RESUMEN
BACKGROUND: Temporal lobe epilepsy (TLE), a prevalent neurological disorder, is associated with hippocampal oxidative stress and inflammation. A recent study reveals that the long noncoding RNA ILF3 divergent transcript (ILF3-AS1) level is elevated in the hippocampus of TLE patients; however, the functional roles of ILF3-AS1 in TLE and underlying mechanisms deserve further investigation. Hence, this study aimed to elucidate whether ILF3-AS1 is involved in the pathogenesis of TLE by regulating oxidative stress and inflammation and to explore its underlying mechanism in vitro. METHODS: Human hippocampal neurons were subjected to a magnesium-free (Mg2+-free) solution to establish an in vitro model of TLE. The potential binding sites between ILF3-AS1 and miRNA were predicted by TargetScan/Starbase and confirmed by dual luciferase reporter assay. Cell viability and damage were assessed by cell counting kit-8 and lactate dehydrogenase assay kits, respectively. Levels of reactive oxygen species, malondialdehyde, and superoxide dismutase were determined by commercial kits. Levels of Interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-alpha were quantified by enzyme-linked immunosorbent assay. The expressions of gene and protein were determined by quantitative real-time polymerase chain reaction and Western blot analysis. RESULTS: In Mg2+-free-treated hippocampal neurons, both ILF3-AS1 and HMGB1 were highly up-regulated, whereas miR-504-3p was down-regulated. ILF3-AS1 knockdown ameliorated Mg2+-free-induced cellular damage, oxidative stress, and inflammatory response. Bioinformatics analysis revealed that miR-504-3p was a target of ILF3-AS1 and was negatively regulated by ILF3-AS1. MiR-504-3p inhibitor blocked the protection of ILF3-AS1 knockdown against Mg2+-free-induced neuronal injury. Further analysis presented that ILF3-AS1 regulated HMGB1 expression by sponging miR-504-3p. Moreover, HMGB1 overexpression reversed the protective functions of ILF3-AS1 knockdown. CONCLUSION: Our findings indicate that ILF3-AS1 contributes to Mg2+-free-induced hippocampal neuron injuries, oxidative stress, and inflammation by targeting the miR-504-3p/HMGB1 axis. These results provide a novel mechanistic understanding of ILF3-AS1 in TLE and suggest potential therapeutic targets for the treatment of epilepsy.
Asunto(s)
Epilepsia del Lóbulo Temporal , Proteína HMGB1 , Hipocampo , Inflamación , MicroARNs , Estrés Oxidativo , ARN Largo no Codificante , Estrés Oxidativo/fisiología , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo/metabolismo , Inflamación/metabolismo , Inflamación/genética , Neuronas/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Proteínas del Factor Nuclear 90/genéticaRESUMEN
Hyperglycemia accelerates calcification of atherosclerotic plaques in diabetic patients, and the accumulation of advanced glycation end products (AGEs) is closely related to the atherosclerotic calcification. Here, we show that hyperglycemia-mediated AGEs markedly increase vascular smooth muscle cells (VSMCs) NF90/110 activation in male diabetic patients with atherosclerotic calcified samples. VSMC-specific NF90/110 knockout in male mice decreases obviously AGEs-induced atherosclerotic calcification, along with the inhibitions of VSMC phenotypic changes to osteoblast-like cells, apoptosis, and matrix vesicle release. Mechanistically, AGEs increase the activity of NF90, which then enhances ubiquitination and degradation of AGE receptor 1 (AGER1) by stabilizing the mRNA of E3 ubiquitin ligase FBXW7, thus causing the accumulation of more AGEs and atherosclerotic calcification. Collectively, our study demonstrates the effects of VSMC NF90 in mediating the metabolic imbalance of AGEs to accelerate diabetic atherosclerotic calcification. Therefore, inhibition of VSMC NF90 may be a potential therapeutic target for diabetic atherosclerotic calcification.
Asunto(s)
Aterosclerosis , Proteína 7 que Contiene Repeticiones F-Box-WD , Productos Finales de Glicación Avanzada , Ratones Noqueados , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteínas del Factor Nuclear 90 , Receptor para Productos Finales de Glicación Avanzada , Animales , Masculino , Ratones , Productos Finales de Glicación Avanzada/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Proteínas del Factor Nuclear 90/metabolismo , Proteínas del Factor Nuclear 90/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Receptor para Productos Finales de Glicación Avanzada/genética , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/genética , Ratones Endogámicos C57BL , Ubiquitinación , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Hiperglucemia/metabolismo , Hiperglucemia/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Placa Aterosclerótica/genética , ApoptosisRESUMEN
Acquired resistance represents a critical clinical challenge to molecular targeted therapies such as tyrosine kinase inhibitors (TKIs) treatment in hepatocellular carcinoma (HCC). Therefore, it is urgent to explore new mechanisms and therapeutics that can overcome or delay resistance. Here, a US Food and Drug Administration (FDA)-approved pleuromutilin antibiotic is identified that overcomes sorafenib resistance in HCC cell lines, cell line-derived xenograft (CDX) and hydrodynamic injection mouse models. It is demonstrated that lefamulin targets interleukin enhancer-binding factor 3 (ILF3) to increase the sorafenib susceptibility of HCC via impairing mitochondrial function. Mechanistically, lefamulin directly binds to the Alanine-99 site of ILF3 protein and interferes with acetyltransferase general control non-depressible 5 (GCN5) and CREB binding protein (CBP) mediated acetylation of Lysine-100 site, which disrupts the ILF3-mediated transcription of mitochondrial ribosomal protein L12 (MRPL12) and subsequent mitochondrial biogenesis. Clinical data further confirm that high ILF3 or MRPL12 expression is associated with poor survival and targeted therapy efficacy in HCC. Conclusively, this findings suggest that ILF3 is a potential therapeutic target for overcoming resistance to TKIs, and lefamulin may be a novel combination therapy strategy for HCC treatment with sorafenib and regorafenib.
Asunto(s)
Carcinoma Hepatocelular , Resistencia a Antineoplásicos , Neoplasias Hepáticas , Mitocondrias , Proteínas del Factor Nuclear 90 , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Humanos , Animales , Ratones , Resistencia a Antineoplásicos/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas del Factor Nuclear 90/metabolismo , Proteínas del Factor Nuclear 90/genética , Línea Celular Tumoral , Diterpenos/farmacología , Compuestos Policíclicos/farmacología , Compuestos Policíclicos/uso terapéutico , Homeostasis/efectos de los fármacos , Sorafenib/farmacología , Modelos Animales de Enfermedad , Limoninas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzofuranos , NaftoquinonasRESUMEN
Intimal hyperplasia is a complicated pathophysiological phenomenon attributable to in-stent restenosis, and the underlying mechanism remains unclear. Interleukin enhancer-binding factor 3 (ILF3), a double-stranded RNA-binding protein involved in regulating mRNA stability, has been recently demonstrated to assume a crucial role in cardiovascular disease; nevertheless, its impact on intimal hyperplasia remains unknown. In current study, we used samples of human restenotic arteries and rodent models of intimal hyperplasia, we found that vascular smooth muscle cell (VSMC) ILF3 expression was markedly elevated in human restenotic arteries and murine ligated carotid arteries. SMC-specific ILF3 knockout mice significantly suppressed injury induced neointimal formation. In vitro, platelet-derived growth factor type BB (PDGF-BB) treatment elevated the level of VSMC ILF3 in a dose- and time-dependent manner. ILF3 silencing markedly inhibited PDGF-BB-induced phenotype switching, proliferation, and migration in VSMCs. Transcriptome sequencing and RNA immunoprecipitation sequencing depicted that ILF3 maintained its stability upon binding to the mRNA of the high-mobility group box 1 protein (HMGB1), thereby exerting an inhibitory effect on the transcription of dual specificity phosphatase 16 (DUSP16) through enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Therefore, the results both in vitro and in vivo indicated that the loss of ILF3 in VSMC ameliorated neointimal hyperplasia by regulating the STAT3/DUSP16 axis through the degradation of HMGB1 mRNA. Our findings revealed that vascular injury activates VSMC ILF3, which in turn promotes intima formation. Consequently, targeting specific VSMC ILF3 may present a potential therapeutic strategy for ameliorating cardiovascular restenosis.
Asunto(s)
Proteína HMGB1 , Hiperplasia , Ratones Noqueados , Músculo Liso Vascular , Miocitos del Músculo Liso , Proteínas del Factor Nuclear 90 , Estabilidad del ARN , Factor de Transcripción STAT3 , Túnica Íntima , Animales , Humanos , Masculino , Ratones , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteína HMGB1/metabolismo , Proteína HMGB1/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Neointima/metabolismo , Neointima/patología , Proteínas del Factor Nuclear 90/metabolismo , Proteínas del Factor Nuclear 90/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Factor de Transcripción STAT3/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/patologíaRESUMEN
BACKGROUND: Sepsis is a systemic inflammatory response which is frequently associated with acute lung injury (ALI). Activating transcription factor 3 (ATF3) promotes M2 polarization, however, the biological effects of ATF3 on macrophage polarization in sepsis remain undefined. METHODS: LPS-stimulated macrophages and a mouse model of cecal ligation and puncture (CLP)-induced sepsis were generated as in vitro and in vivo models, respectively. qRT-PCR and western blot were used to detect the expression of ATF3, ILF3, NEAT1 and other markers. The phenotypes of macrophages were monitored by flow cytometry, and cytokine secretion was measured by ELISA assay. The association between ILF3 and NEAT1 was validated by RIP and RNA pull-down assays. RNA stability assay was employed to assess NEAT1 stability. Bioinformatic analysis, luciferase reporter and ChIP assays were used to study the interaction between ATF3 and ILF3 promoter. Histological changes of lung tissues were assessed by H&E and IHC analysis. Apoptosis in lungs was monitored by TUNEL assay. RESULTS: ATF3 was downregulated, but ILF3 and NEAT1 were upregulated in PBMCs of septic patients, as well as in LPS-stimulated RAW264.7 cells. Overexpression of ATF3 or silencing of ILF3 promoted M2 polarization of RAW264.7 cells via regulating NEAT1. Mechanistically, ILF3 was required for the stabilization of NEAT1 through direct interaction, and ATF3 was a transcriptional repressor of ILF3. ATF3 facilitated M2 polarization in LPS-stimulated macrophages and CLP-induced septic lung injury via ILF3/NEAT1 axis. CONCLUSION: ATF3 triggers M2 macrophage polarization to protect against the inflammatory injury of sepsis through ILF3/NEAT1 axis.
Asunto(s)
Factor de Transcripción Activador 3 , Macrófagos , ARN Largo no Codificante , Sepsis , Animales , Humanos , Ratones , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Lipopolisacáridos , Macrófagos/metabolismo , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Células RAW 264.7 , Sepsis/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Survivin is a novel attractive target for cancer therapy; however, it is considered undruggable because it lacks enzymatic activities. Herein, we describe our efforts toward the discovery of a novel series of 4,11-dioxo-4,11-dihydro-1H-anthra[2,3-d]imidazol-3-ium derivatives as survivin inhibitors by targeting ILF3/NF110. Intensive structural modifications led us to identify a lead compound AQIM-I, which remarkably inhibited nonsmall cell lung cancer cells A549 with an IC50 value of 9 nM and solid tumor cell proliferation with more than 700-fold selectivity against human normal cells. Further biological studies revealed that compound AQIM-I significantly inhibited survivin expression and colony formation and induced ROS production, apoptosis, cell cycle arrest, DNA damage, and autophagy. Furthermore, the promoter-luciferase reporter assay showed that AQIM-I attenuated the survivin promoter activity enhanced by the overexpression of ILF3/NF110 in a concentration-dependent manner, and specific binding (KD = 163 nM) of AQIM-I to ILF3/NF110 was detected by surface plasmon resonance.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Survivin/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Apoptosis , Proteínas Inhibidoras de la Apoptosis , Línea Celular Tumoral , Proliferación Celular , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismoRESUMEN
Non-small cell lung cancer (NSCLC) is a highly lethal disease worldwide. We found the pseudogene-derived lncRNA PTTG3P is upregulated in NSCLC and associated with larger tumor size, advanced staging, and poor prognosis. This study investigated the oncogenic roles and mechanisms of PTTG3P in NSCLC. We demonstrate that PTTG3P promoted NSCLC cell proliferation, migration, tumorigenesis, and metastasis while inhibiting apoptosis in vitro and in vivo. Mechanistically, PTTG3P formed an RNA-protein complex with ILF3 to maintain MAP2K6 and E2F1 mRNA stability, two oncogenic factors involved in NSCLC progression. RNA-seq revealed MAP2K6 and E2F1 were downregulated upon PTTG3P knockdown. RIP and RNA stability assays showed PTTG3P/ILF3 interaction stabilized MAP2K6 and E2F1 transcripts. Interestingly, E2F1 transcriptionally upregulated PTTG3P by binding its promoter, forming a positive feedback loop. Knockdown of E2F1 or PTTG3P attenuated their mutual regulatory effects on cell growth and migration. Thus, a PTTG3P/ILF3/E2F1 axis enhances oncogene expression to promote NSCLC pathogenesis. Our study reveals PTTG3P exerts oncogenic functions in NSCLC via mRNA stabilization and a feedback loop, highlighting its potential as a prognostic biomarker and therapeutic target.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Retroalimentación , Neoplasias Pulmonares/genética , Transformación Celular Neoplásica , Carcinogénesis/genética , Estabilidad del ARN/genética , Proteínas del Factor Nuclear 90/genética , Factor de Transcripción E2F1/genéticaRESUMEN
BACKGROUND: Proteasome 26S subunit, non-ATPase 3 (PSMD3) has been reported to participate in various human cancers. Nevertheless, the function of PSMD3 in lung cancer (LC) remains unclear. METHODS: RT-qPCR and western blot were used to detect the expression of PSMD3 in LC tissues form TCGA database and clinical samples, and LC cell lines. To study the effect of PSMD3 on LC cell proliferation, migration, invasion, and apoptosis, siRNAs targeting PSMD3 were synthesized and overexpressed plasmids were constructed. CCK-8 assay, Transwell assay, and etc. were used to evaluate the results. Tumor xenograft model was used to evaluate the function of PSMD3 on tumor growth. CO-IP and MS were used to scan the proteins that bind with PSMD3. The interaction between PSMD3 and ILF3 in lung cancer cells were studied using IF staining, CHX protein stability, and ubiquitination assay. Additionally, the effect of ILF3 on cell progression and LC tumor growth was demonstrated by conducting a recovery assay using siILF3 and an ILF3 inhibitor YM155. RESULTS: We observed that PSMD3 was significantly overexpressed in LC tissues and cells, which indicated a poor prognosis. Meanwhile, we found that PSMD3 promoted cell proliferation, migration, and invasion of LC cells. We also determined that PSMD3 stabilized the protein expression of ILF3 and the deubiquitination of ILF3 in lung cancer cells. Furthermore, animal experiments showed that the ILF3 inhibitor YM155 could suppress tumor growth with the presence of PSMD3. CONCLUSIONS: PSMD3 collectively regulated the stability of ILF3 protein and facilitated the ubiquitination of endogenous ILF3 in LC, which ultimately promoted the progression of LC cells. The PSMD3/ ILF3 axis could potentially be used as a novel strategy for both diagnosis and treatment of LC.
Asunto(s)
Neoplasias Pulmonares , Proteínas del Factor Nuclear 90 , Complejo de la Endopetidasa Proteasomal , Animales , Humanos , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Transducción de Señal , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Skin is the first line of the body to resist pathogen invasion. A potentially fatal infection may result from problems with wound healing. Small molecule drugs like astragaloside IV (AS-IV) show pro-healing activities, but the mechanisms are not fully understood. Using real-time quantitative PCR and a western blot assay, the amount of gene expression was evaluated. The proliferation and migration of keratinocytes were determined by MTS and wound healing assay, respectively. The binding of lncRNA H19 to RBP protein ILF3 and the binding of ILF3 protein to CDK4 mRNA were confirmed by RNA immunoprecipitation. Treatment with AS-IV enhanced the expression of lncRNA H19, ILF3, and CDK4 and improved the proliferation and migration of keratinocytes HaCaT. Additionally, apoptosis of keratinocytes was attenuated by AS-IV. Further studies showed that both lncRNA H19 and ILF3 were important for AS-IV-mediated keratinocyte growth and migration. In addition, lncRNA H19 recruited ILF3 to increase CDK4 mRNA level and enhanced cell proliferation. We discovered a lncRNA H19/ILF3/CDK4 axis that is activated by AS-IV to promote keratinocyte migration and proliferation. These results elucidate the mechanism of action of AS-IV and justify its application in further application in wound healing treatment.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina , Queratinocitos , Proteínas del Factor Nuclear 90 , ARN Largo no Codificante , Proliferación Celular/genética , Queratinocitos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , Células HaCaT , Humanos , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Quinasa 4 Dependiente de la Ciclina/genéticaRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) is an essential hub that integrates nutrient signals and coordinates metabolism to control cell growth. Amino acid signals are detected by sensor proteins and relayed to the GATOR2 and GATOR1 complexes to control mTORC1 activity. Here we perform genome-wide CRISPR/Cas9 screens, coupled with an assay for mTORC1 activity based on fluorescence-activated cell sorting analysis of pS6, to identify potential regulators of mTORC1-dependent amino acid sensing. We then focus on interleukin enhancer binding factor 3 (ILF3), one of the candidate genes from the screen. ILF3 tethers the GATOR complexes to lysosomes to control mTORC1. Adding a lysosome-targeting sequence to the GATOR2 component WDR24 bypasses the requirement for ILF3 to modulate amino-acid-dependent mTORC1 signalling. ILF3 plays an evolutionarily conserved role in human and mouse cells, and in worms to regulate the mTORC1 pathway, control autophagy activity and modulate the ageing process.
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Aminoácidos , Serina-Treonina Quinasas TOR , Animales , Humanos , Ratones , Aminoácidos/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: Although immune checkpoint blockade (ICB) therapy has brought survival benefits to patients with specific cancer types, most of cancer patients remain refractory to the ICB therapy, which is largely attributed to the immunosuppressive tumor microenvironment. Thereby, it is urgent to profile key molecules and signal pathways responsible for modification of tumor microenvironment. METHODS: Multiple databases of esophageal squamous cell carcinoma (ESCC) were integratively analyzed to screen candidate genes responsible for infiltration of CD8+ T cells. Expression of pescadillo ribosomal biogenesis factor 1 (PES1) in clinical ESCC samples was examined by qRT-PCR, western blotting, and immunohistochemistry. The mechanisms of PES1 were investigated via RNA sequencing and mass spectrometry followed by immunoprecipitation and proximity ligation assay. The clinical and therapeutic significance of PES1 in ESCC was comprehensively investigated using ESCC cells and mouse model. RESULTS: PES1 was significantly upregulated and correlated with poor prognosis in ESCC patients. PES1 knockdown decreased ESCC cell growth in vitro and in vivo and enhanced the efficacy of ICB therapy in mouse model, which was established through subcutaneous inoculation with ESCC cells. Analyses on RNA sequencing and mass spectrometry suggested that PES1 expression was negatively correlated with IL15 and ILF3 was one of the PES1-associated proteins. It has been known that ILF3 interacts with and stabilizes IL15 mRNA to increase IL15 protein level. Our data further indicated that PES1 interfered with the interaction between ILF3 and IL15 mRNA and impaired ILF3-mediated stabilization of IL15 mRNA, which eventually reduced the protein level of IL15. Interestingly, the inhibitory effect of ICB therapy boosted by PES1 knockdown dramatically antagonized by knockdown of IL15, which suppressed the tumor-infiltrated CD8+ T cells in ESCC. Finally, we confirmed the relationships among PES1, IL15, and CD8+ T cell infiltration in 10 locally advanced ESCC patients receiving ICB neoadjuvant therapy and demonstrated that ICB therapy would be more effective in those with low expression of PES1. CONCLUSIONS: Altogether, our findings herein provided novel insights on biological function and clinical significance of PES1 and suggested that high expression of PES1 could suppress ILF3-IL15 axis-mediated immunosurveillance and promote resistance to ICB through restraining tumor-infiltrated CD8+ T cells.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Animales , Ratones , Linfocitos T CD8-positivos , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/terapia , Inmunoterapia , Interleucina-15/farmacología , Interleucina-15/uso terapéutico , Microambiente Tumoral , Proteínas de Unión al ARN/metabolismo , Proteínas del Factor Nuclear 90/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) is a gastrointestinal tumor with high clinical incidence. Long non-coding RNAs (lncRNAs) play vital roles in modulating the growth and epithelial-mesenchymal transition (EMT) of HCC. However, the underlying mechanism of lncRNA KDM4A antisense RNA 1 (KDM4A-AS1) in HCC remains elusive. In our study, the role of KDM4A-AS1 in HCC was systematically investigated. The levels of KDM4A-AS1, interleukin enhancer-binding factor 3 (ILF3), Aurora kinase A (AURKA), and E2F transcription factor 1 (E2F1) were determined by RT-qPCR or western blot. ChIP and dual luciferase reporter experiments were performed to detect the binding relationship between E2F1 and KDM4A-AS1 promoter sequence. RIP and RNA-pull down confirmed the interaction of ILF3 with KDM4A-AS1/AURKA. Cellular functions were analyzed by MTT, flow cytometry, wound healing and transwell assays. IHC was performed to detect Ki67 in vivo. We found that KDM4A-AS1 was increased in HCC tissues and cells. Elevated KDM4A-AS1 level was correlated to poor prognosis of HCC. Knockdown of KDM4A-AS1 inhibited the proliferation, migration, invasion and EMT of HCC cells. ILF3 bound to KDM4A-AS1 and AURKA. KDM4A-AS1 maintained the stability of AURKA mRNA by recruiting ILF3. E2F1 transcriptionally activated KDM4A-AS1. Overexpressed KDM4A-AS1 reversed the contribution of E2F1 depletion to AURKA expression and EMT in HCC cells. KDM4A-AS1 promoted tumor formation in vivo through the PI3K/AKT pathway. These results revealed that E2F1 transcriptionally activated KDM4A-AS1 to regulate HCC progression via the PI3K/AKT pathway. E2F1 and KDM4A-AS1 may serve as good prognostic targets for HCC treatment.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , ARN Largo no Codificante , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba , ARN Mensajero , Transición Epitelial-Mesenquimal/genética , Fosfatidilinositol 3-Quinasas/genética , Proliferación Celular/genética , MicroARNs/genética , Línea Celular Tumoral , ARN Largo no Codificante/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismoRESUMEN
The regulatory role of circRNAs in cancer metastasis has become a focused issue in recent years. To date, however, the discovery of novel functional circRNAs and their regulatory mechanisms via binding with RBPs in bladder cancer (BC) are still lacking. Here, we screened out circSLC38A1 based on our sequencing data and followed validation with clinical tissue samples and cell lines. Functional assays showed that circSLC38A1 promoted BC cell invasion in vitro and lung metastasis of mice in vivo. By conducting RNA pull-down, mass spectrum, and RIP assays, circSLC38A1 was found to interact with Interleukin enhancer-binding factor 3 (ILF3), and stabilize ILF3 protein via modulating the ubiquitination process. By integrating our CUT&Tag-seq and RNA-seq data, TGF-ß2 was identified as the functional target of the circSLC38A1-ILF3 complex. In addition, m6A methylation was enriched in circSLC38A1 and contributed to its upregulation. Clinically, circSLC38A1 was identified in serum exosomes of BC patients and could distinguish BC patients from healthy individuals with a diagnostic accuracy of 0.878. Thus, our study revealed an essential role and clinical significance of circSLC38A1 in BC via activating the transcription of TGF-ß2 in an ILF3-dependent manner, extending the understanding of the importance of circRNA-mediated transcriptional regulation in BC metastasis.
Asunto(s)
Proteínas del Factor Nuclear 90 , ARN Circular , Factor de Crecimiento Transformador beta2 , Neoplasias de la Vejiga Urinaria , Animales , Ratones , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Oncogenes , ARN Circular/genética , Factor de Crecimiento Transformador beta2/metabolismo , Neoplasias de la Vejiga Urinaria/patología , HumanosRESUMEN
SIGNIFICANCE: Secretion of TNFα by tumor-associated macrophages stimulates cancer cells to upregulate lncRNA MALR, which induces ILF3 liquid-liquid phase separation and activation of HIF1α signaling to promote cancer progression.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , ARN Largo no Codificante , Humanos , Carcinoma de Células Escamosas de Esófago/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Neoplasias Esofágicas/genética , Macrófagos , Regulación Neoplásica de la Expresión Génica , Proliferación Celular , Proteínas del Factor Nuclear 90/genéticaRESUMEN
Interleukin enhancer-binding factor 3 (ILF3) as an RNA-binding protein that plays a critical role in the process of cancer and antiviral responses. However, no researcher has focused on the pan-cancer analysis of ILF3, and the effect of ILF3 on tumor immunity is still largely unclear. This study synthetically analyzed the relationship between the expression of ILF3 across various cancers and prognosis, microsatellite instability (MSI), tumor mutational burden (TMB), tumor immune cell infiltration, and common immune checkpoint molecules by multiple bioinformatics databases. Experimentally, we detected the mRNA abundance of ILF3 and immune checkpoint molecules in liver hepatocellular carcinoma (LIHC) tissues. The functions of ILF3 on hepatocellular carcinoma (HCC) cells were verified by western blot assay and cytotoxicity assay. We found that ILF3 was aberrantly expressed and associated with the prognosis in several types of tumors. The ILF3 expression was significantly correlated with infiltrating levels of immune cells and immune molecules in certain cancers, particularly in LIHC. Detection of clinical liver cancer tissues confirmed the positive correlation between ILF3 and immune checkpoint molecules, including programmed cell death 1 (PD-1), programmed cell death ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), and T cell immunoglobulin domain and mucin domain-3 (TIM3). Furthermore, reduced PD-L1 and increased sensitivity of HCC cells to T cells cytotoxicity were found in ILF3-knockdown cells. This work suggested ILF3 may be used as a prognostic marker for various tumors to predict the response to immunotherapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas del Factor Nuclear 90 , Humanos , Antígeno B7-H1/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/tratamiento farmacológico , Proteínas de Punto de Control Inmunitario/metabolismo , Inmunoterapia , Interleucinas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , Proteínas del Factor Nuclear 90/genética , PronósticoRESUMEN
BACKGROUND: The biological function of lncRNA ELF3-AS1 remains largely unknown in cancers. The cause of SNAI2 overexpression in tumor metastasis remains largely unclear. The molecular mechanisms underlying the high co-expression of antisense lncRNAs and adjacent protein-coding genes remains unclear. METHODS: RNA-seq, CHIP and dual-luciferase reporter assay were performed to identify lncRNAs regulated by SNAI2. MicroRNA-seq and RNA-seq studies were conducted to reveal the biological function of ELF3-AS1 in GC. RNA pulldown and CHIRP assays were conducted to identify the protein that interacts with ELF3-AS1. RESULTS: A total of 123 lncRNAs were identified to be regulated by SNAI2 in GC by RNA sequencing. The ELF3 gene and antisense lncRNA ELF3-AS1 were both transcriptionally repressed by SNAI2 or SNAI1. Down-regulation of ELF3-AS1 and ELF3 predicted poor prognosis in GC. Nuclear localized lncRNA ELF3-AS1 negatively regulated GC cell cycle progression via suppressing G1/S transition and histone synthesis. ELF3-AS1 mainly inhibited GC metastasis by repressing SNAI2 signaling. Additionally, ELF3-AS1 modulated ELF3 mRNA stability by RNA-RNA interaction. The RNA duplexes formed by ELF3 mRNA and lncRNA ELF3-AS1 directly interacted with the double-stranded RNA (dsRNA) binding protein complex ILF2/ILF3 (NF45/NF90). In turn, the ILF2/ILF3 complex dynamically regulated the expression of ELF3-AS1 and ELF3 by affecting the dsRNA stability. CONCLUSIONS: The SNAI2-ELF3-AS1 feedback loop regulates ELF3 expression at transcriptional and post-transcriptional levels and drives gastric cancer metastasis by maintaining SNAI2 overexpression. The ILF2/ILF3 complex plays a critical role in regulating dsRNA stability. In addition, our work provides a direct evidence that head-to-head antisense lncRNAs can share promoters with neighboring coding genes, which make their expression subject to similar transcriptional regulation, leading to high co-expression.
Asunto(s)
ARN Largo no Codificante , Neoplasias Gástricas , Humanos , ARN Largo no Codificante/genética , Neoplasias Gástricas/genética , Retroalimentación , Estabilidad del ARN , Regulación hacia Abajo , ARN Bicatenario , Proteínas de Unión al ADN , Factores de Transcripción/genética , Proteínas Proto-Oncogénicas c-ets , Proteínas del Factor Nuclear 90/genética , Proteína del Factor Nuclear 45RESUMEN
Double-stranded RNA-binding proteins (dsRBPs) are major players in the regulation of gene expression patterns. Among them, Nuclear Factor 90 (NF90) has a plethora of well-known functions in viral infection, transcription, and translation as well as RNA stability and degradation. In addition, NF90 has been identified as a regulator of microRNA (miRNA) maturation by competing with Microprocessor for the binding of pri-miRNAs in the nucleus. NF90 was recently shown to control the biogenesis of a subset of human miRNAs, which ultimately influences, not only the abundance, but also the expression of the host gene and the fate of the mRNA target repertoire. Moreover, recent evidence suggests that NF90 is also involved in RNA-Induced Silencing Complex (RISC)-mediated silencing by binding to target mRNAs and controlling their translation and degradation. Here, we review the many, and growing, functions of NF90 in RNA biology, with a focus on the miRNA pathway and RISC-mediated gene silencing.
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MicroARNs , Proteínas del Factor Nuclear 90 , Humanos , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , MicroARNs/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estabilidad del ARN , BiologíaRESUMEN
The regulatory network between arsenic, genes and signaling pathways has been reported in arsenic carcinogenesis. Studies on circRNA represent a growing field, but the extent to circRNA potential mechanisms remains poorly understood. So this study we explore the systematic function of hsa_circ_0005050 in mediating the cell apoptosis and proliferation. We demonstrated that hsa_circ_0005050 was highly expressed in subjects who are long-term exposed to arsenic, and could be induced by NaAs2O3 in A549 and 16HBE. Knockdown of hsa_circ_0005050 promotes A549 cell viability, whereas exerts the opposite effects in 16HBE. Mechanistically, hsa_circ_0005050 regulates the p53 and NF-κB signaling pathway involved in the apoptosis and proliferation. And we found that hsa_circ_0005050 could directly bind to the RNA binding protein ILF3 and mutually influence each other's formation. Upon si-hsa_circ_0005050, ILF3 export to the cytoplasm resulting the formation of a ternary complex ILF3-p65-IκBA, breaks the balance of p53 and NF-κB pathway and induces A549 apoptosis and leads to 16HBE proliferation. As a result of these investigations, suggestions were identified for future research.
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Arsénico , Humanos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , FN-kappa B/metabolismo , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , ARN Circular/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Carcinogénesis/inducido químicamente , Intoxicación por ArsénicoRESUMEN
Hypertension is characterized by sympathetic hyperactivity, which is related to the overexcitation of the presympathetic neurons in the rostral ventrolateral medulla (RVLM). Nitric oxide (NO) has been reported to be a vital neuromodulator involved in central cardiovascular regulation. However, the mechanism of interleukin-enhanced binding factor 3 (ILF3) participating in blood pressure (BP) regulation is still unclear. Therefore, this study aims to clarify the role of ILF3 within the rostral ventrolateral medulla (RVLM) in regulating NO in hypertension. It was found that the expression level of ILF3 was significantly increased in the RVLM of spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats through microarray gene expression analysis, Western blot, and immunofluorescence. Overexpression of ILF3 by injecting constructed adenovirus into the RVLM increased the BP and renal sympathetic nerve activity (RSNA) of the WKY rats, significantly decreasing NO production and neuronal nitric oxide synthase (nNOS) expression. Knockdown of ILF3 in the RVLM of SHR significantly reduced BP but increased NO production and the neuronal nitric oxide synthase (nNOS) expression. Furthermore, it was found that the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway was activated via Western blotting in the RVLM after overexpression of ILF3, whereas it was attenuated after knockdown of ILF3 in SHR. In addition, inhibition of PI3K by intracisternal infusion of the PI-103 attenuated the increase in Akt phosphorylation and decrease in nNOS expression and NO production caused by overexpressing ILF3, which ultimately blunted high BP induced by overexpressing ILF3. Taken together, this current study suggests that ILF3 participates in high BP via reducing NO production in the RVLM through PI3K/Akt pathway.
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Hipertensión , Proteínas Proto-Oncogénicas c-akt , Ratas , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ratas Endogámicas WKY , Fosfatidilinositol 3-Quinasa/metabolismo , Bulbo Raquídeo/metabolismo , Presión Sanguínea , Ratas Endogámicas SHR , Interleucinas/metabolismo , Proteínas del Factor Nuclear 90/metabolismoRESUMEN
BACKGROUND: Nuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity. RESULTS: Here, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3' UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased. CONCLUSIONS: These findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.