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The application of wireless power transmission technology is becoming more and more extensive. However, in practical applications, the problem of reducing the energy transmission efficiency caused by the offset of the coupling coil needs to be solved urgently. Changing the coil structure is a widely adopted method to deal with this problem. Based on the characteristics of the existing magnetic coupling resonant wireless power transmission system and the principle of the anti-offset coil, this paper innovatively designs a new type of quadrilateral overlapping wireless power transfer coupling coil, which has a strong anti-offset capability. The new type of coil model was built in the simulation and experiment, and the relevant parameters were measured. Experimental results verify that the proposed coil structure has an excellent anti-offset capability.
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Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets, such as protein-protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale by using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced the reaction volumes, reagent consumption, and synthesis time. As a proof-of-concept, we assembled a 2700-member target-focused macrocyclic library that we could subsequently assay in the same microtiter synthesis plates, saving the need for additional transfers and deconvolution schemes. We screened the library against the MDM2-p53 protein-protein interaction and generated micromolar and sub-micromolar inhibitors. Our approach based on acoustic liquid transfer provides a general strategy for the development of macrocycle ligands.
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Compuestos Macrocíclicos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Acústica , Humanos , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications.
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Alquinos/química , Azidas/química , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular , Química Clic , Reacción de Cicloadición , Escherichia coli , Humanos , Ratones , Péptidos/síntesis química , Solubilidad , Triazoles/síntesis química , Triazoles/farmacología , Agua/químicaRESUMEN
BACKGROUND: Since the inhibitory immune checkpoint receptors have been described to benefit the OSCC patients clinically, it is unknown that whether their expression in tumor immune microenvironment (TME) can determine the clinical outcome in response to nimotuzumab therapy. METHODS: We examined the expression patterns of immune checkpoint receptors (including TIM-3, LAG-3, PD-L1, and CTLA-4) and an immunoregulatory enzyme called IDO in 36 OSCC patients during nimotuzumab therapy by immunohistochemistry. Then, we divided the recruited patients into clinical responders and non-responders according to computed tomography (CT) scan and analyzed the relationship between the immunological parameters and clinical outcome. RESULTS: We observed that nimotuzumab therapy significantly increased the expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 in the TME, and the expression of LAG-3 and PD-L1 before nimotuzumab therapy was inversely correlated with the overall survival. In clinical non-responders, we found the expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 was significantly increased during nimotuzumab therapy, and the expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 before nimotuzumab therapy was negatively correlated with the overall survival. However, in clinical responders, neither of those showed significant. CONCLUSIONS: It suggests that these immune checkpoint receptors and IDO could be considered as biomarkers to reflect immune status in the tumor microenvironment during nimotuzumab therapy. Blockade of these immune checkpoint receptors might enhance nimotuzumab-based cancer immunotherapy, thus potentially improving clinical outcomes of OSCC patients.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Boca/tratamiento farmacológico , Antígenos CD/genética , Antígeno B7-H1/genética , Antígeno CTLA-4/genética , Carcinoma de Células Escamosas/genética , Femenino , Receptor 2 Celular del Virus de la Hepatitis A/genética , Humanos , Inmunoterapia , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/genética , Pronóstico , Microambiente Tumoral , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
There is a lack of current treatment options for ovarian clear cell carcinoma (CCC) and the cancer is often resistant to platinum-based chemotherapy. Hence there is an urgent need for novel therapeutics. The transcription factor hepatocyte nuclear factor 1ß (HNF1ß) is ubiquitously overexpressed in CCC and is seen as an attractive therapeutic target. This was validated through shRNA-mediated knockdown of the target protein, HNF1ß, in five high- and low-HNF1ß-expressing CCC lines. To inhibit the protein function, cell-permeable, non-helical constrained proteomimetics to target the HNF1ß-importinâ α protein-protein interaction were designed, guided by X-ray crystallographic data and molecular dynamics simulations. In this way, we developed the first reported series of constrained peptide nuclear import inhibitors. Importantly, this general approach may be extended to other transcription factors.
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Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Péptidos de Penetración Celular/química , Cristalografía por Rayos X , Femenino , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Estructura Molecular , Neoplasias Ováricas/química , Unión Proteica/efectos de los fármacosRESUMEN
The exploitation of synthetic lethality by small-molecule targeting of pathways that maintain genomic stability is an attractive chemotherapeutic approach. The Ctf4/AND-1 protein hub, which links DNA replication, repair, and chromosome segregation, represents a novel target for the synthetic lethality approach. Herein, we report the design, optimization, and validation of double-click stapled peptides encoding the Ctf4-interacting peptide (CIP) of the replicative helicase subunit Sld5. By screening stapling positions in the Sld5 CIP, we identified an unorthodox i,i+6 stapled peptide with improved, submicromolar binding to Ctf4. The mode of interaction with Ctf4 was confirmed by a crystal structure of the stapled Sld5 peptide bound to Ctf4. The stapled Sld5 peptide was able to displace the Ctf4 partner DNA polymeraseâ α from the replisome in yeast extracts. Our study provides proof-of-principle evidence for the development of small-molecule inhibitors of the human CTF4 orthologue AND-1.
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Péptidos/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , ADN Polimerasa I/química , ADN Polimerasa I/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Compuestos de Diazonio/química , Polarización de Fluorescencia , Inestabilidad Genómica , Humanos , Simulación de Dinámica Molecular , Péptidos/síntesis química , Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.
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Benzofenonas/química , Reactivos de Enlaces Cruzados/química , Péptidos/química , Etiquetas de Fotoafinidad , Proteínas Proto-Oncogénicas c-mdm2/química , Benzofenonas/síntesis química , Química Clic , Reactivos de Enlaces Cruzados/síntesis química , Ligandos , Estructura MolecularRESUMEN
Peptide stapling is a strategy for constraining short peptides typically in an alpha-helical conformation. Stapling is carried out by covalently linking the side-chains of two amino acids, thereby forming a peptide macrocycle. There is an expanding repertoire of stapling techniques based on different macrocyclisation chemistries. In this tutorial review, we categorise and analyse key examples of peptide stapling in terms of their synthesis and applicability to biological systems.
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Péptidos/química , Química Clic , Ciclización , Reacción de Cicloadición , Lactamas/química , Procesos FotoquímicosRESUMEN
Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This inâ situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces inâ situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.
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Compuestos Macrocíclicos/química , Péptidos/química , Medios de Cultivo , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/químicaRESUMEN
Background: Endothelial dysfunction is the early stage of carotid atherosclerosis (CAS) in patients with hypertension. It is worth identifying the potential hub genes of endothelial dysfunction to elucidate pathological mechanism in the progression of the disease. Method: We obtained gene expression profiles of GSE43292 from the Gene Expression Omnibus (GEO) database. Hub genes associated with CAS were identified through weighted gene correlation network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) regression. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore potential biological mechanisms and signaling pathways. Finally, in vitro experiments on human umbilical vein endothelial cells (HUVECs) were conducted to validate these hub genes. Results: The microarray dataset GSE43292 included 32 CAS plaques samples and corresponding macroscopically intact tissues from patients with hypertension. A total of 161 differentially expressed genes were discovered. Through WGCNA analysis, the gray60 module emerged as the most significant module associated with clinical features. The GO and KEGG enrichment analyses of genes in the gray60 module highlighted the substantial involvement of immune response-related signaling pathways. Two key hub genes (CCR1 and NCKAP1L) were pinpointed via LASSO regression. We found a significant increase in the mRNA expression level of the hub genes in oxidized low density lipoprotein (ox-LDL) treated HUVECs. Conclusions: Our study indicated that the hub genes related to immune responses are involved in the development of CAS. Two hub genes (CCR1 and NCKAP1L) of endothelial dysfunction were identified. These genes may provide a valuable therapeutic target of CAS in patients with hypertension.
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Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.
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Background: Previous studies have reported inconsistent results on the causal association between habitual tea consumption and the risk of cardiovascular disease (CVD). This study is aim to determine the association between habitual tea intake and CVD using two-sample Mendelian randomization (MR) analysis. Methods: The genetically predicted causation between tea consumption and 7 common cardiovascular diseases (atrial fibrillation, hypertension, acute myocardial infarction, coronary atherosclerosis, peripheral vascular disease, angina, and heart failure) was evaluated using MR analysis model. We performed a total of 9 MR analysis methods to analyze the final results. The IVW methods was used as the primary outcome. The other MR analysis method (simple mode, weighted mode, simple median, weighted median, penalized weighted median, MR Egger, and MR-Egger (bootstrap)) were performed as the complement to IVW. Also, the robustness of the MR analysis results was assessed using a leave-one-out analysis. Results: The IVW analysis methods indicated that there is no causal association between tea consumption and risk of CVD (AF: OR, 0.997, 95% CI, 0.992-1.0001, p = 0.142; hypertension: OR, 0.976, 95% CI, 0.937-1.017, p = 0.242; AMI: OR, 0.996, 95% CI, 0.991-1.000, p = 0.077; CA: OR, 1.001, 95% CI, 0.993-1.009, p = 0.854; PVD: OR, 1.002, 95% CI, 1.000-1.005, p = 0.096; angina: OR, 0.999, 95% CI, 0.993-1.006, p = 0.818; HF: OR, 0.999, 95% CI, 0.996-1.002, p = 0.338). The other MR analysis method and further leave-one-out sensitivity analysis suggested the results were robust. Conclusion: This MR study indicated that there was no genetically predicted causal association between habitual tea intake and risk of CVD.
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Combining different compound classes gives molecular hybrids that can offer access to novel chemical space and unique properties. Peptides provide ideal starting points for such molecular hybrids, which can be easily modified with a variety of molecular entities. The addition of small molecules can improve the potency, stability and cell permeability of therapeutically relevant peptides. Furthermore, they are often applied to create peptide-based tools in chemical biology. In this review, we discuss general methods that allow the discovery of this compound class and highlight key examples of peptide-small molecule hybrids categorised by the application and function of the small molecule entity.
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The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and ß-(1-azulenyl)-L-alanine (AzAla) were incorporated into inâ vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.
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Alanina/análogos & derivados , Colorantes Fluorescentes/química , Imagen Óptica , Péptidos Cíclicos/química , Sesquiterpenos/química , Triptófano/análogos & derivados , Alanina/química , Azulenos/química , Línea Celular Tumoral , Humanos , Estructura Molecular , Permeabilidad , Triptófano/químicaRESUMEN
Coagulation factor XI (FXI) has emerged as a promising target for the development of safer anticoagulation drugs that limit the risk of severe and life-threatening bleeding. Herein, we report the first cyclic peptide-based FXI inhibitor that selectively and potently inhibits activated FXI (FXIa) in human and animal blood. The cyclic peptide inhibitor (Ki = 2.8 ± 0.5 nM) achieved anticoagulation effects that are comparable to that of the gold standard heparin applied at a therapeutic dose (0.3-0.7 IU/mL in plasma) but with a substantially broader estimated therapeutic range. We extended the plasma half-life of the peptide via PEGylation and demonstrated effective FXIa inhibition over extended periods in vivo. We validated the anticoagulant effects of the PEGylated inhibitor in an ex vivo hemodialysis model with human blood. Our work shows that FXI can be selectively targeted with peptides and provides a promising candidate for the development of a safe anticoagulation therapy.
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Anticoagulantes/química , Factor XIa/antagonistas & inhibidores , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Animales , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Coagulación Sanguínea/efectos de los fármacos , Factor XIa/metabolismo , Semivida , Humanos , Isomerismo , Modelos Biológicos , Tiempo de Tromboplastina Parcial , Biblioteca de Péptidos , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Polietilenglicoles/química , Conejos , Diálisis RenalRESUMEN
Combinatorial cyclization of hundreds to thousands of random linear peptides by structurally diverse chemical linkers offers access to large macrocyclic compound libraries. A bottleneck in the development of such libraries is the preparation of large numbers of short random linear peptides. Herein, we present a tag-based strategy that is not dependent on a throughput-limiting chromatographic purification step and thus enables parallel production of short peptides. In brief, peptides are synthesized on solid phase as conjugates with a disulfide-linked Cys-Gly-Arg-Trp tetra-peptide tag. The charged arginine residue in the tag allows for purification of the peptides by diethyl ether-precipitation and the tryptophan allows for quantification of the product by absorption measurement. Addition of a reducing agent releases the short peptides from the tag. The released sulfhydryl group in the peptide can readily be used for cyclization of the peptide library with bis-electrophilic linker reagents.
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Disulfuros/química , Oligopéptidos/química , Cromatografía Liquida , Indicadores y Reactivos/química , Espectrometría de Masas , Oligopéptidos/síntesis química , Oligopéptidos/aislamiento & purificación , Biblioteca de PéptidosRESUMEN
We report a novel divinyltriazine linker for the stapling of two cysteine residues to form macrocyclic peptides from their unprotected linear counterparts. The stapling reaction occurred rapidly under mild conditions on a range of unprotected peptide sequences. The resulting constrained peptides displayed greater stability in a serum stability assay when compared to their linear counterparts.
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Herein, we describe the development of a novel staple with an electrophilic warhead to enable the generation of stapled peptide covalent inhibitors of the p53-MDM2 protein-protein interaction (PPI). The peptide developed showed complete and selective covalent binding resulting in potent inhibition of p53-MDM2 PPI.
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Inhibidores Enzimáticos/química , Péptidos Cíclicos/química , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Sulfonas/química , Sitios de Unión/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Humanos , Lisina/química , Simulación de Dinámica Molecular , Muramidasa/química , Péptidos Cíclicos/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/química , Saccharomyces cerevisiae/química , Sulfonas/síntesis químicaRESUMEN
Stapled peptides have great potential as modulators of protein-protein interactions (PPIs). However, there is a vast landscape of chemical features that can be varied for any given peptide, and identifying a set of features that maximizes cellular uptake and subsequent target engagement remains a key challenge. Herein, we present a systematic analysis of staple functionality on the peptide bioactivity landscape in cellular assays. Through application of a "toolbox" of diversified dialkynyl linkers to the stapling of MDM2-binding peptides via a double-click approach, we conducted a study of cellular uptake and p53 activation as a function of the linker. Minor changes in the linker motif and the specific pairing of linker with peptide sequence can lead to substantial differences in bioactivity, a finding which may have important design implications for peptide-based inhibitors of other PPIs. Given the complexity of the structure-activity relationships involved, the toolbox approach represents a generalizable strategy for optimization when progressing from in vitro binding assays to cellular efficacy studies.
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Péptidos/química , Alquinos/química , Secuencia de Aminoácidos , Línea Celular , Permeabilidad de la Membrana Celular , Humanos , Estructura Molecular , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND & AIM: Oral mucosal melanoma (OMM) is a kind of malignancy with extremely rare morbidity. It exhibits a poorer biological behavior and clinical outcome compared with cutaneous melanoma. lncRNAs are endogenous cellular RNA transcripts with no protein-coding potential and are associated with oncogenesis through cis- or trans-acting mechanisms. Despite increased evidence that proved lncRNAs have vital roles in tumorigenesis of mucosal melanoma, little is known about their functions in the progress of lymph node dissemination of OMM. METHOD: Here, we constructed a lncRNA and mRNA microarray using six metastatic lymph nodes and paired-matched non-metastatic lymph nodes. Then, we performed RT-PCR to validate the microarray data both in primary and metastases. We further constructed lncRNA and mRNA co-expressing networks and analyzed the biological functions by Gene Ontology (GO) and pathway analyses for dysregulated lncRNAs and mRNAs. Cis- and trans-regulation analysis were also performed to explore the specific mechanism of lncRNAs in OMM. RESULT: Our results showed that 570 lncRNAs were upregulated with 292 lncRNAs downregulated in the metastatic OMM tissues. The results of RT-PCR were consistent with our microarray dataset both in primary and metastases. Gene Ontology (GO) and pathway analyses indicated that they play an important role in the melanin biosynthetic process, new growing cell tip and lysosomes in metastatic OMM. In the cis-regulation analysis, we observed metastasis-associated gene, PLEKHA5, the cis gene of lnc-AEBP2-1_1 and lnc-AEBP2-2_1, and microphthalmia-associated transcription factor (MITF), the cis gene of SAMMSON_3, SAMMSON_5 and lnc-MITF-5_1. In the trans-regulation analysis, CTBP2 and SUZ12 regulated lncRNA expression in the core TF-lncRNA-gene network. CONCLUSION: Our results suggest that lncRNAs may be involved in the metastasis of OMM, and further investigation is needed to focus on the biological functions and the underlining molecular mechanisms exerted by these dysregulated lncRNAs in OMM.