RESUMEN
BACKGROUND: The role of ELAVL1 in the progression of various tumors has been demonstrated. Our research aims to investigate how ELAVL1 controls the glycolytic process in nasopharyngeal carcinoma cells through the HMGB3/ß-catenin pathway. METHODS: The expression of ELAVL1 was detected in clinical tumor samples and nasopharyngeal carcinoma cell lines. A subcutaneous tumor model was established in nude mice to investigate the role of ELAVL1 in tumor progression. The relationship between HMGB3 and ELAVL1 was validated by RNA pull down and RIP assays. TOPFlash/FOPFlash reporter assay was used to detect ß-catenin activity. Assay kits were utilized to measure glucose consumption, lactate production, and G6PD activity in nasopharyngeal carcinoma cells. Western blot was conducted to detect the expression of glycolysis-related proteins. The glycolytic capacity was analyzed through extracellular acidification rate (ECAR). RESULTS: In both clinical samples and nasopharyngeal carcinoma cell lines, the expression levels of ELAVL1 mRNA and protein were found to be upregulated. Knockdown of ELAVL1 significantly inhibited the in vivo proliferation of nasopharyngeal carcinoma and suppressed the glycolytic capacity of nasopharyngeal carcinoma cells. ELAVL1 interacts with HMGB3, leading to an increase in the stability of HMGB3 mRNA. Overexpression of HMGB3 elevated the reduced ß-catenin activity caused by sh-ELAVL1 and reversed the inhibitory effect of sh-ELAVL1 on cellular glycolytic capacity. Treatment with ß-catenin inhibitor (FH535) effectively suppressed the promotion of glycolytic capacity induced by HMGB3 overexpression. CONCLUSIONS: ELAVL1 promotes glycolysis in nasopharyngeal carcinoma cells by interacting with HMGB3 to stabilize HMGB3 mRNA, thereby activating ß-catenin pathway. Therefore, targeting the ELAVL1-HMGB3-ß-catenin axis has the potential to be a novel approach for treating nasopharyngeal carcinoma.
Asunto(s)
Proteína 1 Similar a ELAV , Glucólisis , Proteína HMGB3 , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , beta Catenina , Animales , Femenino , Humanos , Ratones , beta Catenina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Regulación Neoplásica de la Expresión Génica , Proteína HMGB3/metabolismo , Proteína HMGB3/genética , Ratones Desnudos , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patología , Transducción de SeñalRESUMEN
BACKGROUND: Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. METHODS: Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. RESULTS: In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. CONCLUSION: Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.
Asunto(s)
Movimiento Celular , Proliferación Celular , Proteína 1 Similar a ELAV , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ubiquitina Tiolesterasa , Humanos , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Células Cultivadas , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Vasos Retinianos/patología , Vasos Retinianos/metabolismo , AngiogénesisRESUMEN
Prostate cancer (PCa) currently ranks second in male tumor mortality. Targeting immune checkpoint in tumor as immunotherapy is a new direction for tumor treatment. However, targeting PD-1/PD-L1 and CTLA4 to treat PCa has poor immunotherapeutic efficacy because PCa is known as a cold tumor. Understanding the mechanism of immunosuppression in PCa can promote the use of immunotherapy to treat PCa. ELAVL1 is highly expressed in many tumors, participates in almost all tumor biological activities and is an oncogene. ELAVL1 is also involved in the development and differentiation of T and B lymphocytes. However, the relationship between ELAVL1 and tumor immunity has not yet been reported. In recent years, ELAVL1 has been shown to regulate downstream targets in an m6A -dependent manner. PD-L1 has been shown to have m6A sites in multiple tumors that are regulated by m6A. In this study, ELAVL1 was highly expressed in PCa, and PCa with high ELAVL1 expression is immunosuppressive. Knocking down ELAVL1 reduced PD-L1 expression in PCa. Moreover, PD-L1 was shown to have an m6A site, and its m6A level was upregulated in PCa. ELAVL1 interacts with PD-L1 mRNA and promotes PD-L1 RNA stability via m6A, ultimately inhibiting the infiltration of CD4-positive T cells. In addition, androgen receptor (AR) was shown to be regulated with ELAVL1, and knocking down AR could also affect the expression of PD-L1. Therefore, ELAVL1 can directly or indirectly regulate the expression of PD-L1, thereby affecting the infiltration of CD4-positive T cells in PCa and ultimately leading to immune suppression.
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Antígeno B7-H1 , Linfocitos T CD4-Positivos , Proteína 1 Similar a ELAV , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Próstata , Estabilidad del ARN , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/inmunología , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Línea Celular Tumoral , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , ARN Mensajero/genéticaRESUMEN
Hu antigen R (HuR) plays a key role in regulating genes critical to the pathogenesis of diabetic nephropathy (DN). This study investigates the therapeutic potential of niclosamide (NCS) as an HuR inhibitor in DN. Uninephrectomized mice were assigned to four groups: normal control; untreated db/db mice terminated at 14 and 22 weeks, respectively; and db/db mice treated with NCS (20 mg/kg daily via i.p.) from weeks 18 to 22. Increased HuR expression was observed in diabetic kidneys from db/db mice, which was mitigated by NCS treatment. Untreated db/db mice exhibited obesity, progressive hyperglycemia, albuminuria, kidney hypertrophy and glomerular mesangial matrix expansion, increased renal production of fibronectin and a-smooth muscle actin, and decreased glomerular WT-1+-podocytes and nephrin expression. NCS treatment did not affect mouse body weight, but reduced blood glucose and HbA1c levels and halted the DN progression observed in untreated db/db mice. Renal production of inflammatory and oxidative stress markers (NF-κBp65, TNF-a, MCP-1) and urine MDA levels increased during disease progression in db/db mice but were halted by NCS treatment. Additionally, the Wnt1-signaling-pathway downstream factor, Wisp1, was identified as a key downstream mediator of HuR-dependent action and found to be markedly increased in db/db mouse kidneys, which was normalized by NCS treatment. These findings suggest that inhibition of HuR with NCS is therapeutic for DN by improving hyperglycemia, renal inflammation, and oxidative stress. The reduction in renal Wisp1 expression also contributes to its renoprotective effects. This study supports the potential of repurposing HuR inhibitors as a novel therapy for DN.
Asunto(s)
Nefropatías Diabéticas , Reposicionamiento de Medicamentos , Proteína 1 Similar a ELAV , Niclosamida , Animales , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Ratones , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Masculino , Niclosamida/farmacología , Niclosamida/uso terapéutico , Riñón/metabolismo , Riñón/patología , Riñón/efectos de los fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Glucemia/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Crimean-Congo Hemorrhagic Fever virus (CCHFV) is a tick-borne pathogen that causes severe acute fever disease in humans and requires a biosafety level 4 laboratory for handling. Hazara virus (HAZV), belonging to the same virus genus as CCHFV, does not exhibit pathogenesis in humans. To investigate host RNA-binding proteins (RBPs) that regulate CCHFV replication, we generated a series of mutant RAW264.7 cells by CRISPR/Cas9 system and these cells were infected with HAZV. The viral titers in the supernatant of these cells was investigated, and HuR (ELAVL1) was identified. HuR KO RAW264.7 cells reduced HAZV replication. HuR is an RBP that enhances mRNA stability by binding to adenyl-uridine (AU)-rich regions in their 3' non-coding region (NCR). HuR regulates innate immune response by binding to host mRNAs of signaling molecules. The expression of cytokine genes such as Ifnb, Il6, and Tnf was reduced in HuR KO cells after HAZV infection. Although HuR supports the innate immune response during HAZV infection, we found that innate immune activation by HAZV infection did not affect its replication. We then investigated whether HuR regulates HAZV genome RNA stability. HAZV RNA genome was precipitated with an anti-HuR antibody, and HAZV genome RNA stability was lowered in HuR KO cells. We found that HuR associated with HAZV RNA and stabilized it to enhance HAZV replication. Furthermore, HuR-deficiency reduced CCHFV minigenome replication. CCHFV is a negative-strand RNA virus and positive-strand RNA is produced during replication. HuR was associated with positive-strand RNA rather than negative-strand RNA, and AU-rich region in 3'-NCR of S segment was responsible for immunoprecipitation with anti-HuR antibody and minigenome replication. Additionally, HuR inhibitor treatment reduced CCHFV minigenome replication. Our results indicate that HuR aids replication of the CCHFV minigenome by associating with the AU-rich region in the 3'-NCR.
Asunto(s)
Proteína 1 Similar a ELAV , Genoma Viral , Virus de la Fiebre Hemorrágica de Crimea-Congo , ARN Viral , Replicación Viral , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Animales , Ratones , ARN Viral/genética , ARN Viral/metabolismo , Células RAW 264.7 , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Humanos , Sistemas CRISPR-CasRESUMEN
Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here, we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium.NEW & NOTEWORTHY Our results indicate that intestinal epithelial tissue-specific transgenic miR-195 expression decreases the levels of SOX9 expression, along with reduced numbers of Paneth cells. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restores Paneth cell development ex vivo. miR-195 inhibits SOX9 translation by preventing binding of HuR to Sox9 mRNA. These findings suggest that interaction between miR-195 and HuR controls Paneth cell function via SOX9 in the intestinal epithelium.
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Proteína 1 Similar a ELAV , Mucosa Intestinal , MicroARNs , Células de Paneth , Factor de Transcripción SOX9 , Animales , MicroARNs/genética , MicroARNs/metabolismo , Células de Paneth/metabolismo , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genética , Mucosa Intestinal/metabolismo , Ratones , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Ratones Transgénicos , Humanos , Organoides/metabolismo , Biosíntesis de Proteínas , Ratones Endogámicos C57BLRESUMEN
Age-related macular degeneration (AMD) is a common retinal pathology characterized by degeneration of macula's retinal pigment epithelium (RPE) and photoreceptors, visual impairment, or loss. Compared to wet AMD, dry AMD is more common, but lacks cures; therefore, identification of new potential therapeutic targets and treatments is urgent. Increased oxidative stress and declining antioxidant, detoxifying systems contribute to the pathophysiologic mechanisms underlying AMD. The present work shows that the Embryonic Lethal Abnormal Vision-Like 1/Human antigen R (ELAVL1/HuR) and the Vascular Endothelial Growth Factor (VEGF) protein levels are higher in the RPE of both dry and wet AMD patients compared to healthy subjects. Moreover, increased HuR protein levels are detected in the retina, and especially in the RPE layer, of a dry AMD model, the nuclear factor erythroid 2-related factor 2 (Nrf2) / peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) double knock-out mouse. The crosstalk among Nrf2, HuR and VEGF has been also studied in ARPE-19 cells in basal and stressful conditions related to the AMD context (i.e., oxidative stress, autophagy impairment, Nrf2 deficit), offering new evidence of the mutual influence between Nrf2 and HuR, of the dependence of VEGF expression and secretion by these two factors, and of the increased susceptibility of cells to stressful conditions in Nrf2- or HuR-impaired contexts. Overall, this study shows evidence of the interplay among Nrf2, HuR and VEGF, essential factors for RPE homeostasis, and represents an additional piece in the understanding of the complex pathophysiologic mechanisms underlying AMD.
Asunto(s)
Proteína 1 Similar a ELAV , Factor 2 Relacionado con NF-E2 , Epitelio Pigmentado de la Retina , Factor A de Crecimiento Endotelial Vascular , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Atrofia Geográfica/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/fisiopatología , Degeneración Macular/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Degeneración Macular Húmeda/metabolismo , Degeneración Macular Húmeda/genéticaRESUMEN
Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)-an mRNA-binding protein-is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to AREVegf-a. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR. Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.
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Proteína 1 Similar a ELAV , Naftoquinonas , Factor A de Crecimiento Endotelial Vascular , Humanos , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Naftoquinonas/farmacología , Animales , Ratones , Línea Celular Tumoral , Ratones Desnudos , Glioma/metabolismo , Glioma/tratamiento farmacológico , Glioma/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: An increasing number of studies have demonstrated the association of circular RNAs (circRNAs) with the pathological processes of various diseases and their involvement in the onset and progression of multiple cancers. Nevertheless, the functional roles and underlying mechanisms of circRNAs in the autophagy regulation of gastric cancer (GC) have not been fully elucidated. METHODS: We used transmission electron microscopy and the mRFP-GFP-LC3 dual fluorescent autophagy indicator to investigate autophagy regulation. The cell counting kit-8 assay, colony formation assay, 5-ethynyl-2'-deoxyuridine incorporation assay, Transwell assay, and Western blot assay were conducted to confirm circPTPN22's influence on GC progression. Dual luciferase reporter assays validated the binding between circPTPN22 and miR-6788-5p, as well as miR-6788-5p and p21-activated kinase-1 (PAK1). Functional rescue experiments assessed whether circPTPN22 modulates PAK1 expression by competitively binding miR-6788-5p, affecting autophagy and other biological processes in GC cells. We investigated the impact of circPTPN22 on in vivo GC tumors using a nude mouse xenograft model. Bioinformatics tools predicted upstream regulatory transcription factors and binding proteins of circPTPN22, while chromatin immunoprecipitation and ribonucleoprotein immunoprecipitation assays confirmed the binding status. RESULTS: Upregulation of circPTPN22 in GC has been shown to inhibit autophagy and promote cell proliferation, migration, and invasion. Mechanistically, circPTPN22 directly binds to miR-6788-5p, subsequently regulating the expression of PAK1, which activates protein kinase B (Akt) and extracellular signal-regulated kinase (Erk) phosphorylation. This modulation ultimately affects autophagy levels in GC cells. Additionally, runt-related transcription factor 1 (RUNX1) negatively regulates circPTPN22 expression, while RNA-binding proteins such as FUS (fused in sarcoma) and ELAVL1 (recombinant ELAV-like protein 1) positively regulate its expression. Inhibition of the autophagy pathway can increase FUS expression, further upregulating circPTPN22 in GC cells, thereby exacerbating the progression of GC. CONCLUSION: Under the regulation of the transcription factor RUNX1 and RNA-binding proteins FUS and ELAVL1, circPTPN22 activates the phosphorylation of Akt and Erk through the miR-6788-5p/PAK1 axis, thereby modulating autophagy in GC cells. Inhibition of autophagy increases FUS, which in turn upregulates circPTPN22, forming a positive feedback loop that ultimately accelerates the progression of GC.
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Autofagia , Movimiento Celular , Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteína 1 Similar a ELAV , MicroARNs , ARN Circular , Proteína FUS de Unión a ARN , Neoplasias Gástricas , Quinasas p21 Activadas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Neoplasias Gástricas/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Autofagia/genética , MicroARNs/genética , MicroARNs/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Proliferación Celular/genética , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Movimiento Celular/genética , Línea Celular Tumoral , Animales , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Ratones , Invasividad Neoplásica , Ratones Endogámicos BALB CRESUMEN
Glutaminase (GLS) is directly related to cell growth and tumor progression, making it a target for cancer treatment. The RNA-binding protein HuR (encoded by the ELAVL1 gene) influences mRNA stability and alternative splicing. Overexpression of ELAVL1 is common in several cancers, including breast cancer. Here we show that HuR regulates GLS mRNA alternative splicing and isoform translation/stability in breast cancer. Elevated ELAVL1 expression correlates with high levels of the glutaminase isoforms C (GAC) and kidney-type (KGA), which are associated with poor patient prognosis. Knocking down ELAVL1 reduces KGA and increases GAC levels, enhances glutamine anaplerosis into the TCA cycle, and drives cells towards glutamine dependence. Furthermore, we show that combining chemical inhibition of GLS with ELAVL1 silencing synergistically decreases breast cancer cell growth and invasion. These findings suggest that dual inhibition of GLS and HuR offers a therapeutic strategy for breast cancer treatment.
Asunto(s)
Neoplasias de la Mama , Proteína 1 Similar a ELAV , Glutaminasa , Glutaminasa/metabolismo , Glutaminasa/genética , Glutaminasa/antagonistas & inhibidores , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , ARN Mensajero/metabolismo , ARN Mensajero/genética , Regulación Neoplásica de la Expresión Génica , Empalme Alternativo , Proliferación Celular , Glutamina/metabolismo , Estabilidad del ARNRESUMEN
BACKGROUND: Endometrial cancer (EC) is the sixth most frequent cancer in women worldwide and has higher fatality rates. The pathophysiology of EC is complex, and there are currently no reliable methods for diagnosing and treating the condition. Long non-coding RNA (lncRNA), according to mounting evidence, is vital to the pathophysiology of EC. HOTAIR is regarded as a significant prognostic indicator of EC. ZBTB7A decreased EC proliferation and migration, according to recent studies, however the underlying mechanism still needs to be clarified. METHODS: The research utilized RT-qPCR to measure HOTAIR expression in clinical EC tissues and various EC cell lines. Kaplan-Meier survival analysis was employed to correlate HOTAIR levels with patient prognosis. Additionally, the study examined the interaction between ZBTB7A and HOTAIR using bioinformatics tools and ChIP assays. The experimental approach also involved manipulating the expression levels of HOTAIR and ZBTB7A in EC cell lines and assessing the impact on various cellular processes and gene expression. RESULTS: The study found significantly higher levels of HOTAIR in EC tissues compared to adjacent normal tissues, with high HOTAIR expression correlating with poorer survival rates and advanced cancer characteristics. EC cell lines like HEC-1 A and KLE showed higher HOTAIR levels compared to normal cells. Knockdown of HOTAIR in these cell lines reduced proliferation, angiogenesis, and migration. ZBTB7A was found to be inversely correlated with HOTAIR, and its overexpression led to a decrease in HOTAIR levels and a reduction in malignant cell behaviors. The study also uncovered that HOTAIR interacts with ELAVL1 to regulate SOX17, which in turn activates the Wnt/ß-catenin pathway, promoting malignant behaviors in EC cells. CONCLUSION: HOTAIR is a critical regulator in EC, contributing to tumor growth and poor prognosis. Its interaction with ZBTB7A and regulation of SOX17 via the Wnt/ß-catenin pathway underlines its potential as a therapeutic target.
Asunto(s)
Proliferación Celular , Proteína 1 Similar a ELAV , Neoplasias Endometriales , ARN Largo no Codificante , Factores de Transcripción SOXF , Humanos , ARN Largo no Codificante/genética , Femenino , Neoplasias Endometriales/genética , Neoplasias Endometriales/patología , Neoplasias Endometriales/metabolismo , Factores de Transcripción SOXF/genética , Factores de Transcripción SOXF/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Pronóstico , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Movimiento Celular/genética , Animales , Ratones , Persona de Mediana Edad , Vía de Señalización Wnt/genética , AngiogénesisRESUMEN
Human antigen R (HuR) is an RNA binding protein mainly involved in maintaining the stability and controlling the translation of mRNAs, critical for immune response, cell survival, proliferation and apoptosis. Although HuR is a nuclear protein, its mRNA translational-related function occurs at the cytoplasm, where the oligomeric form of HuR is more abundant. However, the regulation of nucleo-cytoplasmic transport of HuR and its connection with protein oligomerization remain unclear. In this work, we describe the phosphorylation of Tyr5 as a new hallmark for HuR activation. Our biophysical, structural and computational assays using phosphorylated and phosphomimetic HuR proteins demonstrate that phosphorylation of Tyr5 at the disordered N-end stretch induces global changes on HuR dynamics and conformation, modifying the solvent accessible surface of the HuR nucleo-cytoplasmic shuttling (HNS) sequence and releasing regions implicated in HuR dimerization. These findings explain the preferential cytoplasmic accumulation of phosphorylated HuR in HeLa cells, aiding to comprehend the mechanisms underlying HuR nucleus-cytoplasm shuttling and its later dimerization, both of which are relevant in HuR-related pathogenesis.
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Citoplasma , Proteína 1 Similar a ELAV , Multimerización de Proteína , Humanos , Citoplasma/metabolismo , Fosforilación , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Células HeLa , Núcleo Celular/metabolismoRESUMEN
Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.
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Citoplasma , Proteína 1 Similar a ELAV , Inflamación , Poli(ADP-Ribosa) Polimerasa-1 , ARN Mensajero , Proteínas Quinasas p38 Activadas por Mitógenos , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Humanos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Citoplasma/metabolismo , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , ARN Mensajero/metabolismo , ARN Mensajero/genética , Fosforilación , Regulación de la Expresión Génica , Animales , Poli ADP Ribosilación/genética , Células HEK293 , Núcleo Celular/metabolismo , RatonesRESUMEN
Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the underlying mechanisms remain to be further studied. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3' UTR of Dgat2 mRNA and intron 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3' UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat-diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.
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Dieta Alta en Grasa , Proteína 1 Similar a ELAV , Absorción Intestinal , Triglicéridos , Animales , Humanos , Ratones , Regiones no Traducidas 3'/genética , Aciltransferasas , Diacilglicerol O-Acetiltransferasa/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/metabolismo , Grasas de la Dieta/farmacología , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/metabolismo , Obesidad/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Triglicéridos/metabolismo , Triglicéridos/biosíntesisRESUMEN
MicroRNAs (miRNAs) can positively regulate gene expression through an unconventional RNA activation mechanism involving direct targeting 3' untranslated regions (UTRs). Our prior study found miR-93-5p activates mitogen-activated protein kinase kinase kinase 2 (MAP3K2) in hepatocellular carcinoma (HCC) via its 3'UTR. However, the underlying mechanism remains elusive. Here, we identified two candidate AU-rich element (ARE) motifs (ARE1 and ARE2) adjacent to the miR-93-5p binding site located within the MAP3K2 3'UTR using AREsite2. Luciferase reporter and translation assays validated that only ARE2 participated in MAP3K2 activation. Integrative analysis revealed that human antigen R (HuR), an ARE2-associated RNA-binding protein (RBP), physically and functionally interacted with the MAP3K2 3'UTR. Consequently, an HuR-ARE2 complex was shown to facilitate miR-93-5p-mediated upregulation of MAP3K2 expression. Furthermore, bioinformatics analysis and studies of HCC cells and specimens highlighted an oncogenic role for HuR and positive HuR-MAP3K2 expression correlation. HuR is also an enhancing factor in the positive feedback circuit comprising miR-93-5p, MAP3K2, and c-Jun demonstrated in our prior study. The newly identified HuR-ARE2 involvement enriches the mechanism of miR-93-5p-driven MAP3K2 activation and suggests new therapeutic strategies warranted for exploration in HCC.
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Regiones no Traducidas 3' , Carcinoma Hepatocelular , Proteína 1 Similar a ELAV , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , MAP Quinasa Quinasa Quinasa 2 , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Regiones no Traducidas 3'/genética , MAP Quinasa Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa Quinasa 2/genética , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Línea Celular Tumoral , Biosíntesis de ProteínasRESUMEN
Circular RNAs (circRNAs) have been discovered to serve as vital regulators in atherosclerosis (AS). However, the role and mechanism of circ_0002331 in AS process are still unclear. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL to establish an in vitro model for AS. The expression levels of circ_0002331, Cyclin D2 (CCND2) and ELAVL1 were analyzed by quantitative real-time PCR. Cell proliferation, apoptosis, migration, invasion and angiogenesis were assessed by EdU assay, flow cytometry, transwell assay and tube formation assay. The protein levels of CCND2, ELAVL1, and autophagy-related markers were detected using western blot analysis. IL-8 level was analyzed by ELISA. The relationship between ELAVL1 and circ_0002331 or CCND2 was analyzed by RIP assay and RNA pull-down assay. Moreover, FISH assay was used to analyze the co-localization of ELAVL1 and CCND2 in HUVECs. Our data showed that circ_0002331 was obviously downregulated in AS patients and ox-LDL-induced HUVECs. Overexpression of circ_0002331 could promote proliferation, migration, invasion and angiogenesis, while inhibit apoptosis, autophagy and inflammation in ox-LDL-induced HUVECs. Furthermore, CCND2 was positively regulated by circ_0002331, and circ_0002331 could bind with ELAVL1 to promote CCND2 mRNA stability. Besides, CCND2 overexpression suppressed ox-LDL-induced HUVECs dysfunction, and its knockdown also reversed the regulation of circ_0002331 on ox-LDL-induced HUVECs dysfunction. In conclusion, circ_0002331 might be a potential target for AS treatment, which could improve ox-LDL-induced dysfunction of HUVECs via regulating CCND2 by binding with ELAVL1.
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Apoptosis , Aterosclerosis , Movimiento Celular , Proliferación Celular , Ciclina D2 , Proteína 1 Similar a ELAV , Células Endoteliales de la Vena Umbilical Humana , Lipoproteínas LDL , Estabilidad del ARN , ARN Circular , ARN Mensajero , Transducción de Señal , Humanos , ARN Circular/metabolismo , ARN Circular/genética , Lipoproteínas LDL/toxicidad , Lipoproteínas LDL/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Ciclina D2/metabolismo , Ciclina D2/genética , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Células Cultivadas , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Regulación de la Expresión Génica , Estudios de Casos y Controles , Autofagia , Masculino , Persona de Mediana Edad , FemeninoRESUMEN
BACKGROUND AND AIMS: Long noncoding RNAs (lncRNAs) play important roles in the progression of atherosclerosis. In this study, we identified an uncharacterized lncRNA, Liver Expressions by PSRC1 Induced Specifically (LEPIS). This study aimed to clarify the mechanism though which LEPIS affects atherosclerosis (AS). METHODS: The expression of LEPIS and its potential target, tropomodulin 4 (TMOD4), was increased in the livers of ApoE-/- mice fed a high-fat diet (HFD). An ApoE-/- mouse model in which LEPIS or TMOD4 was overexpressed in the liver was established. The plaque load in the aorta was assessed, plasma was collected to measure blood lipid levels, and the liver was collected to study cholesterol metabolism. RESULTS: We found that both LEPIS and TMOD4 increased the AS burden and reduced hepatic cholesterol levels. A further study revealed that LEPIS and TMOD4 affected the expression of genes related to hepatic cholesterol homeostasis, including proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR), which are closely related to hypercholesterolemia. Mechanistically, human antigen R (HuR), an RNA-binding protein (RBP), was shown to be critical for the regulation of TMOD4 by LEPIS. Furthermore, we found that verexpression of LEPIS promoted the shuttling of HuR from the nucleus to the cytoplasm, enhanced the stability of TMOD4 mRNA, and in turn promoted the expression of TMOD4. In addition, TMOD4 was found to affect intracellular cholesterol levels through PCSK9. CONCLUSIONS: These results suggest that the LEPIS-HuR-TMOD4 axis is a potential intervention target for dysregulated hepatic cholesterol homeostasis and AS and may provide the basis for further reductions in the circulating LDL-C concentration and arterial plaque burden.
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Aterosclerosis , Colesterol , Modelos Animales de Enfermedad , Homeostasis , Hígado , Ratones Noqueados para ApoE , Animales , Humanos , Masculino , Ratones , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Colesterol/metabolismo , Colesterol/sangre , Dieta Alta en Grasa , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Hígado/metabolismo , Ratones Endogámicos C57BL , Placa Aterosclerótica , Proproteína Convertasa 9/metabolismo , Proproteína Convertasa 9/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Rapid eye movement sleep (REMS) maintains brain excitability at least by regulating Na-K ATPase activity. Although REMS deprivation (REMSD)-associated elevated noradrenaline (NA) increases Na-K ATPase protein expression, its mRNA transcription did not increase. We hypothesized and confirmed both in vivo as well as in vitro that elevated mRNA stability explains the apparent puzzle. The mRNA stability was measured in control and REMSD rat brain with or without in vivo treatment with α1-adrenoceptor (AR) antagonist, prazosin (PRZ). Upon REMSD, Na-K ATPase α1-, and α2-mRNA stability increased significantly, which was prevented by PRZ. To decipher the molecular mechanism of action, we estimated NA-induced Na-K ATPase mRNA stability in Neuro-2a cells under controlled conditions and by transcription blockage using Actinomycin D (Act-D). NA increased Na-K ATPase mRNA stability, which was prevented by PRZ and propranolol (PRP, ß-AR antagonist). The knockdown assay confirmed that the increased mRNA stabilization was induced by elevated cytoplasmic abundance of Human antigen R (HuR) and involving (Phospholipase C) PLC-mediated activation of Protein Kinase C (PKC). Additionally, using cell-impermeable Enz-link sulfo NHS-SS-Biotin, we observed that NA increased Na-K ATPase α1-subunits on the Neuro-2a cell surface. We conclude that REMSD-associated elevated NA, acting on α1- and ß-AR, increases nucleocytoplasmic translocation of HuR and increases Na-K ATPase mRNA stability, resulting in increased Na-K ATPase protein expression. The latter then gets translocated to the neuronal membrane surface involving both PKC and (Protein Kinase A) PKA-mediated pathways. These findings may be exploited for the amelioration of REMSD-associated chronic disorders and symptoms.
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Proteína 1 Similar a ELAV , Norepinefrina , Proteína Quinasa C , Estabilidad del ARN , ATPasa Intercambiadora de Sodio-Potasio , Animales , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Ratas , Norepinefrina/metabolismo , Proteína Quinasa C/metabolismo , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Estabilidad del ARN/efectos de los fármacos , Masculino , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Mensajero/biosíntesis , Ratones , Transducción de Señal/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Línea Celular Tumoral , Ratas Wistar , HumanosRESUMEN
Transient receptor potential melastatin 7 (TRPM7) is a cation channel that plays a role in the progression of rheumatoid arthritis (RA), yet its involvement in synovial hyperplasia and inflammation has not been determined. We previously reported that TRPM7 affects the destruction of articular cartilage in RA. Herein, we further confirmed the involvement of TRPM7 in fibroblast-like synoviocyte (FLS) proliferation, metastasis and inflammation. We observed increased TRPM7 expression in FLSs derived from human RA patients. Pharmacological inhibition of TRPM7 protected primary RA-FLSs from proliferation, metastasis and inflammation. Furthermore, we found that TRPM7 contributes to RA-FLS proliferation, metastasis and inflammation by increasing the intracellular Ca2+ concentration. Mechanistically, the PKCα-HuR axis was demonstrated to respond to Ca2+ influx, leading to TRPM7-mediated RA-FLS proliferation, metastasis and inflammation. Moreover, HuR was shown to bind to IL-6 mRNA after nuclear translocation, which could be weakened by TRPM7 channel inhibition. Additionally, adeno-associated virus 9-mediated TRPM7 silencing is highly effective at alleviating synovial hyperplasia and inflammation in adjuvant-induced arthritis rats. In conclusion, our findings unveil a novel regulatory mechanism involved in the pathogenesis of RA and suggest that targeting TRPM7 might be a potential strategy for the prevention and treatment of RA.
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Artritis Experimental , Artritis Reumatoide , Proliferación Celular , Interleucina-6 , Proteína Quinasa C-alfa , Sinoviocitos , Canales Catiónicos TRPM , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/genética , Artritis Reumatoide/patología , Artritis Reumatoide/metabolismo , Animales , Sinoviocitos/metabolismo , Sinoviocitos/patología , Humanos , Interleucina-6/metabolismo , Interleucina-6/genética , Proteína Quinasa C-alfa/metabolismo , Proteína Quinasa C-alfa/genética , Artritis Experimental/patología , Artritis Experimental/metabolismo , Masculino , Ratas , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína 1 Similar a ELAV/metabolismo , Proteína 1 Similar a ELAV/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Células Cultivadas , Inflamación/metabolismo , Inflamación/patología , Ratas Sprague-Dawley , Femenino , Transducción de SeñalRESUMEN
Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1ß-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1ß-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1ß induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1ß-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1ß-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1ß-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1ß. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1ß-induced IL-8 production through this non-transcriptional mechanism.