extensive dysregulation of miRNA expression has been reported and may contribute to diverse oncogenic processes. Depending on the cellular context, target genes, and regulatory environment, individual miRNAs can exert either tumor‐promoting or tumor‐suppressive effects. However, the specific targets and regulatory networks of many GBM‐associated miRNAs remain incompletely understood. Further studies are therefore warranted to elucidate their potential diagnostic and prognostic significance and to evaluate their feasibility as therapeutic targets in GBM. Mechanistically, miRNAs may affect GBM progression through the regulation of angiogenesis, metabolic pathways and related enzymes, as well as the differentiation and biological properties of glioma stem cells [40]. Their extensive regulatory capacity is further supported by evidence suggesting that miRNAs may regulate approximately 3% of genes associated with glioma and nearly 30% of protein‐coding genes, while a single miRNA may potentially influence the expression of up to 100 GBM‐related mRNAs [40]. Through these broad regulatory interactions, miRNAs participate in multiple biological processes relevant to GBM pathogenesis, including tumor‐cell proliferation, migration, invasion, and therapeutic resistance. Nevertheless, much of the available evidence has been generated from preclinical studies, and additional clinical validation is required to establish the translational and clinical significance of these findings. Comprehensive characterization of miRNA expression profiles, targets, and regulatory networks may facilitate the identification of novel therapeutic opportunities and support the development of miRNA‐based strategies for GBM treatment.

3.2. Regulatory Mechanisms

The regulatory role of miRNAs in modulating MGMT expression occurs primarily through posttranscriptional regulation. By binding specifically to the 3′ untranslated region (3′UTR) of MGMT transcripts, miRNAs induce mRNA degradation or translational repression. Additionally, certain miRNAs operate indirectly by targeting key components of relevant signaling pathways, such as the Wnt/β‐catenin pathway or specific transcription factors, which subsequently diminishes the transcriptional activation of MGMT.

Although the prognostic and therapeutic relevance of MGMT promoter methylation in GBM was established [41], it later became clear that methylation alone does not fully govern MGMT protein abundance. Kreth et al., in 2013, reported two MGMT transcript variants, and both were detectable in GBM [42]. Normal brain specimens contained a 440‐base pair (bp) short form, featuring a canonical poly(A) signal and a 105‐nucleotide (nt) 3′UTR. By contrast, GBM specimens expressed a longer variant of roughly 850 bp, whose alternative poly(A) signal spanned 522 nt. When 63 clinical specimens were examined—53 glioblastoma and 10 anaplastic astrocytoma cases—transcript size was found to correlate with MGMT expression [42]. In particular, transcripts carrying 3′UTRs of standard length tended to accompany elevated MGMT levels, while those with elongated 3′UTRs were linked to reduced MGMT expression [42]. Computational inspection of putative miRNA‐binding sites further indicated that an extended 3′UTR increases the likelihood that an mRNA will be targeted by miRNAs. Building on these observations, the present discussion next examines, in greater depth, miRNAs that can modulate MGMT expression in GBM.

3.2.1. miRNA‐142‐3p

Lee and colleagues identified an inverse association in GBM cell lines between MGMT gene expression and miR‐142‐3p abundance [43]. Lines with elevated MGMT transcripts displayed reduced miR‐142‐3p levels, whereas diminished MGMT expression coincided with higher miR‐142‐3p amounts [43]. Upon forcing miR‐142‐3p expression, MGMT mRNA remained largely unchanged, yet MGMT protein declined. Such discordance implies direct targeting of the MGMT 3′UTR by miR‐142‐3p, an idea subsequently supported by luciferase reporter assays [43]. Moreover, miR‐142‐3p‐overexpressing cells became more responsive to alkylating drugs, including TMZ and carmustine (BCNU), with the BCNU effect appearing especially strong [43].

Interleukin 6, an oncogenic cytokine, has been reported to reduce miR‐142‐3p expression, which points to a possible tumor‐suppressive role for this miRNA [44]. Independent investigations have lent additional support to this function. Wei et al. [45] showed that exosomal miR‐142‐3p derived from M1‐polarized macrophages curtails GBM proliferation and induces apoptosis by targeting HMGB1 and influencing the PD‐1/PD‐L1 immune checkpoint, thereby offering in vitro evidence of its tumor‐suppressive activity in GBM. Collectively, miR‐142‐3p controls MGMT protein levels and increases alkylating agent sensitivity in vitro, indicating promise as a biomarker for tailored GBM treatment.

3.2.2. miRNA‐181d‐5p

Zhang et al. were among the first to elucidate the miRNA‐mediated regulatory mechanism of MGMT. By integrating genome‐wide miRNA profiling with subsequent functional and mechanistic investigations, they identified miR‐181d, currently designated miR‐181d‐5p, as a key posttranscriptional regulator of MGMT [46, 47]. Their studies demonstrated that miR‐181d directly interacts with the extended 3′UTR of MGMT transcripts, thereby contributing to the regulation of MGMT expression. In vitro, ectopic expression of miR‐181d led to a significant reduction in both MGMT mRNA and protein abundance and consequently increased the susceptibility of tumor cells to TMZ [46]. Consistent with these experimental observations, clinical analyses revealed that higher miR‐181d expression was associated with more favorable survival outcomes among patients with GBM [46]. Moreover, miR‐181d expression was increased following treatment with either TMZ or radiotherapy, with an even greater induction observed when the two modalities were administered in combination. Taken together, these findings indicate that miR‐181d may contribute to the therapeutic response of GBM to chemoradiotherapy and suggest its potential utility as a biomarker for treatment response.

Subsequent investigations have further examined the relationship between miR‐181d and MGMT and have generally supported the regulatory model proposed by Zhang et al. Notably, miR‐181d appears to function within a broader miRNA regulatory network, in which interactions with other miRNAs, including miR‐409‐3p [48], miR‐648 [49], and miR‐661 [47], may cooperatively enhance the suppression of MGMT expression. These observations suggest that the regulation of MGMT by miR‐181d is not an isolated event but may involve coordinated interactions among multiple miRNAs, highlighting the importance of miRNA‐mediated posttranscriptional control in determining MGMT abundance.

Overall, available evidence supports a tumor‐suppressive role for miR‐181d in GBM. By directly targeting MGMT transcripts, miR‐181d can reduce MGMT expression and thereby enhance the sensitivity of GBM cells to TMZ. Its association with treatment response and patient survival further indicates that miR‐181d may have potential value as both a regulatory molecule and a biomarker of therapeutic sensitivity in GBM.

3.2.3. miR‐182‐5p

Accumulating evidence indicates that miR‐182 is involved in the regulation of GBM progression and may exert tumor‐suppressive effects on several malignant phenotypes. By modulating the expression of multiple genes implicated in tumor biology, miR‐182 has been reported to restrain glioma‐cell proliferation and migratory capacity, thereby limiting tumor progression [50]. Feng et al. demonstrated that increased miR‐182 expression in GBM cells targets neuropilin and consequently suppresses both cellular proliferation and migration [50]. In addition to these effects, Cheng et al. [51] identified miR‐182‐5p as a posttranscriptional regulator of MGMT. Specifically, miR‐182‐5p binds to the 3′UTR of MGMT and reduces the stability and translational efficiency of its mRNA, resulting in decreased MGMT protein abundance. Importantly, enforced expression of miR‐182‐5p increased the cytotoxic response of glioma cells to TMZ, as evidenced by diminished cell viability and enhanced apoptotic cell death. This increased TMZ sensitivity was associated with the suppression of MGMT protein expression, supporting a role for miR‐182‐5p‐mediated MGMT regulation in determining the response of GBM cells to alkylating chemotherapy [51].

Collectively, these findings suggest that miR‐182 may exert pleiotropic antitumor effects in GBM through the regulation of genes involved in tumor‐cell behavior and therapeutic resistance. Its ability to modulate MGMT expression and enhance TMZ responsiveness further highlights its potential relevance to GBM treatment. Nevertheless, additional preclinical investigations and well‐designed clinical studies are required to establish the therapeutic feasibility of targeting miR‐182, determine effective and tumor‐specific delivery approaches, and evaluate its compatibility with existing treatment regimens. Further clarification of these aspects may facilitate the development of miR‐182‐based therapeutic strategies and potentially improve treatment responses and outcomes in patients with GBM.

3.2.4. miR‐198

Aberrant miR‐198 expression has been observed in GBM, with its abundance reportedly reduced by approximately 3‐ to 5‐fold in tumor tissues relative to normal brain specimens [52]. This marked downregulation suggests that miR‐198 may be involved in the molecular pathogenesis of GBM. Nie et al. further demonstrated that miR‐198 expression was significantly decreased in GBM tissues and that lower miR‐198 levels were associated with an unfavorable prognosis in patients with GBM [53]. Functional studies showed that restoration of miR‐198 expression increased the sensitivity of GBM cells to TMZ in both in vitro and in vivo models. Mechanistically, MGMT was identified as a direct target of miR‐198, and increased miR‐198 expression suppressed MGMT protein production, thereby enhancing the responsiveness of GBM cells to TMZ. Conversely, enforced expression of MGMT attenuated the miR‐198‐mediated increase in TMZ sensitivity, providing further evidence that the miR‐198/MGMT regulatory axis contributes to chemotherapeutic response. Consistent with these experimental findings, analysis of clinical GBM specimens revealed elevated MGMT protein expression and an inverse relationship between MGMT abundance and miR‐198 levels. Collectively, these observations support a tumor‐suppressive role for miR‐198 in GBM and indicate that its downregulation may contribute to TMZ resistance through impaired posttranscriptional repression of MGMT. Thus, miR‐198 may have potential utility as a biomarker for disease prognosis and therapeutic response, as well as a candidate target for strategies aimed at improving TMZ sensitivity in GBM [53].

3.2.5. miR‐221/222

The miR‐221/222 cluster has been extensively investigated across multiple malignancies and is frequently reported to be dysregulated, with increased expression observed in GBM, prostate cancer, papillary thyroid carcinoma, hepatocellular carcinoma, and pancreatic cancer [54]. In GBM, Gillies and Lorimer demonstrated that miR‐221/222 is aberrantly upregulated and contributes to malignant cellular behavior through the suppression of the cell‐cycle inhibitor p27 [55]. Subsequent studies have identified a broader range of downstream targets and signaling pathways affected by miR‐221/222, including the Akt pathway, phosphatase and tensin homolog, tissue inhibitor of metalloproteinases‐3, and the matrix metalloproteinases MMP‐2 and MMP‐9 [54, 56]. Consistent with these observations, in vitro overexpression of miR‐221/222 increased the abundance of p‐Akt, MMP‐2, and MMP‐9, thereby promoting GBM‐cell proliferation and invasive behavior. Corresponding in vivo experiments further supported these findings, with miR‐221/222 overexpression associated with increased tumor volume and morphological alterations indicative of malignant progression [54].

Beyond its effects on tumor‐associated signaling pathways, miR‐221/222 has also been implicated in the posttranscriptional regulation of MGMT. A putative miR‐221/222 recognition site was identified within the 3′UTR of MGMT and subsequently validated through in vitro experiments [57]. Ectopic expression of miR‐221/222 decreased MGMT expression in human GBM cell lines and, notably, increased the susceptibility of these cells to TMZ treatment [57]. These findings suggest that miR‐221/222 can influence TMZ responsiveness through direct regulation of MGMT, despite its broader association with oncogenic phenotypes in GBM.

In summary, miR‐221/222 exhibits predominantly oncogenic characteristics in GBM and has been reported to be associated with unfavorable survival outcomes. At the same time, its direct suppression of MGMT may increase the sensitivity of GBM cells to TMZ in vitro. This apparent context‐dependent effect highlights the complexity of miRNA‐mediated regulation in GBM and suggests that the biological and therapeutic consequences of miR‐221/222 may depend on the specific molecular pathways and cellular contexts involved.

3.2.6. miR‐214‐5p

miR‐214‐5p has been implicated as a tumor‐suppressive miRNA in several types of cancer, where it can restrict malignant phenotypes such as cellular proliferation, migration, and invasion [58, 59]. Its involvement in TMZ resistance in glioma was previously insufficiently characterized; however, studies using LN‐18/TR cells have provided evidence that miR‐214‐5p participates in the regulation of the β‐catenin/MGMT signaling axis. Treatment with miR‐214‐5p mimics reduced the expression of both β‐catenin and MGMT, whereas inhibition of miR‐214‐5p produced the opposite effect. Furthermore, suppression of miR‐214‐5p prevented the C3G‐mediated decrease in β‐catenin and MGMT expression. These observations suggest that C3G may facilitate the miR‐214‐5p‐dependent repression of β‐catenin, subsequently contributing to the downregulation of MGMT and the attenuation of TMZ resistance.

The functional relationship between C3G, miR‐214‐5p, and TMZ‐induced apoptosis was further investigated in LN‐18/TR cells. Both C3G treatment and miR‐214‐5p overexpression promoted apoptosis in response to TMZ, whereas inhibition of miR‐214‐5p substantially diminished the proapoptotic effect induced by C3G. These findings indicate that the ability of C3G to enhance TMZ‐induced apoptosis is at least partly dependent on miR‐214‐5p. Collectively, the study provides evidence for a potential molecular mechanism through which C3G may counteract TMZ resistance by modulating miR‐214‐5p and its downstream β‐catenin/MGMT pathway, thereby potentially reducing the contribution of MGMT to chemoresistance in glioma cells [60].

Overall, the available evidence supports a TMZ‐sensitizing role for C3G that is associated with increased miR‐214‐5p expression. Mechanistically, miR‐214‐5p may suppress β‐catenin through direct interaction with CTNNB1, with subsequent reduction of MGMT expression contributing to enhanced TMZ responsiveness. Thus, the C3G/miR‐214‐5p/β‐catenin/MGMT regulatory axis may represent a potential therapeutic avenue for overcoming TMZ resistance in glioma. Further studies are needed to validate this mechanism and determine whether C3G can be effectively translated into a therapeutic strategy for TMZ‐resistant gliomas.

3.2.7. miR‐29c

miR‐29c has been implicated in the regulation of MGMT expression through an indirect mechanism involving the transcription factor specificity protein 1 (SP1), and increased miR‐29c expression has been associated with enhanced responsiveness of glioma cells to TMZ [61, 62]. Clinically, higher miR‐29c abundance has also been correlated with more favorable outcomes, suggesting that miR‐29c may have potential relevance as a therapeutic target in glioma. More broadly, members of the miR‐29 family have been reported to influence epigenetic regulation by suppressing DNA methylation of tumor‐suppressor genes, which may contribute to reduced tumor‐cell proliferation and increased chemosensitivity [63]. However, the biological functions of individual miR‐29 family members may differ according to cellular context. For example, Li et al. [64] reported that miR‐29b promoted oncogenic phenotypes through suppression of tumor‐suppressive molecules, including phosphatase and tensin homolog. These findings highlight the functional complexity of the miR‐29 family and indicate that its members may exert distinct effects in tumor development and therapeutic response.

Mechanistically, accumulating evidence indicates that miR‐29c can modulate MGMT expression indirectly by targeting SP1, a ubiquitously expressed zinc‐finger transcription factor involved in the transcriptional regulation of numerous genes [65]. Given the established association between elevated MGMT expression and resistance to TMZ, modulation of the miR‐29c/SP1/MGMT regulatory axis may represent a potential strategy for improving TMZ responsiveness in glioma. Further investigation is required to clarify the molecular details of this pathway and to determine whether pharmacological or miRNA‐based approaches targeting this regulatory network can effectively overcome TMZ chemoresistance.

3.2.8. miR‐370‐3p

Peng et al. reported that miR‐370‐3p expression is markedly reduced in human glioma tissues across different tumor grades, with the lowest levels detected in GBM specimens and patient‐derived GBM cell lines [66]. Functionally, restoration of miR‐370‐3p expression significantly decreased glioma‐cell viability and proliferation and altered cell‐cycle distribution, particularly by reducing the proportion of cells in the S and G2/M phases [66]. Mechanistic analysis identified β‐catenin as a direct posttranscriptional target of miR‐370‐3p through its interaction with the 3′UTR of β‐catenin mRNA. Given the established involvement of β‐catenin in Wnt signaling and the regulation of tumor‐cell proliferation and migration, suppression of this pathway may contribute to the antitumor activity of miR‐370‐3p [66]. Consistent with these observations, Lulli et al. [67] demonstrated that increased miR‐370‐3p expression impaired the proliferative, migratory, and clonogenic capacities of GBM stem‐like cells in vitro and significantly attenuated tumor growth in vivo.

In addition to its effects on β‐catenin signaling, miR‐370‐3p has been implicated in the regulation of MGMT and the response of GBM cells to TMZ. An inverse association between miR‐370‐3p and MGMT mRNA expression has been reported, suggesting a potential role for this miRNA in modulating MGMT‐dependent chemoresistance. Nadaradjane et al. proposed that miR‐370‐3p might have value as a biomarker for treatment monitoring and therapeutic response in GBM [68]. However, circulating miR‐370‐3p levels varied during standard treatment and were not significantly associated with overall survival. Interestingly, patients who maintained relatively high miR‐370‐3p expression before disease recurrence appeared to have longer survival, indicating that the clinical significance of this miRNA may depend on its temporal expression pattern [68]. Functional experiments further showed that miR‐370‐3p overexpression reduced both MGMT mRNA and protein abundance, resulting in increased TMZ‐induced cytotoxicity and greater chemosensitivity of tumor cells. In mouse xenograft models, combined administration of TMZ and miR‐370‐3p markedly suppressed subcutaneous tumor growth and was accompanied by reduced MGMT expression in excised tumor tissues [68].

Forkhead box protein M1 (FOXM1) represents another downstream target of miR‐370‐3p and is a key transcriptional regulator involved in cell‐cycle progression. Increased miR‐370‐3p expression was associated with decreased FOXM1 levels, providing an additional mechanism through which this miRNA may interfere with cell‐cycle progression and promote tumor‐cell death [68]. Thus, the biological effects of miR‐370‐3p in GBM appear to involve the coordinated regulation of multiple oncogenic pathways, including β‐catenin, MGMT, and FOXM1.

Collectively, current evidence supports miR‐370‐3p as a potential tumor‐suppressive miRNA in GBM. Through suppression of MGMT expression, miR‐370‐3p may increase the responsiveness of GBM cells to TMZ, including tumors with an MGMT‐unmethylated phenotype. Although its circulating expression does not appear to consistently predict overall survival, the therapeutic effects observed in preclinical models suggest that miR‐370‐3p may have potential as a TMZ‐sensitizing molecule. Further studies are needed to clarify its clinical biomarker value, establish the molecular basis of its regulation of MGMT and FOXM1, and determine whether miR‐370‐3p‐based interventions can be effectively translated into therapeutic strategies for TMZ‐resistant GBM.

3.2.9. miR‐409‐3p

Khalil et al. reported that miR‐409‐3p was approximately fivefold more abundant in GBM tissues than in normal brain specimens and that its expression was inversely associated with MGMT levels [48]. Analysis of clinical samples showed a reciprocal pattern, with miR‐409‐3p enrichment observed in tumors characterized by low MGMT expression, whereas specimens with elevated MGMT levels generally displayed reduced miR‐409‐3p abundance. Consistent with these observations, ectopic introduction of miR‐409‐3p into the T98G glioma cell line, which exhibits relatively high MGMT expression, resulted in a substantial reduction in both MGMT transcript and protein levels. These findings indicate that miR‐409‐3p can negatively regulate MGMT, potentially through a combination of enhanced mRNA degradation and impaired protein synthesis [48]. Notably, simultaneous transfection of miR‐409‐3p and miR‐181d produced a stronger inhibitory effect on MGMT expression than either miRNA alone, suggesting that these miRNAs may cooperate in the posttranscriptional regulation of MGMT [48].

Given the inverse relationship between miR‐409‐3p and MGMT abundance, modulation of this miRNA may have implications for overcoming alkylating‐agent resistance, particularly in GBM characterized by an MGMT‐unmethylated phenotype. Nevertheless, the broader molecular functions of miR‐409‐3p in GBM remain incompletely defined, as additional downstream targets have yet to be fully characterized. Available evidence suggests that miR‐409‐3p may also participate in pathways associated with angiogenesis and metastatic behavior, indicating that its biological effects are likely to extend beyond MGMT regulation. Further studies are therefore warranted to delineate its target network and to clarify whether miR‐409‐3p‐based modulation can be therapeutically exploited to enhance the efficacy of alkylating chemotherapy in GBM.

3.2.10. miR‐486‐3p

Prior research indicates that miR‐486 frequently exhibits reduced expression across multiple types of malignancies, leading to its classification as a miRNA with tumor‐suppressive activity [69, 70]. According to Plaisier and colleagues, elevated miR‐486‐3p levels may increase neuronal tumor responsiveness to chemotherapeutic agents [71]. Allicin, which constitutes the main bioactive compound in garlic, has long been consumed as a nutritional supplement; in addition, its possible antineoplastic effects have attracted considerable research interest [72]. Evidence indicates that allicin is capable of promoting cancer cell death while markedly restraining tumor cell proliferation [73]. Such bioactivity gives allicin notable therapeutic potential against diverse malignancies, including GBM [74]. The experimental findings showed that allicin exposure led to a pronounced elevation of miR‐486‐3p within GBM. Increased miR‐486‐3p expression strengthens GBM chemosensitivity to TMZ, an effect confirmed through both in vitro and in vivo studies. Additional mechanistic work identified MGMT as a direct downstream target of miR‐486‐3p; moreover, miR‐486‐3p upregulation markedly repressed MGMT protein translation. In addition, ectopic MGMT expression counteracted the miR‐486‐3p‐mediated increase in TMZ chemosensitivity [75].

Collectively, allicin raises miR‐486‐3p abundance and improves GBM cell susceptibility to TMZ. This implies that allicin might be developed as a beneficial adjunct to TMZ therapy, thereby potentially improving clinical outcomes. Furthermore, miR‐486‐3p represents a candidate molecular target for boosting GBM treatment responses, and additional rigorous studies are warranted.

3.2.11. miR‐603

Guo et al. have found that miR‐603 was upregulated in GBM samples and promoted cell proliferation as well as cell cycle progression. The targets of miR‐603 include Wnt inhibitory factor 1 and catenin beta‐1, which promote cell proliferation and migration by activating the Wnt/β‐catenin signaling pathway [76]. Therefore, miR‐603 is considered an oncogenic miRNA.

miR‐603 is suppressed through its interaction with the 3′UTR region of MGMT [77]. In vitro experiments have demonstrated that transfection of miR‐603 significantly reduced MGMT mRNA and protein expression while enhancing cellular sensitivity to TMZ. Similar results were observed in vivo [77]. Kushwaha et al. [77] have also found that the combined use of miR‐181d and miR‐603 exhibited the most significant regulatory effect on MGMT expression compared with their individual applications. From a therapeutic innovation perspective, miR‐603 represents a potential candidate molecule for inhibiting MGMT and suppressing Wnt/β‐catenin signaling pathway activation. Abate et al. proposed that miR‐603 and miR‐221 can target MGMT, thereby enhancing the efficacy of TMZ in GBM treatment, while self‐assembled nanoparticles (SANPs) were employed to deliver miRNAs into the brain [78]. Specifically, the researchers developed SANPs to co‐deliver miR‐603 and miR‐221, aiming to enhance the efficacy of TMZ in GBM treatment by preventing the development of chemotherapy resistance [78]. The study has found that encapsulating miRNA with SANP significantly enhanced the delivery of miRNA into the brain in an orthotopic GBM model. In the same experimental model, chemoresistance to TMZ was observed, whereas tumor growth inhibition was noted when TMZ treatment was combined with SANP co‐encapsulating miR‐603 and miR‐221. These data clearly demonstrate that two miRNAs, miR‐603 and miR‐221, modulate MGMT levels, which are considered the primary factors contributing to chemotherapy resistance in GBM and are currently a major cause of poor prognosis [78].

In summary, SANP demonstrates therapeutic potential for brain disorders by delivering miRNAs to the brain. More importantly, this study reveals the role of miRNAs in overcoming chemotherapy resistance in tumor treatment, potentially enabling more effective antitumor combination therapies.

3.2.12. miR‐648, miR‐181d, and miR‐767‐3p

Kreth et al. identified two MGMT subtypes, one containing a long UTR and the other containing a short UTR, and used target prediction software to determine the miRNA binding sites within the MGMT UTR [42]. They hypothesized that these miRNAs are expressed in human GBM and exhibit a negative correlation with MGMT expression. In human GBM samples, six miRNAs (miR‐184, miR‐183, miR‐661, miR‐370, miR‐767‐3p, and miR‐648) exclusively bind to the long UTR, two (miR‐1197 and miR‐655) bind to the short UTR, and one (miR‐181‐d) binds to both UTRs. They hypothesized that these miRNAs are expressed in human GBM and negatively correlate with MGMT expression [47].

The two UTRs were cloned into a reporter vector containing dual luciferases and co‐transfected with miRNAs. They observed that short UTR‐bound miRNAs (miR‐181d, miR‐665, and miR‐1197) exhibited no regulatory activity [47]. This indicates that the short UTR of MGMT is not regulated by these miRNAs. Among the seven miRNAs potentially regulating the long UTR, five (miR‐661, miR‐370, miR‐181d, miR‐767‐3p, and miR‐648) significantly suppressed luciferase activity. However, only three miRNAs (miR‐181d, miR‐767‐3p, and miR‐648) reduced MGMT protein expression, with miR‐648 exhibiting the most pronounced decrease in MGMT protein levels. In the qPCR experiment, only two miRNAs (miR‐181d and miR‐767‐3p) significantly reduced MGMT mRNA expression, indicating that these two miRNAs regulate MGMT expression by directly degrading mRNA transcripts, whereas miR‐648 may function through translational repression [47]. MGMT can be synergistically downregulated by miR‐181d and miR‐648 at both the posttranscriptional and translational levels [49]. We have found that MGMT expression was negatively correlated with miR‐648 in patients with MGMT promoter methylation. Similarly, Kushwaha et al. [77] have reported that transfection of miR‐648 did not necessarily affect MGMT expression.

Further studies have revealed that miR‐767‐3p and miR‐648 were significantly upregulated in human GBM samples, and co‐transfection of these three miRNAs (miR‐181d, miR‐767‐3p, and miR‐648) markedly enhanced the sensitivity to TMZ treatment [47]. These data are consistent with the findings of Jesionek‐Kupnicka et al. [49] who identified an association between MGMT and expression of miR‐181d and miR‐648.

Current evidence indicates that miRNA‐mediated regulation of MGMT extends beyond solitary targeting. Instead, multiple miRNAs function cooperatively (e.g., miR‐181d‐5p and miR‐409‐3p) to synergistically degrade MGMT transcripts. Furthermore, the dual functionality of certain miRNAs—targeting MGMT for DNA repair while simultaneously modulating immune checkpoints (like PD‐L1) or proliferation pathways (like Wnt/β‐catenin)—highlights their central role in the broader glioblastoma network. Analytically, this suggests that future clinical applications should prioritize multiplexed miRNA panels over single‐miRNA assays for a more accurate prediction of temozolomide sensitivity.

4. lncRNAs and MGMT

lncRNAs are a class of non‐coding RNA molecules with transcripts exceeding 200 nucleotides in length [79]. The underlying mechanism involves lncRNAs acting as competing endogenous RNAs (ceRNAs) that bind to miRNAs, thereby relieving miRNA‐mediated suppression of specific mRNA and subsequently modulating target mRNA expression [80]. lncRNAs are important regulators of gene expression and cellular behavior. They can modulate transcription through DNA methylation, histone modifications, and chromatin remodeling, and can also participate in posttranscriptional regulatory networks [81, 82]. Through these mechanisms, lncRNAs influence cancer‐cell proliferation, apoptosis, epithelial‐mesenchymal transition, migration, invasion, metastasis, stemness, and chemoresistance. Studies have shown that aberrant expression of lncRNAs is widespread in tumors and that dysregulated lncRNAs can function as oncogenes or tumor suppressors to drive tumor progression by shaping the cancer epigenome [81]. Aberrant expression of lncRNAs has emerged as a critical regulatory factor in the initiation and progression of GBM [83]; therefore, these lncRNAs may serve as diagnostic and prognostic biomarkers, as well as therapeutic targets.

4.1. lncRNAs in GBM

Driven by the development of increasingly sensitive RNA‐detection and high‐throughput sequencing technologies, the number of annotated long non‐coding RNAs (lncRNAs) has expanded rapidly and now exceeds the number of protein‐coding genes in the human genome. Most lncRNAs are predominantly transcribed by RNA polymerase II and subsequently undergo a range of co‐ and posttranscriptional modifications, including 5′ capping, alternative splicing, polyadenylation, and diverse chemical RNA modifications. lncRNAs have been identified in several cellular compartments, including the nucleus, nucleolus, cytoplasm, and mitochondria, where their subcellular localization underpins distinct regulatory functions, ranging from chromatin remodeling and transcriptional control in the nucleus to posttranscriptional and translational regulation in the cytoplasm and mitochondria. A growing body of evidence supports a mechanistic association between lncRNA dysregulation and the development and progression of glioblastoma, highlighting the potential of lncRNAs as both therapeutic targets and molecular biomarkers that may contribute to early detection, diagnosis, prognostic stratification, and monitoring of the disease [83].

Aberrant expression of lncRNAs has been extensively demonstrated to be closely associated with the occurrence, progression, and therapeutic resistance of GBM [84]. They regulate the malignant biological behaviors of GBM through various molecular mechanisms, including epigenetic modifications, ceRNA network regulation, signaling pathway intervention, and tumor microenvironment remodeling [85]. Functionally, lncRNAs can act as both oncogenic factors and tumor suppressors, exerting profound effects on GBM processes such as proliferation, invasion, stemness, metabolic reprogramming, and immune evasion [86]. These findings suggest that lncRNAs hold promise as novel biomarkers for the early diagnosis, prognostic evaluation, and therapeutic response monitoring of GBM, thereby providing more precise clinical management tools and approaches. The potential role of lncRNAs in the initiation, progression, and metastasis of GBM is not only associated with significant changes in expression levels but is also closely linked to their genomic locations. lncRNAs can regulate neighboring or distant coding genes through cis‐ or trans‐acting mechanisms [87].

These findings further underscore the complex and multifaceted roles of lncRNAs in GBM pathogenesis. By regulating gene expression and signaling networks involved in tumor growth, invasion, stemness, and treatment resistance, lncRNAs may provide promising targets for future therapeutic strategies. Overall, lncRNAs play critical roles in the initiation, progression, and therapeutic response of GBM.

4.2. Regulatory Mechanisms

lncRNAs systematically regulate MGMT expression through two distinct biological mechanisms: ceRNA networks in the cytoplasm and epigenetic mechanisms in the nucleus. As ceRNAs, lncRNAs act as molecular sponges that sequester specific miRNAs, thereby relieving the posttranscriptional suppression of MGMT. At the epigenetic level, nuclear lncRNAs recruit chromatin‐remodeling complexes (e.g., PRC2) or modulate histone acetylation at the promoter region to directly control MGMT transcription.

Research has found that lncRNA can act as a specific “sponge” for miRNA, attenuating the regulatory effect of miRNA on mRNA, and plays a crucial biological role in the occurrence, progression, and metastasis of GBM [88]. Among them, lncRNAs can influence the chemosensitivity of GBM by regulating MGMT expression [89]. This suggests that targeting these lncRNAs may provide novel strategies for overcoming chemoresistance in GBM. Subsequently, this article provides a detailed discussion of lncRNAs capable of regulating MGMT expression in GBM.

4.2.1. lncRNA Urothelial Carcinoma‐Associated 1 (UCA1)

Urothelial carcinoma‐associated 1 (UCA1) is a primate‐specific lncRNA expressed in human trophoblasts, where it contributes to the expansion of human trophoblast stem cells. Nevertheless, aberrant expression of UCA1 can disrupt trophoblast syncytialization and simultaneously stimulate interferon‐related signaling pathways [90]. Beyond its physiological functions, UCA1 has been characterized as an oncogenic lncRNA in several malignancies and has been implicated in the regulation of tumor‐cell proliferation, metastatic behavior, and apoptotic processes [91, 92, 93]. In GBM, both UCA1 and MGMT were reported to be expressed at elevated levels in tumor tissues and cell models. Experimental depletion of UCA1 or enforced expression of miR‐182‐5p increased the responsiveness of GBM cells to TMZ, as reflected by changes in cell viability, apoptosis, MGMT protein abundance, and DNA damage‐associated markers. Moreover, during TMZ exposure, increased miR‐182‐5p expression substantially counteracted the changes in MGMT abundance and TMZ sensitivity caused by UCA1 silencing [51].

Mechanistic investigations indicate that miR‐182‐5p recognizes the 3′‐UTRs of both UCA1 and MGMT mRNA, thereby contributing to posttranscriptional suppression of MGMT expression. Previous studies have additionally implicated miR‐182 in the regulation of CXCL1 production, glycolytic metabolism, and invasive properties of glioma cells through interactions with glioma‐associated stromal cells [94]. Alterations in miR‐182 signaling have also been associated with changes in glioma‐cell proliferation and migratory capacity [95]. Importantly, increasing miR‐182‐5p levels potentiated the antitumor effects of TMZ while reducing MGMT protein expression, providing evidence that the miR‐182‐5p/MGMT regulatory relationship may participate in UCA1‐mediated TMZ responses.

Collectively, these observations indicate that suppression of miR‐182‐5p can diminish the increase in TMZ sensitivity produced by UCA1 depletion, supporting a regulatory connection between UCA1, miR‐182‐5p, and MGMT. In clinical tissue specimens, miR‐182‐5p abundance showed an inverse association with both UCA1 and MGMT expression, whereas UCA1 levels were positively associated with MGMT expression. These expression patterns, together with the experimental findings, support the involvement of the UCA1/miR‐182‐5p/MGMT regulatory axis in glioma. One proposed mechanism is that UCA1 acts as a ceRNA, reducing the availability of miR‐182‐5p and consequently weakening miR‐182‐5p‐mediated repression of MGMT. The resulting increase in MGMT expression may contribute to reduced TMZ responsiveness in GBM cells.

4.2.2. lncRNA HOX Transcript Antisense RNA (HOTAIR)

HOTAIR is a well‐characterized oncogenic lncRNA that contributes to tumor progression by enhancing cellular proliferation and motility and by suppressing apoptotic processes [96]. In normal brain tissue, HOTAIR expression is minimal; however, its abundance is markedly increased in glioma specimens and TMZ‐resistant GBM cells. Notably, HOTAIR carried by serum‐derived exosomes has been implicated in the acquisition of TMZ resistance by GBM cells. Conversely, silencing HOTAIR can restore TMZ responsiveness in both in vitro and in vivo models [97]. Consistent with these observations, elevated HOTAIR levels in GBM are correlated with an unfavorable response to TMZ therapy. Experimental suppression of HOTAIR increases the susceptibility of GBM cells to TMZ, whereas forced HOTAIR expression promotes a resistant phenotype [98]. Transcriptomic analyses have further demonstrated that lncRNA expression patterns in brain tumor cells differ substantially from those observed in control parental brain cancer stem cells [99]. Among these dysregulated transcripts, HOTAIR is one of the most strongly elevated lncRNAs detected in GBM cell lines, supporting its potential involvement in glioma development and its possible clinical relevance.

Mechanistically, accumulating evidence suggests that HOTAIR contributes to TMZ resistance by increasing MGMT protein abundance through the HOTAIR/miR‐214‐3p/β‐catenin regulatory axis. In GBM patients exhibiting high HOTAIR expression, genes associated with the Wnt/β‐catenin signaling cascade are preferentially enriched, and activation of this pathway has been linked to unfavorable clinical outcomes and reduced TMZ responsiveness in glioma [100]. β‐catenin signaling therefore appears to be an important mediator of HOTAIR‐associated TMZ resistance, with MGMT functioning as a downstream molecular effector that influences the therapeutic response to TMZ. In addition, a significant relationship has been reported between HOTAIR abundance and the methylation status of the MGMT promoter. Specifically, tumors harboring a methylated MGMT promoter tend to display lower HOTAIR expression than those lacking promoter methylation [98].

Overall, the available evidence supports an association between increased HOTAIR expression and TMZ resistance in GBM. This effect may be mediated, at least in part, through activation of the miR‐214/β‐catenin/MGMT signaling axis, thereby contributing to diminished sensitivity to TMZ treatment.

4.2.3. lncRNA X‐Inactive Specific Transcript (XIST)

The long non‐coding RNA XIST is upregulated and functions as an oncogene in GBM [101], ovarian cancer [102], and non‐small cell lung cancer [103]. A previous study has revealed that XIST is upregulated in glioma tissues, and the knockdown of XIST inhibits glioma cell migration, invasion, and proliferation while promoting apoptosis [104]. Furthermore, XIST knockdown enhances the sensitivity of TMZ‐resistant GBM to TMZ. XIST suppresses miR‐29c expression by directly targeting TMZ‐resistant GBM cells, whereas miR‐29c inhibition promotes glioma cell proliferation and chemoresistance. The effects of XIST knockdown on the indicated TMZ‐resistant GBM can be partially reversed by miR‐29c inhibition. These data indicate that XIST can directly bind to miR‐29c, suppressing its expression and thereby influencing the proliferation of glioma cells and their chemoresistance to TMZ [101].

The study found that XIST knockdown significantly reduced the protein levels of mutS homolog 6 (MSH6), SP1, and MGMT, whereas miR‐29c inhibition markedly increased their expression. Moreover, inhibition of miR‐29c partially reversed the suppressive effects of XIST knockdown on these proteins. These results indicate that the XIST/miR‐29c axis co‐regulates SP1 and MGMT expression in TMZ‐resistant glioma cell lines. In glioma tissues, miR‐29c expression was downregulated, whereas SP1 and MGMT mRNA levels were elevated. XIST expression was negatively correlated with miR‐29c expression but positively correlated with SP1 and MGMT expression. Collectively, these findings suggest that targeting XIST to restore miR‐29c expression may suppress TMZ resistance in glioma cells and represent a promising approach for improving the efficacy of TMZ‐based chemotherapy [101].

4.2.4. lnc‐TALC

The study identified a previously unreported lncRNA in GBM, termed lnc‐TALC, which is highly expressed in TMZ‐resistant GBM. lnc‐TALC is located at the AL358975 locus and consists of two exons, with a total length of 418 nucleotides. Mechanistically, lnc‐TALC competitively binds to miR‐20b‐3p, thereby promoting c‐Met expression, which is associated with TMZ resistance. In TMZ‐resistant GBM cells, the phosphorylated AKT/FOXO3 axis regulates the expression of lnc‐TALC. Further studies revealed that miR‐20b‐3p and miR‐335‐3p may simultaneously target the 3′UTR of both lnc‐TALC and MET mRNA. Furthermore, luciferase reporter assays, RNA immunoprecipitation (RIP), and MS2‐RIP experiments confirmed the direct interaction between miR‐20b‐3p and the target sites within the 3′UTR regions of lnc‐TALC and MET mRNA. lnc‐TALC mediates the acetylation of H3K9, H3K27, and H3K36 in the MGMT promoter region through the c‐Met/Stat3/p300 axis, thereby increasing MGMT expression. The inhibition or overexpression of miR‐20b‐3p promotes or suppresses MGMT expression, respectively, revealing the critical role of lnc‐TALC in the regulation of MGMT expression [105]. In clinical patients, lnc‐TALC plays a critical role in TMZ resistance and GBM recurrence, suggesting that lnc‐TALC in GBM may serve as a therapeutic target to overcome TMZ resistance, thereby enhancing the clinical efficacy of TMZ chemotherapy [105].

In summary, AKT‐mediated lncRNA lnc‐TALC promotes MGMT expression and induces TMZ resistance in GBM by sequestering miR‐20b‐3p, activating c‐Met, and remodeling histone H3 acetylation in the MGMT promoter region. lnc‐TALC may serve as a potential therapeutic target to overcome TMZ resistance, thereby enhancing the clinical efficacy of TMZ chemotherapy in patients with GBM.

4.2.5. lncRNA Polycomb Repressive Complex 2 (PRC2)‐ and DEAD‐Box Helicase 5 (DDX5)‐Associated lncRNA (PRADX)

Li et al. identified a lncRNA named PRC2‐ and DDX5‐associated lncRNA (PRADX, ENST00000449248.1), which is upregulated in GBM and colon adenocarcinoma. This lncRNA predominantly localizes in the nucleus of tumor cells and interacts with the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) protein through its 5′‐terminal sequence. Furthermore, PRADX enhances H3K27 trimethylation (H3K27me3) at the UBXN1 gene promoter region by recruiting the PRC2/DDX5 complex, thereby promoting nuclear factor‐κB activity through UBXN1 suppression. Knockdown of PRADX significantly inhibited tumor cell viability and clonal growth in vitro [106]. In a xenograft model, PRADX knockdown suppressed tumor growth and tumorigenesis and prolonged the survival of tumor‐bearing mice, indicating the role of PRADX as a cancer driver and its potential as a therapeutic target.

One study has revealed that the lncRNA PRADX is overexpressed in mesenchymal GBM and can modulate the tumor microenvironment, thereby promoting tumor invasion and metastasis [107]. PRADX binds to EZH2 and recruits PRC2 to the MGMT promoter region, facilitating the formation of H3K27me3, leading to increased MGMT gene expression. The study identified a small‐molecule inhibitor therapeutic agent, EPIC‐0307, that selectively disrupts the PRADX‐EZH2 interaction, demonstrating its potent antitumor effects. The drug also upregulated multiple tumor suppressors in various GBM cell models and modulated DNA repair pathways, as well as in nude mouse GBM models. Furthermore, EPIC‐0307 enhanced the chemosensitivity of TMZ by modulating DNA damage repair factors and MGMT expression in GBM cells [108]. In summary, EPIC‐0307, a potential chemotherapeutic agent targeting the PRADX‐EZH2 axis in GBM treatment, can more effectively combat GBM when combined with TMZ and overcome TMZ resistance in patients undergoing chronic therapy (Figure 1).

FIGURE 1.

Integrated network of lncRNA‐mediated signaling axes converging on MGMT upregulation and temozolomide resistance in glioblastoma. (1) HOTAIR/miR‐214/β‐catenin/MGMT axis: Overexpressed lncRNA HOTAIR functions as a ceRNA by sponging miR‐214‐3p, which relieves the repression of β‐catenin. Stabilized/activated β‐catenin subsequently promotes MGMT transcription. (2) XIST/miR‐29c/Sp1/MGMT axis: LncRNA XIST sequesters miR‐29c, derepressing the transcription factor Sp1. Sp1 then binds to the MGMT promoter to enhance its transcriptional output. (3) lnc‐TALC/miR‐20b‐3p/c‐Met/Stat3/p300 axis: Lnc‐TALC sponges miR‐20b‐3p, leading to c‐Met pathway activation. This triggers downstream AKT phosphorylation (p‐AKT), which inactivates FOXO3 via phosphorylation (p‐FOXO3). The resultant signaling cascade involving Stat3 and p300 promotes histone acetylation at the MGMT promoter, driving transcriptional activation. (4) PRADX/PRC2/H3K27me3 axis: Nuclear lncRNA PRADX recruits the Polycomb repressive complex 2 (PRC2) to the MGMT promoter. The catalytic subunit EZH2 within PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), a repressive mark that paradoxically correlates with transcriptional activation of MGMT in this context. All four axes ultimately converge on elevated MGMT protein expression, which repairs TMZ‐induced O⁶‐methylguanine (O⁶‐meG) DNA lesions, thereby conferring chemoresistance. Arrows indicate activation or stimulation; T‐bars indicate inhibition; dashed lines indicate indirect or multi‐step regulation. Created by the authors using BioRender. Original figure, not adapted from any published source.

The reviewed studies demonstrate that lncRNAs govern MGMT expression through two primary analytical frameworks: the cytoplasmic ceRNA network (e.g., UCA1 and XIST acting as miRNA sponges) and nuclear epigenetic remodeling (e.g., PRADX recruiting the PRC2 complex). Unlike miRNAs, which generally act as direct suppressors, these lncRNAs predominantly function as oncogenic drivers that relieve MGMT suppression. Synthesizing these findings reveals that targeting lncRNAs represents a promising preclinical strategy that warrants further investigation, as silencing a single upstream lncRNA may simultaneously affect multiple downstream chemoresistance pathways. However, these findings remain confined to preclinical models and require clinical validation before therapeutic utility can be established.

5. circRNAs and MGMT

circRNAs are a class of single‐stranded ncRNA molecules characterized by covalent closed‐loop structures. Unlike linear RNAs, circRNAs lack polyadenylated tails or 5′ and 3′ termini, rendering them resistant to degradation by ribonuclease R (RNase R) [109, 110], relatively stable compared with mRNA. Over the past decade, circRNA research has evolved into a mature field of study, revealing the functional impact of this unique RNA molecular family in cancer. Although their biogenesis and abundance may differ from their homologous linear mRNAs, circRNAs exhibit similar molecular interaction profiles and resultant cellular functions during tumorigenesis. However, compared with linear RNA variants, the diversity of circular RNA variants increases tenfold. Coupled with their high cellular stability, circular RNAs can influence every stage of tumorigenesis [111]. circRNAs are generally expressed at low levels and exhibit tissue‐specific functions [112, 113, 114]. Numerous studies have demonstrated that circRNAs are aberrantly expressed in various cancers, including gliomas [115, 116], and are closely associated with the proliferation, migration, and invasion of tumor cells [117, 118].

It has been reported that circRNAs play a crucial role in regulating chemoresistance in cancer [119, 120]. The primary biological function of circRNA is to regulate gene expression at the transcriptional or posttranscriptional level [121]. Specifically, circRNAs can function as miRNA sponges [122], adsorbing miRNAs to modulate their activity or acting as RNA‐binding proteins [123] or protein scaffolds [124], involved in the regulation of various biological processes. Additionally, circRNAs can also regulate the translation process [125] and affect the conversion of precursors into proteins or polypeptides [126]. As ceRNAs, circRNAs are widely recognized to play a crucial role in the malignant progression of tumors [127]. Therefore, modulating circRNA expression can influence tumor biological behavior in preclinical models, suggesting that circRNAs may represent promising therapeutic targets. However, it is important to note that the vast majority of current evidence is derived from in vitro and in vivo studies, and the translational potential of circRNA‐targeted therapies for improving patient outcomes in GBM remains to be validated in clinical settings.

5.1. circRNA in GBM

CircRNAs are abundantly expressed in brain tissue and have important biological functions, particularly in cancer. Accumulating evidence indicates that circRNAs contribute substantially to glioma progression. Numerous circRNAs are aberrantly expressed in human gliomas and may participate in gliomagenesis by regulating glioma‐cell proliferation, invasion, metastasis, and cell‐cycle progression. Consequently, circRNAs may have potential value in the diagnosis, prognosis, and treatment of glioma [128]. Circular RNAs are important regulators of GBM tumorigenesis. Many act as endogenous RNA or miRNA sponges, competitively sequestering miRNAs and thereby altering the expression of downstream target genes that contribute to GBM progression. Although their precise roles in GBM development and prognosis remain incompletely understood, aberrant circRNA expression and tumor‐specific distribution appear to be common features. Individual circRNAs may be either upregulated or downregulated in GBM cells and tissues and exert distinct effects through diverse molecular mechanisms [129].

In summary, owing to their stable circular structure, high abundance in tumor tissues, and relative tissue specificity, circRNAs are closely involved in the initiation and progression of GBM. Their dysregulated expression may provide novel biomarkers for early diagnosis and prognostic evaluation and may also identify potential therapeutic targets for GBM.

5.2. Regulatory Mechanisms

The mechanisms by which circRNAs regulate MGMT are primarily anchored in the ceRNA network. Functioning as highly stable miRNA sponges, circRNAs sequester target miRNAs to upregulate MGMT expression. Furthermore, this ceRNA activity directly intersects with intracellular signaling pathways that promote glioma cell proliferation and resistance, effectively linking posttranscriptional sponging to broader oncogenic signaling networks.

In recent years, growing evidence has demonstrated that aberrantly expressed circRNAs play critical roles in GBM tumorigenesis and progression [130]. circRNAs primarily function as miRNA sponges and regulate the expression of downstream mRNAs through a circRNA‐miRNA‐mRNA (ceRNA) network [131]. This study indicates that circRNAs can influence tumor‐cell sensitivity to chemotherapeutic agents by regulating MGMT expression, with important implications for cancer therapy. This review provides a detailed discussion of circRNAs that regulate MGMT expression in GBM.

5.2.1. circWDR62

WDR62 comprises 32 exons and encodes a 1523‐residue protein associated with the minus ends of microtubule mitotic spindles. This protein is highly expressed in the forebrain during neurogenesis, particularly in the ventricular and subventricular zones, where it plays a crucial role in neuronal cell proliferation and migration [132]. Studies have found that the host gene WDR62 is significantly upregulated in most tumors and is associated with poor prognosis in various cancers [133, 134]. circWDR62 expression is elevated in TMZ‐resistant glioma cells and in exosomes derived from these cells. Its high expression is associated with poor glioma prognosis, whereas circWDR62 knockdown significantly suppresses TMZ resistance and malignant glioma progression. Mechanistically, circWDR62 acts as a competing endogenous RNA (ceRNA) that sequesters miR‐370‐3p. Geng et al. discovered that circWDR62 might exert its regulatory function by acting as a sponge for miR‐370‐3p. circRNA can function as a ceRNA to regulate the expression of target genes. The dual‐luciferase reporter gene assay demonstrated that miR‐370‐3p can directly bind to MGMT [25]. The study also confirmed that MGMT is a direct target gene of the circWDR62/miR‐370‐3p axis in glioma. circWDR62 promotes TMZ resistance and enhances tumor cell proliferation, migration, and invasion by directly targeting miR‐370‐3p and upregulating MGMT expression.

In summary, circWDR62 is encapsulated in exosomes, and exosomal circWDR62 acts as a sponge for miR‐370‐3p to regulate MGMT, thereby promoting TMZ resistance and malignant progression of glioma both in vitro and in vivo. This reveals a novel direction for the development of TMZ resistance in gliomas and provides a potential therapeutic target for TMZ‐resistant gliomas (Figure 2).

FIGURE 2.

Schematic overview of non‐coding RNA‐mediated regulatory mechanisms targeting MGMT expression and modulating temozolomide sensitivity in glioblastoma. (A) miRNA‐mediated direct regulation: MicroRNAs (e.g., miR‐181d‐5p, miR‐142‐3p, miR‐182‐5p, miR‐198, miR‐370‐3p, miR‐486‐3p) directly bind to complementary sequences within the 3′ untranslated region (3′UTR) of MGMT mRNA. This interaction triggers either mRNA degradation or translational repression, leading to reduced MGMT protein levels and enhanced TMZ‐induced cytotoxicity (indicated by green checkmark). (B) lncRNA‐mediated ceRNA regulation: Long non‐coding RNAs such as UCA1 and XIST function as competing endogenous RNAs (ceRNAs) by sponging specific miRNAs (e.g., miR‐182‐5p, miR‐29c). This sequestration relieves miRNA‐mediated suppression of MGMT mRNA, resulting in elevated MGMT protein expression and diminished TMZ sensitivity (indicated by red cross). (C) lncRNA‐mediated epigenetic regulation: Nuclear‐localized lncRNAs including HOTAIR and PRADX recruit chromatin‐modifying complexes such as Polycomb repressive complex 2 (PRC2) to the MGMT promoter region. This recruitment facilitates histone H3 lysine 27 trimethylation (H3K27me3), an epigenetic mark that promotes MGMT transcriptional activation. The subsequent increase in MGMT protein levels contributes to TMZ resistance (indicated by red cross). Arrows indicate activation or promotion; T‐bars indicate inhibition or repression. Green and red symbols denote TMZ sensitivity and resistance, respectively. Created by the authors using BioRender. Original figure, not adapted from any published source.

Although research on circRNAs in MGMT regulation is less extensive than that on miRNAs or lncRNAs, their unique structural stability and enrichment in exosomes (e.g., circWDR62) present a distinct analytical advantage. They not only modulate intracellular ceRNA networks to upregulate MGMT but also mediate intercellular communication within the tumor microenvironment, actively propagating chemoresistance to neighboring sensitive cells. This specific characteristic positions circRNAs as highly promising, stable liquid biopsy biomarkers for monitoring acquired temozolomide resistance in real‐time.

6. Clinical Translation

6.1. Therapeutic Strategies

The identification of ncRNA‐MGMT regulatory axes provides a theoretical framework for overcoming TMZ resistance. Transitioning from established mechanisms to clinical application necessitates advanced delivery platforms. Recent developments using SANPs to co‐deliver miR‐603 and miR‐221 directly address this limitation, successfully preventing TMZ resistance in orthotopic GBM models [78]. Furthermore, targeting upstream components of the ceRNA network, such as using small‐molecule inhibitors (e.g., EPIC‐0307) to disrupt the interaction between lncRNA PRADX and EZH2, prevents the recruitment of PRC2 to the MGMT promoter [108]. This pharmacological intervention demonstrates the preclinical feasibility of precision epigenetic remodeling, highlighting a potential experimental direction that requires rigorous clinical validation before it can complement generic chemotherapy.

6.2. Tumor Microenvironment

Evaluating the ncRNA‐MGMT network strictly within the confines of tumor cell DNA repair is insufficient. A novel perspective emerges when integrating these mechanisms with the immunosuppressive TME [51]. For example, lncRNA UCA1 promotes TMZ resistance by sequestering miR‐182‐5p. Restoring miR‐182‐5p not only directly suppresses MGMT but also impairs glioma‐associated stromal cell glycolysis and CXCL1 secretion [94]. Similarly, exosomal transfer of circWDR62 propagates TMZ resistance to neighboring sensitive cells by sponging miR‐370‐3p [25]. Targeting these specific ncRNAs offers a dual therapeutic advantage: disabling the primary DNA repair defense while simultaneously disrupting the intercellular communication that sustains the immunosuppressive TME.

7. Conclusions

Despite advances in surgical techniques and chemoradiotherapy, survival outcomes for glioma remain unsatisfactory. Chemotherapy resistance, particularly resistance to TMZ, is a major cause of treatment failure. Elucidating the mechanisms underlying TMZ resistance in GBM and developing strategies to overcome it are therefore essential for improving therapeutic efficacy and patient prognosis. Growing evidence indicates that non‐coding RNAs contribute to chemotherapy resistance in tumor cells. This review systematically summarizes how miRNAs, lncRNAs, and circRNAs affect GBM sensitivity to TMZ by directly or indirectly regulating MGMT expression. The study found that multiple miRNAs (such as miR‐142‐3p, miR‐181d‐5p, miR‐182‐5p, etc.) can directly target the 3′ untranslated region (3′UTR) of MGMT mRNA, promoting its degradation or inhibiting translation, thereby reducing MGMT protein levels and enhancing the efficacy of TMZ. Additionally, certain miRNAs, such as miR‐29c, can indirectly regulate MGMT expression by targeting transcription factors including Sp1. The expression of these miRNAs is closely associated with prognosis in patients with GBM, supporting their potential utility as biomarkers and therapeutic targets. Moreover, miRNA combinations, such as miR‐181d plus miR‐603, may suppress MGMT expression more effectively than either miRNA alone, providing a rationale for combination‐based therapeutic strategies (Table 1).

TABLE 1.

Summary of major ncRNAs, mechanisms, and their impact on MGMT expression and TMZ resistance.

ncRNA	Molecular target/Mechanism	Effect on MGMT expression	Association with TMZ resistance	Supporting evidence	Ref.
miR‐142‐3p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro	[40]
miR‐181d‐5p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, Clinical	[41]
miR‐182‐5p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro	[42]
miR‐198	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, In vivo, Clinical	[43]
miR‐221/222	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro	[44]
miR‐214‐5p	Indirectly targets beta‐catenin	Downregulation	Sensitizes (Decreases resistance)	In vitro	[45]
miR‐29c	Indirectly targets Sp1	Downregulation	Sensitizes (Decreases resistance)	In vitro, Clinical	[46]
miR‐370‐3p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, In vivo	[47]
miR‐409‐3p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, Clinical	[48]
miR‐486‐3p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, In vivo	[49]
miR‐603	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, In vivo	[50]
miR‐648	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro, Clinical	[51]
miR‐767‐3p	Directly binds MGMT 3′UTR	Downregulation	Sensitizes (Decreases resistance)	In vitro	[51]
lncRNA UCA1	Sponges miR‐182‐5p (ceRNA)	Upregulation	Promotes resistance	In vitro, Clinical	[42]
lncRNA HOTAIR	Activates miR‐214/beta‐catenin/MGMT	Upregulation	Promotes resistance	In vitro, Clinical	[90]
lncRNA XIST	Activates miR‐29c/Sp1/MGMT	Upregulation	Promotes resistance	In vitro, Clinical	[91]
lnc‐TALC	c‐Met/Stat3/p300 axis	Upregulation	Promotes resistance	In vitro, In vivo, Clinical	[92]
lncRNA PRADX	Promotes methylation via PRC2	Upregulation	Promotes resistance	In vitro, In vivo	[93]
circWDR62	Sponges miR‐370‐3p via exosomes	Upregulation	Promotes resistance	In vitro, In vivo	[25]

Abbreviations: 3′ UTR, 3′ untranslated region; ceRNA, competing endogenous RNA; circRNA, circular RNA; GBM, glioblastoma; HOTAIR, HOX transcript antisense RNA; lncRNA, long non‐coding RNA; MGMT, O6‐methylguanine‐DNA methyltransferase; ncRNA, non‐coding RNA; PRADX, PRC2 and DDX5‐associated lncRNA; PRC2, polycomb repressive complex 2; Sp1, specificity protein 1; TMZ, temozolomide; UCA1, urothelial carcinoma‐associated 1; XIST, X‐inactive specific transcript.

lncRNAs regulate MGMT expression through competing endogenous RNA (ceRNA) mechanisms, in which they sequester miRNAs and through recruitment of epigenetic regulatory complexes such as PRC2. For example, UCA1 sponges miR‐182‐5p, relieving its inhibitory effect on MGMT expression. HOTAIR promotes TMZ resistance by activating the miR‐214‐3p/beta‐catenin/MGMT signaling axis. Aberrant expression of these lncRNAs is closely associated with GBM progression and chemoresistance, suggesting that lncRNA‐targeted interventions may offer a strategy to reverse TMZ resistance.

CircRNAs can function as miRNA sponges within competing endogenous RNA networks, thereby regulating downstream gene expression. For example, circWDR62 promotes TMZ resistance by sequestering miR‐370‐3p and consequently increasing MGMT expression. Their high stability and tissue‐specific expression profiles make circRNAs promising candidates for GBM diagnosis and therapy.

Although substantial progress has been made in understanding ncRNA‐mediated regulation of MGMT, important challenges remain. The ncRNA regulatory network is highly complex, and a single ncRNA may exert oncogenic or tumor‐suppressive effects depending on the biological context. In addition, limited delivery efficiency and stability of ncRNA‐based therapeutics remain major barriers to clinical translation. Future studies should clarify the context‐specific mechanisms of ncRNAs and develop effective delivery platforms, such as nanoparticle‐based systems, to facilitate their therapeutic application.

Author Contributions

Junfeng Zhao: methodology, writing – review and editing, writing – original draft. Jiaxuan Chen: data curation, formal analysis, methodology. Yingrui Bai: software, visualization. Ying Li: investigation, data curation, validation. Yiming Han: software, investigation, funding acquisition. Chuankun Han: conceptualization. Yintao Li: writing – review and editing, resources, project administration, supervision.

Funding

This work was supported by the National Natural Science Foundation of China (grant number: 82373044) and the Natural Science Foundation of Shandong Province (grant numbers: ZR2023LSW023).

Ethics Statement

The authors have nothing to report.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.