The Biological Roles and Molecular Mechanisms of Long Non-Coding RNA MEG3 in the Hallmarks of Cancer

Simple Summary MEG3 is a class of lncRNA, which is considered a tumor suppressor. It is lost or decreased in different biological processes of various human tumors and is closely related to various diseases. MEG3 can modulate the expression of target genes through transcription, translation, post-translational modification and epigenetic regulation. Studies have shown that MEG3 dysfunction has been linked to a poor prognosis and drug resistance. MEG3 mediates the hallmarks of cancer through a variety of mechanisms, acting as a tumor suppressor to limit tumor growth. Hence, MEG3 is a potential prognostic marker and antitumor therapeutic target. Abstract Long non-coding RNAs (lncRNAs) are critical regulators in various biological processes involved in the hallmarks of cancer. Maternally expressed gene 3 (MEG3) is lncRNA that regulates target genes through transcription, translation, post-translational modification, and epigenetic regulation. MEG3 has been known as a tumor suppressor, and its downregulation could be found in various cancers. Furthermore, clinical studies revealed that impaired MEG3 expression is associated with poor prognosis and drug resistance. MEG3 exerts its tumor suppressive effect by suppressing various cancer hallmarks and preventing cells from acquiring cancer-specific characteristics; as it could suppress tumor cells proliferation, invasion, metastasis, and angiogenesis; it also could promote tumor cell death and regulate tumor cell metabolic reprogramming. Hence, MEG3 is a potential prognostic marker, and overexpressing MEG3 might become a potential antitumor therapeutic strategy. Herein, we summarize recent knowledge regarding the role of MEG3 in regulating tumor hallmarks as well as the underlying molecular mechanisms. Furthermore, we also discuss the clinical importance of MEG3, as well as their potential in tumor prognosis and antitumor therapeutic strategies.


Introduction
Cancer is the main cause of death globally and a significant impediment to extending life expectancy. In 2020, there was an estimated 19.3 million new cases of cancer and nearly 10 million cancer-related mortality globally [1]. While cancers that are accessible for early identification are slowing down, other prevalent malignancies are making significant progress [2]. Hence, there is an urgent need to find novel prognostic biomarkers and tumor therapeutic targets to combat cancer.
Tumorigenesis as well as the malignant transformation from benign tumors to malignant cancers is a complex process due to aberrant gene expressions. Distinct from normal cells, tumor cells have gained special characteristics, which are known as "hallmarks of

MEG3 Could Sponge miRNAs
LncRNAs can function as ceRNAs that bind to target miRNAs like a sponge and prevent miRNA from binding to its target mRNA, thus affecting the mRNA abundance of the target gene and their protein levels [30,31]. Numerous studies have reported that MEG3 can function as ceRNA by sponging and sequestering miRNAs, such as miR-21, miR-181a, and miR-421, from their target genes [32][33][34][35][36][37][38][39][40][41][42]. Similar to the targets of the miR-NAs it regulates, MEG3 possesses microRNA response elements (MREs). Through these MREs, MEG3 binds to the miRNA binding sites competitively with the corresponding target mRNAs, thereby removing the target mRNAs and eliminating the inhibitory effect of miRNA on them. The lncRNA-miRNA-mRNA forms a complex network of action, whose homeostasis is crucial for maintaining normal physiological conditions. Meanwhile, disruption of this homeostasis is closely related to diseases including cancers [43].

MEG Regulations on Target Genes Transcription
Besides as a ceRNA, MEG3 can regulate its targets through transcriptional as well as post-translational regulations ( Figure 2). For example, MEG3 could promote p53 expression by promoting its transcriptional activity and post-translational modification. MEG3 could enhance p53 transcriptional activity, thereby increasing p53 expression level and negatively regulating the cell cycle [44,45] Furthermore, MEG3 could also decrease the level of murine double minute 2 (MDM2), an E3 ubiquitin ligase that enhances p53 ubiquitination/proteasomal degradation, leading to p53 protein stabilization and transcriptional activation of p53 downstream targets [46]. Meanwhile, Weng et al. also found that MEG3, through its 732-1174 nucleic acid region, binds directly to Clusterin (CLU) protein and impedes CLU's interactions with its target proteins, such as vascular endothelial growth factor (VEGF) or matrix metalloproteinase (MMP-9) [47]. MEG3 affects the stability of proteins by regulating their post-translational modifications. Zhang et al. showed that MEG3 could suppress the accumulation of the phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein by recruiting ubiquitination enzymes and The 837 kb-long DLK1-MEG3 locus contains the protein-coding genes DIO3, RTL1, and DLK1. The MEG3 gene has ten exons and is 35 kb long. The IG-DMR is 13 kb upstream of the MEG3 gene. The MEG3-DMR overlaps with the MEG3 promoter. IG-DMR: intergenic differentially methylated region.

MEG3 Could Sponge miRNAs
LncRNAs can function as ceRNAs that bind to target miRNAs like a sponge and prevent miRNA from binding to its target mRNA, thus affecting the mRNA abundance of the target gene and their protein levels [30,31]. Numerous studies have reported that MEG3 can function as ceRNA by sponging and sequestering miRNAs, such as miR-21, miR-181a, and miR-421, from their target genes [32][33][34][35][36][37][38][39][40][41][42]. Similar to the targets of the miRNAs it regulates, MEG3 possesses microRNA response elements (MREs). Through these MREs, MEG3 binds to the miRNA binding sites competitively with the corresponding target mRNAs, thereby removing the target mRNAs and eliminating the inhibitory effect of miRNA on them. The lncRNA-miRNA-mRNA forms a complex network of action, whose homeostasis is crucial for maintaining normal physiological conditions. Meanwhile, disruption of this homeostasis is closely related to diseases including cancers [43].

MEG Regulations on Target Genes Transcription
Besides as a ceRNA, MEG3 can regulate its targets through transcriptional as well as post-translational regulations ( Figure 2). For example, MEG3 could promote p53 expression by promoting its transcriptional activity and post-translational modification. MEG3 could enhance p53 transcriptional activity, thereby increasing p53 expression level and negatively regulating the cell cycle [44,45] Furthermore, MEG3 could also decrease the level of murine double minute 2 (MDM2), an E3 ubiquitin ligase that enhances p53 ubiquitination/proteasomal degradation, leading to p53 protein stabilization and transcriptional activation of p53 downstream targets [46]. Meanwhile, Weng et al. also found that MEG3, through its 732-1174 nucleic acid region, binds directly to Clusterin (CLU) protein and impedes CLU's interactions with its target proteins, such as vascular endothelial growth factor (VEGF) or matrix metalloproteinase (MMP-9) [47]. MEG3 affects the stability of proteins by regulating their post-translational modifications. Zhang et al. showed that MEG3 could suppress the accumulation of the phosphorylated signal transducer and activator of transcription 3 (p-STAT3) protein by recruiting ubiquitination enzymes and thus directing pSTAT3 into ubiquitin/proteasomal degradation pathway without affecting its phosphorylation. This in turn suppresses the p-STAT3/c-Myc axis, and subsequently, leads to a decrease in cell proliferation potential [48].

MEG3 Regulates Various Hallmarks of Cancer
Recent studies revealed that MEG3 is associated with hallmarks of cancer, including proliferation, cell death, invasion and metastasis, metabolic reprogramming, and angiogenesis, by regulating various pathways (Table 1). MEG3 inhibits cancer progression through different mechanisms. MEG3 is involved in tumor progression in two ways, such as acting as a sponge for miRNA and regulating its targets through transcriptional as well Several studies have reported that lncRNAs are involved in chromatin remodeling by directing the recruitment of chromatin modifiers to target gene sites, for example, by associating with polycomb repressive complex 2 (PRC2) and inducing the trimethylation of histone H3 lysine 27 (H3K27me3) [49,50]. MEG3 could function as a molecular scaffold linking different proteins and forming large complexes that regulate chromatin structure and gene expression. By interacting with the RNA binding domain of Jumonji and AT-rich interaction domain containing 2 (JARID2), MEG3 stimulates PRC2 and JARID2 assembly, thereby enhancing H3K27me3 recruitment and suppressing the transcription of E-cadherin and miR-200 family [51].

MEG3 Regulates Various Hallmarks of Cancer
Recent studies revealed that MEG3 is associated with hallmarks of cancer, including proliferation, cell death, invasion and metastasis, metabolic reprogramming, and angiogenesis, by regulating various pathways (Table 1). MEG3 inhibits cancer progression through different mechanisms. MEG3 is involved in tumor progression in two ways, such as acting as a sponge for miRNA and regulating its targets through transcriptional as well as post-translational regulations. For example, MEG3 is closely related to the expression level of p53, a tumor suppressor whose mutation could be found in more than 50% of cancer patients [55]. MEG3 can directly interact with the DNA binding domain of p53 thereby enhancing the transcription of numerous p53 target genes [56]. MEG3 can also regulate p53 expression level indirectly by decreasing MDM2 protein level, leading to the decrease in MDM2-mediated p53 ubiquitination/proteasomal degradation, thereby stabilizing p53 protein levels [57,58].
Cancer stem cells (CSCs) are a small population of tumor cells that are usually in the dormant stage and have been assumed to be the main reason for tumorigenesis potential, tumor metastasis, recurrence, and drug resistance [86]. Targeting CSCs has been considered a potential therapeutic strategy for eradicating cancers; however, they are significantly less sensitive to current chemotherapy-and radiotherapy-based antitumor therapeutic strategies, as these strategies target proliferative cells [87]. MEG3 can repress CSC selfrenewal ability and decrease cancer stemness phenotype in oral CSCs by blocking miR-421 [88]. Furthermore, by sponging miR-708, MEG3 enhances SOCS3 expression, thereby decreasing colorectal CSCs stemness by suppressing STAT3 signaling [89].

MEG3 Induces Cell Death
Apoptosis is a programmed cell death controlled by a signaling cascade to maintain a stable internal environment. The elimination of cancer cells by apoptosis has been a key cue in clinical cancer treatment [90]. Apoptosis could be divided into intrinsic and extrinsic apoptotic pathways. The intrinsic apoptotic pathway, also known as mitochondriamediated apoptosis, is regulated by pro-apoptotic B-cell lymphoma 2 (Bcl-2) proteins, antiapoptotic Bcl-2 proteins, and BH3-only proteins, which triggers the activation of executor caspases 3 and 7 by activating caspase 8. Meanwhile, the extrinsic apoptotic pathway is regulated by death receptors, such as the tumor necrosis factor (TNF) receptor, which promotes the cleavage of initiator caspase, caspase 9, subsequently activating executor caspases [91,92].
Autophagy is an intracellular self-destructive form of cell death that transfers cytoplasmic proteins or organelles to the lysosome to fulfill the metabolic and self-renewal needs of organelles and the cell itself [103][104][105]. Previous studies have reported that MEG3 could attenuate autophagy by suppressing the forkhead box O1 (FOXO1) expression, leading to the decrease in autophagy-related proteins microtubule-associated protein light chain 3 II (LC3 II), beclin 1, autophagy related 3 (ATG3), autophagy related 5 (ATG5), and autophagy related 12 (ATG12), as well as the increase in the autophagy substrate p62 [106]. Hence, MEG3 regulation on autophagy needs further investigation.
The link between MEG3 and metastasis has also been confirmed by clinical samples from thyroid cancer (TC) patients showing that MEG3 downregulation was associated with lymph node metastasis. MEG3 could suppress TC cell migration and invasion by downregulating Rac family small GTPase 1 (Rac1) expression by targeting its 3 UTR [117]. Furthermore, MEG3 competitively interacts with miR-27a as the ceRNA of PH domain and leucine-rich repeat protein phosphatase 2 (PHLPP2) mRNA, promoting PHLPP2 protein translation and inhibiting c-Jun phosphorylation and c-Jun-mediated c-Myc mRNA transcription, thereby impairing invasion and lung metastasis of bladder cancer cells [71].

MEG3 Regulation on Tumor Cells Metabolic Reprogramming
Metabolic alteration is a characteristic of tumor cells crucial for supporting their rapid cell growth [3]. Unlike normal cells, which mainly depend on glycolysis followed by oxidative phosphorylation, tumor cells prefer inefficient aerobic glycolysis with a significantly higher turnover rate compared to normal cells even under adequate oxygen availability. This phenomenon is known as the Warburg effect [118,119]. The reprogrammed metabolic network generates intermediates, such as those involved in the glycolysis or tricarboxylic acid (TCA) cycle processes, which benefit cancer cells by helping them meet their energy needs as well as anabolic and redox and building blocks demands in the early stages of cancer development [120]. MEG3 activated by vitamin D can inhibit aerobic glycolysis and lactic acid production in CRC cells by inducing ubiquitin-dependent c-Myc degradation, thereby inhibiting c-Myc target genes expression involved in the glycolysis pathway, such as lactate dehydrogenase A (LDHA), pyruvate kinase muscle 2 (PKM2) and hexokinase 2 (HK2) [72]. Furthermore, MEG3 can promote succinate dehydrogenase (SDH) expression by sponging miR-361-5p, leading to an increase in succinate, a key TCA metabolite, thereby suppressing OSCC progression [73].

MEG3 Suppresses Tumor Angiogenesis
Formation of new blood vessels in tumor tissues from existing blood vessels is crucial for supplying tumor cells with oxygen and nutrient, for adapting to the fluctuating oxygen pressure in their microenvironment, as well as for metastasis [121]. This process involved many angiogenic factors, including vascular endothelial growth factor A (VEGFA), basic fibroblast growth factor (bFGF), and angiogenin. These factors increase endothelial cell development and vascular permeability, resulting in the formation of new blood vessels [122]. The role of MEG3 in tumor angiogenesis remains intriguing. Zhang et al. reported that MEG3 can suppress angiogenesis-related gene VEGFA, placental growth factor (PGF), bFGF, transforming growth factor β1 (TGF-β1) and MMP-9 expression by decreasing phosphorylated levels of AKT and inhibiting AKT pathway, ultimately suppressing angiogenesis in breast cancer [74]. However, Li et al. demonstrated that MEG3 could promote angiogenesis in lung carcinoma, as it could significantly increase the expression of angiogenesis-related factors VEGFA, vascular endothelial growth factor B (VEGFB), bFGF, stromal cell-derived factor-1 (SDF-1), transforming growth factor β (TGF-β), angiogenin, and MMP-9 [75]. The reasons underlying this discrepancy need further investigation.
Analysis of MEG3 expression in glioma patients showed that low expression of MEG3 was associated with poor overall survival rates, advanced WHO grade, low Karnofsky performance score (KPS), isocitrate dehydrogenase (IDH) wild-type, and tumor recurrence [60,125]. Xu et al. revealed that the copy number variation (CNV) levels of MEG3 were positively associated with overall survival and progression-free survival compared to the wild-type in low-grade glioma [123]; Gao et al. revealed, using 63 patients with retinoblastoma, that hypermethylation of MEG3 promoter was highly associated with poor survival, further confirming that MEG3 expression level is negatively correlated with poor prognosis [128]. Meanwhile, using 58 clinical ESCC tissues, Ma et al. found that low MEG3 expression was correlated with tumor size, lymph node metastasis, clinical stage, and poor prognosis [126]. These results were in accordance with other studies involving 48 CRC cases [129]. Furthermore, a negative correlation between MEG3 expression and short overall survival, relapse-free survival, and poor prognosis has also been found in breast cancer, NSCLC, and glioblastoma [76,127,130]. Together, these results show a negative correlation between MEG3 and tumor progression as well as prognosis, indicating the potential of using MEG3 as a biomarker for tumor prognosis.

MEG3 Is a Potential Target for Tumor Therapy
Anti-tumor therapies have been evolving and improving in recent years, yet resistance to chemotherapy, radiotherapy, targeted therapy, and immunotherapy remains a major problem [131]. Cytotoxic anti-tumor drugs such as cisplatin, paclitaxel, and doxorubicin, as well as targeted medicines such as imatinib, have been used for clinical cancer treatment. However, the persistent rise of drug resistance seriously undermines their efficacies [132]. MEG3 can facilitate chemotherapeutic drug sensitivity and radiosensitivity by altering key signaling pathways, making it a novel therapeutic strategy for cancer treatment (Table 3).
MEG3 could also act as an agonist of other antitumor drugs. Through MEG3/miR-4513/phenazine biosynthesis-like domain-containing (PBLD) axis, MEG3 promoted breast cancer cells' sensitivity to paclitaxel [137]. Furthermore, MEG3 suppresses the levels of drug-resistant transporters, including multidrug resistance-associated protein-1 (MRP1), multidrug resistance protein 1 (MDR1), and ATP binding cassette subfamily G member 2 (ABCG2), thus increasing CML cells' sensitivity against imatinib; miR-21 mimics could reverse their levels [138]. Meanwhile, by sponging miR-155, MEG3 upregulated alpha-1,2mannosyltransferase (ALG9) expression, thereby promoting AML cells' sensitivity against adriamycin and vincristine [139]. Moreover, MEG3 could promote pancreatic cancer cells' chemoresistance to gemcitabine [140]. Besides, MEG3 was closely related to 131 I-sensitivity of thyroid carcinoma by sponging miR-182 [141]. Finally, very recent research showed that tumor-targeting therapy of osteosarcoma (OS) can be performed by a highly effective engineered and MEG3-loaded exosome, as a combination of MEG3 and exosome significantly increased MEG3 therapeutic effect [142]. Together, these findings suggest that MEG3 plays a significant role in enhancing chemotherapeutic drug sensitivity and radiosensitivity in a variety of human cancers, making it a potential therapeutic target for cancer treatment.

Conclusions and Perspectives
MEG3 has emerged as a potential tumor suppressor that could regulate various hallmarks of cancer including cell proliferation, cell death, invasion and metastasis, metabolic reprogramming, angiogenesis, and drug resistance (Figure 3). MEG3 expression is downregulated in most malignant tumors, including glioma, HCC, CRC, and breast cancer. As shown in Figure 2, MEG3 regulation on tumor progression occurs through its function as a sponge that adsorbs miRNA, transcription, protein translation and post-translational modifications. However, in some cases, for example in angiogenesis, the role of MEG3 is still unclear, as current studies provide paradoxical results that require further detailed investigation. It is also noteworthy that a recent study showed that MEG3 could promote HCC cell senescence by sponging miR-16-5p, leading to the decrease in vestigial like family member 4 (VGLL4), which is a tumor suppressor and transcriptional cofactor, while increasing the levels of senescence-related markers p21 and p16 [143].
Hence, while more detailed studies are still needed to investigate whether MEG3 could regulate other hallmarks of cancer, such as avoiding immune destruction, genome instability and mutation, non-mutational epigenetic reprogramming, unlocking phenotypic plasticity and polymorphic microbiomes and whether there are exceptions for its tumor suppressive effects in certain hallmarks of cancer, present results demonstrate the tumor suppressive function of MEG3. Furthermore, although detailed investigations are still needed, MEG3 is a potential diagnostic biomarker and anti-tumor therapeutic target.  Acknowledgments: Our intention is to summarize the state of art. However, due to space limitations, we would like to apologize to authors whose works are not cited here. Their contributions should not be consid-ered less important than those that are cited.

Conflicts of Interest:
The author declares no conflict of interest.

Conflicts of Interest:
The authors declare no conflict of interest.