RESUMEN
CagA, a virulence factor of Helicobacter pylori (H. pylori), is known to drive inflammation in gastric epithelial cells and is typically degraded through autophagy. However, the molecular mechanism by which CagA evades autophagy-mediated degradation remains elusive. This study found that H. pylori inhibits autophagic flux by upregulating the expression of AU-rich element RNA-binding factor 1 (AUF1). We confirmed that AUF1 does not affect autophagy initiation but instead hampers lysosomal clearance, as evidenced by treatments with 3-MA, CQ and BafA1. Upregulated AUF1 stabilizes CagA protein levels by inhibiting the autolysosomal degradation of intracellular CagA in H. pylori-infected gastric epithelial cells. Knocking down AUF1 promotes CagA degradation, an effect that can be reversed by the lysosome inhibitor BafA1 and CQ. Transcriptome analysis of AUF1-knockdown gastric epithelial cells infected with H. pylori indicated that AUF1 regulates the expression of lysosomal-associated hydrolase genes, specifically CTSD, to inhibit autolysosomal degradation. Moreover, we observed that knockdown of AUF1 enhanced the stability of CTSD mRNA and identified AUF1 binding to the 3'UTR region of CTSD mRNA. AUF1-mediated downregulation of CTSD expression contributes to CagA stability, and AUF1 overexpression leads to an increase in CagA levels in exosomes, thus promoting extracellular inflammation. In clinical gastric mucosa, the expression of AUF1 and its cytoplasmic translocation are associated with H. pylori-associated gastritis, with CagA being necessary for the translocation of AUF1 into the cytoplasm. Our findings suggest that AUF1 is a novel host-positive regulator of CagA, and dysregulation of AUF1 expression increases the risk of H. pylori-associated gastritis.
Asunto(s)
Antígenos Bacterianos , Autofagia , Proteínas Bacterianas , Células Epiteliales , Mucosa Gástrica , Infecciones por Helicobacter , Helicobacter pylori , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D , Lisosomas , Antígenos Bacterianos/metabolismo , Antígenos Bacterianos/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Helicobacter pylori/metabolismo , Helicobacter pylori/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Lisosomas/metabolismo , Lisosomas/microbiología , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/metabolismo , Inflamación/metabolismo , Inflamación/microbiología , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Línea CelularRESUMEN
Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.
Asunto(s)
Citocromo P-450 CYP2E1 , Etanol , MicroARNs , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP2E1/genética , MicroARNs/metabolismo , MicroARNs/genética , Etanol/toxicidad , Etanol/efectos adversos , Humanos , Animales , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Intestinos/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologíaRESUMEN
Decorin (DCN), a member of the small leucine-rich proteoglycan gene family, is secreted from stromal fibroblasts with non-cell-autonomous anti-breast-cancer effects. Therefore, in the present study, we sought to elucidate the function of decorin in breast stromal fibroblasts (BSFs). We first showed DCN downregulation in active cancer-associated fibroblasts (CAFs) compared to their adjacent tumor counterpart fibroblasts at both the mRNA and protein levels. Interestingly, breast cancer cells and the recombinant IL-6 protein, both known to activate fibroblasts in vitro, downregulated DCN in BSFs. Moreover, specific DCN knockdown in breast fibroblasts modulated the expression/secretion of several CAF biomarkers and cancer-promoting proteins (α-SMA, FAP- α, SDF-1 and IL-6) and enhanced the invasion/proliferation abilities of these cells through activation of the STAT3/AUF1 signaling. Furthermore, DCN-deficient fibroblasts promoted the epithelial-to-mesenchymal transition and stemness processes in BC cells in a paracrine manner, which increased their resistance to cisplatin. These DCN-deficient fibroblasts also enhanced angiogenesis and orthotopic tumor growth in mice in a paracrine manner. On the other hand, ectopic expression of DCN in CAFs suppressed their active features and their paracrine pro-carcinogenic effects. Together, the present findings indicate that endogenous DCN suppresses the pro-carcinogenic and pro-metastatic effects of breast stromal fibroblasts.
Asunto(s)
Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Decorina , Regulación hacia Abajo , Interleucina-6 , Factor de Transcripción STAT3 , Transducción de Señal , Decorina/metabolismo , Decorina/genética , Humanos , Factor de Transcripción STAT3/metabolismo , Femenino , Interleucina-6/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Regulación hacia Abajo/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Fibroblastos/metabolismo , Células del Estroma/metabolismo , Línea Celular Tumoral , Carcinogénesis/patología , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Mama/patología , Mama/metabolismoRESUMEN
Nickel pollution is a recognized factor contributing to lung cancer. Understanding the molecular mechanisms of its carcinogenic effects is crucial for lung cancer prevention and treatment. Our previous research identified the downregulation of a long noncoding RNA, maternally expressed gene 3 (MEG3), as a key factor in transforming human bronchial epithelial cells (HBECs) into malignant cells following nickel exposure. In our study, we found that deletion of MEG3 also reduced the expression of RhoGDIß. Notably, artificially increasing RhoGDIß levels counteracted the malignant transformation caused by MEG3 deletion in HBECs. This indicates that the reduction in RhoGDIß contributes to the transformation of HBECs due to MEG3 deletion. Further exploration revealed that MEG3 downregulation led to enhanced c-Jun activity, which in turn promoted miR-200c transcription. High levels of miR-200c subsequently increased the translation of AUF1 protein, stabilizing SOX2 messenger RNA (mRNA). This stabilization affected the regulation of miR-137, SP-1 protein translation, and the suppression of RhoGDIß mRNA transcription and protein expression, leading to cell transformation. Our study underscores the co-regulation of RhoGDIß expression by long noncoding RNA MEG3, multiple microRNAs (miR-200c and miR-137), and RNA-regulated transcription factors (c-Jun, SOX2, and SP1). This intricate network of molecular events sheds light on the nature of lung tumorigenesis. These novel findings pave the way for developing targeted strategies for the prevention and treatment of human lung cancer based on the MEG3/RhoGDIß pathway.
Asunto(s)
Neoplasias Pulmonares , MicroARNs , ARN Largo no Codificante , Humanos , Proliferación Celular/genética , Transformación Celular Neoplásica/genética , Regulación hacia Abajo , Células Epiteliales/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , MicroARNs/genética , MicroARNs/metabolismo , Níquel , Inhibidor beta de Disociación del Nucleótido Guanina rho/antagonistas & inhibidores , Inhibidor beta de Disociación del Nucleótido Guanina rho/genética , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero , Factores de Transcripción SOXB1/genética , Ribonucleoproteína Nuclear Heterogénea D0/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismoRESUMEN
Alpha-synuclein (SNCA) accumulation plays a central role in the pathogenesis of Parkinson's disease. Determining and interfering with the mechanisms that control SNCA expression is one approach to limiting disease progression. Currently, most of our understanding of SNCA regulation is protein-based. Post-transcriptional mechanisms directly regulating SNCA mRNA expression via its 3' untranslated region (3'UTR) were investigated here. Mass spectrometry of proteins pulled down from murine brain lysates using a biotinylated SNCA 3'UTR revealed multiple RNA-binding proteins, of which HNRNPD/AUF1 was chosen for further analysis. AUF1 bound both proximal and distal regions of the SNCA 3'UTR, but not the 5'UTR or CDS. In the nucleus, AUF1 attenuated SNCA pre-mRNA maturation and was indispensable for the export of SNCA transcripts. AUF1 destabilized SNCA transcripts in the cytosol, primarily those with shorter 3'UTRs, independently of microRNAs by recruiting the CNOT1-CNOT7 deadenylase complex to trim the polyA tail. Furthermore, AUF1 inhibited SNCA mRNA binding to ribosomes. These data identify AUF1 as a multi-tasking protein regulating maturation, nucleocytoplasmic shuttling, stability and translation of SNCA transcripts.
Asunto(s)
Proteínas de Unión al ARN , Ratones , Animales , Regiones no Traducidas 3' , Transporte Activo de Núcleo Celular , Ribonucleoproteína Nuclear Heterogénea D0/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Unión ProteicaRESUMEN
Cervical squamous cell carcinoma (CSCC) is one of the leading causes of cancer death in women worldwide. Patients with advanced cervical carcinoma always have a poor prognosis once resistant to cisplatin due to the lack of effective treatment. It is urgent to investigate the molecular mechanisms of cisplatin resistance. Circular RNAs (circRNAs) are known to exert their regulatory functions in a series of malignancies. However, their effects on CSCC remain to be elucidated. Here, we found that cytoplasmic circARHGAP5, derived from second and third exons of the ARHGAP5 gene, was downregulated in cisplatin-resistant tissues compared with normal cervix tissues and untreated cervical cancer tissues. In addition, experiments from overexpression/knockdown cell lines revealed that circARHGAP5 could inhibit cisplatin-mediated cell apoptosis in CSCC cells both in vitro and in vivo. Mechanistically, circARHGAP5 interacted with AU-rich element RNA-binding protein (AUF1) directly. Overexpression of AUF1 could also inhibit cell apoptosis mediated by cisplatin. Furthermore, we detected the potential targets of AUF1 related to the apoptotic pathway and found that bcl-2-like protein 11 (BIM) was not only negatively regulated by AUF1 but positively regulated by circARHGAP5, which indicated that BIM mRNA might be degraded by AUF1 and thereby inhibited tumor cell apoptosis. Collectively, our data indicated that circARHGAP5 directly bound to AUF1 and prevented AUF1 from interacting with BIM mRNA, thereby playing a pivotal role in cisplatin resistance in CSCC. Our study provides insights into overcoming cancer resistance to cisplatin treatment.
Asunto(s)
Carcinoma de Células Escamosas , Ribonucleoproteína Nuclear Heterogénea D0 , ARN Circular , Neoplasias del Cuello Uterino , Femenino , Humanos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Cisplatino/farmacología , Proteínas Activadoras de GTPasa/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , ARN Circular/genética , ARN Mensajero/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
Coxsackievirus B (CVB) 3C protease (3Cpro) plays a specific cleavage role on AU-rich binding factor (AUF1, also called hnRNP D), which consequently disputes the regulation of AUF1 on downstream molecules. In our study, the iTRAQ approach was first used to quantify the differentially expressed cellular proteins in AUF1-overexpressing HeLa cells, which provides straightforward insight into the role of AUF1 during viral infection. A total of 1,290 differentially expressed proteins (DEPs), including 882 upregulated and 408 downregulated proteins, were identified. The DEPs are involved in a variety of cellular processes via GO terms, protein-protein interactions, and a series of further bioinformatics analyses. Among the DEPs, some demonstrated important roles in cellular metabolism. In particular, DDX5 was further verified to be negatively regulated by AUF1 and increased in CVB-infected cells, which in turn promoted CVB replication. These findings provide potential novel ideas for exploring new antiviral therapy targets.
Asunto(s)
ARN Helicasas DEAD-box , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D , Proteómica , Antivirales , ARN Helicasas DEAD-box/metabolismo , Enterovirus Humano B/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Humanos , Replicación ViralRESUMEN
Mammalian target of rapamycin (mTOR), which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA-binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. We determined that AUF1 is required for phosphorylation of Akt at Thr308, Thr450, and Ser473 and that AUF1 also mediates phosphorylation of the mTORC2-modulated metabolic enzyme glutamine fructose-6-phosphate amidotransferase 1 at Ser243. In addition, AUF1 immunoprecipitation followed by quantitative RT-PCR revealed that the mRNAs of Akt, glutamine fructose-6-phosphate amidotransferase 1, and the mTORC2 component SIN1 associate with AUF1. Furthermore, expression of the p40 and p45, but not the p37 or p42, isoforms of AUF1 specifically mediate Akt phosphorylation. In the absence of AUF1, subcellular fractionation indicated that Akt fails to localize to the membrane. However, ectopic expression of a membrane-targeted allele of Akt is sufficient to allow Akt-Ser473 phosphorylation despite AUF1 depletion. Finally, conditions that enhance mTORC2 signaling, such as acute glutamine withdrawal, augment AUF1 phosphorylation, whereas mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in promoting membrane localization of Akt to facilitate its phosphorylation on this cellular compartment. Targeting AUF1 could have therapeutic benefit for cancers with upregulated mTORC2/Akt signaling.
Asunto(s)
Ribonucleoproteína Nuclear Heterogénea D0 , Proteínas Proto-Oncogénicas c-akt , Proteínas de Unión al ARN , Proliferación Celular , Glutamina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Humanos , Ribonucleoproteína Nuclear Heterogénea D0/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Membrana Celular/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismoRESUMEN
BACKGROUND: The AU-rich element (ARE)-binding factor 1 (AUF1) acts as a switch for septic shock, although its underlying mechanisms remain largely unknown. In this study, we examined the biological significance and potential molecular mechanism of AUF1 in regulating ferroptosis in sepsis-induced acute lung injury (ALI). METHODS: Alveolar epithelial cells (AECs) challenged with ferroptosis-inducing compounds and cecum ligation and puncture (CLP)-induced ALI were used as the in vitro and in vivo model, respectively. The stability of AUF1 and its degradation by ubiquitin-proteasome pathway were examined by cycloheximide chase analysis and co-immunoprecipitation assay. The regulation of AUF1 on nuclear factor E2-related factor 2 (NRF2) and activation transcription factor 3 (ATF3) was explored by RNA immunoprecipitation (RIP), RNA pull-down, and mRNA stability assays. Functionally, the effects of altering AUF1, NRF2 or ATF3 on ferroptosis in AECs or ALI mice were evaluated by measuring cell viability, lipid peroxidation, iron accumulation, and total glutathione level. RESULTS: AUF1 was down-regulated in AECs challenged with ferroptosis-inducing compounds, both on mRNA and protein levels. The E3 ubiquitin ligase FBXW7 was responsible for protein degradation of AUF1 during ferroptosis. By up-regulating NRF2 and down-regulating ATF3, AUF1 antagonized ferroptosis in AECs in vitro. In the CLP-induced ALI model, the survival rate of AUF1 knockout mice was significantly reduced and the lung injuries were aggravated, which were related to the enhancement of lung ferroptosis. CONCLUSIONS: FBXW7 mediates the ubiquitination and degradation of AUF1 in ferroptosis. AUF1 antagonizes ferroptosis by regulating NRF2 and ATF3 oppositely. Activating AUF1 pathway may be beneficial to the treatment of sepsis-induced ALI.
Asunto(s)
Factor de Transcripción Activador 3 , Lesión Pulmonar Aguda , Ferroptosis , Ribonucleoproteína Nuclear Heterogénea D0 , Factor 2 Relacionado con NF-E2 , Sepsis , Factor de Transcripción Activador 3/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/prevención & control , Animales , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , ARN , Sepsis/complicacionesRESUMEN
Active breast cancer-associated fibroblasts (CAFs), the most influential cells in breast tumor microenvironment, express/secrete high levels of the proinvasive/metastatic interleukin-6 (IL-6). Therefore, we have tested here the effect of the IL-6 receptor (IL-6R) inhibitor tocilizumab (TCZ; Actemra) on different active breast CAFs. We have shown that TCZ potently and persistently suppresses the expression of various CAF biomarkers, namely α-SMA, SDF-1 as well as the STAT3 pathway and its downstream target AUF1. TCZ also inhibited the proliferation, migration and invasion abilities of active breast CAF cells. Additionally, TCZ repressed the ability of CAF cells in promoting epithelial-to-mesenchymal transition, and enhancing the migratory/invasive and proliferative capacities of breast cancer cells in vitro. Importantly, these findings were confirmed in orthotopic humanized breast tumors in mice. Furthermore, TCZ suppressed the expression of the pro-angiogenic factor VEGF-A and its transactivator HIF-1α in CAF cells, and consequently inhibited the angiogenic-promoting effect of active CAFs both in vitro and in orthotopic tumor xenografts. These results indicate that inhibition of the IL-6/STAT3/AUF1 pathway by TCZ can normalize active breast CAFs and suppress their paracrine pro-carcinogenic effects, which paves the way toward development of specific CAF-targeting therapy, badly needed for more efficient breast cancer treatments.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ribonucleoproteína Nuclear Heterogénea D0/genética , Humanos , Interleucina-6/metabolismo , Receptores de Interleucina-6/antagonistas & inhibidores , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The functional mechanism is unknown for many genetic variants associated with substance use disorder phenotypes. Rs678849, an intronic variant in the delta-opioid receptor gene (OPRD1), has been found to predict regional brain volume, addiction risk, and the efficacy of buprenorphine/naloxone in treating opioid use disorder. The variant has also been implicated as an expression quantitative trait locus (eQTL) for several genes. OBJECTIVES: The objective of this study was to identify functional differences between the two alleles of rs678849 in vitro. We hypothesized that the two alleles of rs678849 would have different effects on transcriptional activity due to differential interactions with transcription factors. METHODS: 15bp regions containing the C or T alleles of rs678849 were cloned into luciferase constructs and transfected into BE(2)C neuroblastoma cells to test the effect on transcription. Electrophoretic mobility shift assays (EMSA) using nuclear lysates from BE(2)C cell or human postmortem medial prefrontal cortex were used to identify proteins that differentially bound the two alleles. RESULTS: At 24 hours post-transfection, the C allele construct had significantly lower luciferase expression than the T allele construct and empty vector control (ANOVA p < .001). Proteomic analysis and supershift assays identified XRCC6 as a transcription factor specifically binding the C allele, whereas hnRNP D0 was found to specifically bind the T allele. CONCLUSION: These functional differences between the C and T alleles may help explain the psychiatric and neurological phenotype differences predicted by rs678849 genotype and the potential role of the variant as an eQTL.
Asunto(s)
Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Autoantígeno Ku/metabolismo , Variantes Farmacogenómicas , Receptores Opioides delta/genética , Factores de Transcripción/metabolismo , Alelos , Ensayo de Cambio de Movilidad Electroforética , Genotipo , Humanos , Luciferasas de Luciérnaga , Unión Proteica/genética , Sitios de Carácter Cuantitativo/genéticaRESUMEN
Long non-coding RNAs (lncRNAs) have emerged as critical regulators in human disease including atherosclerosis. However, the mechanisms involved in the post-transcriptional regulation of the expression of disease-associated lncRNAs are not fully understood. Gene expression studies revealed that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) lncRNA expression was increased by >2-fold in peripheral blood mononuclear cells (PBMCs) derived from patients with coronary artery disease (CAD) or in carotid artery atherosclerotic plaques. We observed a linear association between NEAT1 lncRNA expression and prevalence of CAD which was independent of age, sex, cardiovascular traditional risk factors and renal function. NEAT1 expression was induced by TNF-α, while silencing of NEAT1 profoundly attenuated the TNF-α-induced vascular endothelial cell pro-inflammatory response as defined by the expression of CXCL8, CCL2, VCAM1 and ICAM1. Overexpression of the RNA editing enzyme adenosine deaminase acting on RNA-1 (ADAR1), but not of its editing-deficient mutant, upregulated NEAT1 levels. Conversely, silencing of ADAR1 suppressed the basal levels and the TNF-α-induced increase of NEAT1. NEAT1 lncRNA expression was strongly associated with ADAR1 in CAD and peripheral arterial vascular disease. RNA editing mapping studies revealed the presence of several inosines in close proximity to AU-rich elements within the AluSx3+/AluJo- double-stranded RNA complex. Silencing of the stabilizing RNA-binding protein AUF1 reduced NEAT1 levels while silencing of ADAR1 profoundly affected the binding capacity of AUF1 to NEAT1. Together, our findings propose a mechanism by which ADAR1-catalyzed A-to-I RNA editing controls NEAT1 lncRNA stability in ASCVD.
Asunto(s)
Adenosina/metabolismo , Elementos Alu/genética , Aterosclerosis/sangre , Enfermedad de la Arteria Coronaria/sangre , Inosina/metabolismo , Placa Aterosclerótica/sangre , Edición de ARN/genética , Estabilidad del ARN/genética , ARN Largo no Codificante/metabolismo , Transducción de Señal/genética , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Aterosclerosis/genética , Sitios de Unión , Células Cultivadas , Estudios de Cohortes , Enfermedad de la Arteria Coronaria/genética , Femenino , Silenciador del Gen , Ribonucleoproteína Nuclear Heterogénea D0/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Placa Aterosclerótica/genética , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , TransfecciónRESUMEN
Long noncoding RNAs (lncRNAs) play an essential role in tumor progression. Few researches focused on the clinical and biological relevance of lncRNAs in peripheral T cell lymphoma (PTCL). In this research, a novel lncRNA (ENST00000503502) was identified overexpressed in the main subtypes of PTCL, and designated as T cell lymphoma-associated lncRNA1 (TCLlnc1). Serum TCLlnc1 was associated with extranodal involvement, high-risk International Prognostic Index, and poor prognosis of the patients. Both in vitro and in vivo, overexpression of TCLlnc1 promoted T-lymphoma cell proliferation and migration, both of which were counteracted by the knockdown of TCLlnc1 using small interfering RNAs. As the mechanism of action, TCLlnc1 directly interacted with transcription activator heterogeneous nuclear ribonucleoprotein D (HNRNPD) and Y-box binding protein-1 (YBX1) by acting as a modular scaffold. TCLlnc1/HNRNPD/YBX1 complex upregulated transcription of TGFB2 and TGFBR1 genes, activated the tumor growth factor-ß signaling pathway, resulting in lymphoma progression, and might be a potential target in PTCL.
Asunto(s)
Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Linfoma de Células T Periférico/metabolismo , ARN Largo no Codificante/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Progresión de la Enfermedad , Femenino , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea D0/genética , Humanos , Células Jurkat , Linfoma de Células T Periférico/sangre , Linfoma de Células T Periférico/genética , Linfoma de Células T Periférico/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , ARN Largo no Codificante/sangre , ARN Largo no Codificante/genética , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
Studies have found that RNA-binding proteins (RBPs) are dysfunctional and play a significant regulatory role in the development of glioma. Based on The Cancer Genome Atlas database and the previous studies, we selected heterogeneous nuclear ribonucleoprotein (HNRNPD) as the research candidate and sought its downstream targeted genes. In the present study, HNRNPD, linc00707, and specific protein 2 (SP2) were highly expressed, while zinc fingers and homeboxes 2 (ZHX2) and miR-651-3p were remarkedly downregulated in glioma tissues and cells. HNRNPD, linc00707, and SP2 knockdown or ZHX2 and miR-651-3p overexpression suppressed glioma cells proliferation, migration, and invasion and vasculogenic mimicry (VM) formation. Knockdown of HNRNPD increased the stability of ZHX2 mRNA. ZHX2 bound to the promoter region of linc00707 and negatively regulate its expression. Linc00707 could bind with miR-651-3p, while miR-651-3p bound to the 3' untranslated region (3'UTR) of SP2 mRNA to negatively regulate its expression. The transcription factor SP2 directly bound to the promoter regions of the VM formation-related proteins MMP2, MMP9, and VE-cadherin, playing a role in promoting transcription in order to regulate the VM formation ability of glioma cells.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Imitación Molecular , Neovascularización Patológica , ARN Largo no Codificante/metabolismo , Factor de Transcripción Sp2/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 3' , Animales , Sitios de Unión , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/patología , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea D0/genética , Proteínas de Homeodominio/genética , Humanos , Ratones Desnudos , MicroARNs/genética , Invasividad Neoplásica , ARN Largo no Codificante/genética , Transducción de Señal , Factor de Transcripción Sp2/genética , Factores de Transcripción/genéticaRESUMEN
Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.
Asunto(s)
Proliferación Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , ARN Circular/metabolismo , Regiones no Traducidas 3' , Sitios de Unión , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HeLa , Humanos , ARN Circular/química , ARN Mensajero/metabolismoRESUMEN
Proper base-pairing of a miRNA with its target mRNA is a key step in miRNA-mediated mRNA repression. RNA remodelling by RNA-binding proteins (RBPs) can improve access of miRNAs to their target mRNAs. The largest isoform p45 of the RBP AUF1 has previously been shown to remodel viral or AU-rich RNA elements. Here, we show that AUF1 is capable of directly promoting the binding of the miRNA let-7b to its target site within the 3'UTR of the POLR2D mRNA. Our data suggest this occurs in two ways. First, the helix-destabilizing RNA chaperone activity of AUF1 disrupts a stem-loop structure of the target mRNA and thus exposes the miRNA target site. Second, the RNA annealing activity of AUF1 drives hybridization of the miRNA and its target site within the mRNA. Interestingly, the RNA remodelling activities of AUF1 were found to be isoform-specific. AUF1 isoforms containing a YGG motif are competent RNA chaperones, whereas isoforms lacking the YGG motif are not. Overall, our study demonstrates that AUF1 has the ability to modulate a miRNA-target site interaction, thus revealing a new regulatory function for AUF1 proteins during post-transcriptional control of gene expression. Moreover, tests with other RBPs suggest the YGG motif acts as a key element of RNA chaperone activity.
Asunto(s)
Empalme Alternativo , Ribonucleoproteína Nuclear Heterogénea D0/genética , MicroARNs/genética , ARN Mensajero/genética , Motivos de Unión al ARN/genética , Regiones no Traducidas 3'/genética , Algoritmos , Secuencia de Aminoácidos , Regulación de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Humanos , Cinética , MicroARNs/metabolismo , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismoRESUMEN
Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.
Asunto(s)
Ribonucleoproteína Nuclear Heterogénea D0/genética , ARN Mensajero/genética , Factores de Transcripción/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Regiones no Traducidas 3' , Animales , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Ratones , Ratones Endogámicos C57BL , Estabilidad del ARNRESUMEN
BACKGROUND: AUF1 is one of the AU-rich binding proteins, which promotes rapid ARE-mRNA degradation. Recently, it has been reported that AUF1 is involved in regulating the antioxidant system because of its capacity to bind specifically to RNA containing oxidized bases and degrade oxidized RNA. Many antioxidant proteins have been reported to be overexpressed in colorectal cancer (CRC), however, the role of AUF1 in the progression of CRC has not been explored. METHODS: The expression level of AUF1 protein in human CRC cell lines and CRC tissues was detected by western blotting and immunohistochemistry (IHC. The effects of AUF1 knockdown on CRC cell proliferation, migration, invasion and changes in the signaling pathways were evaluated using a cell counting kit-8 (CCK-8), Transwell assays and western blotting. Subcutaneous xenograft tumor model was employed to further substantiate the role of AUF1 in CRC. RESULTS: AUF1 protein was upregulated in CRC tissues and CRC cells, and high expression of AUF1 was significantly associated with advanced AJCC stage (P = .001), lymph node metastasis (P = .007), distant metastasis (P = .038) and differentiation (P = .009) of CRC specimens. CRC patients with the high expression of AUF1 had an extremely poor prognosis. The knockdown of AUF1 suppressed CRC cell line proliferation, migration and invasion, inhibited CRC cells tumorigenesis and growth in nude mice, and reduced phosphorylated-ERK1/2 and phosphorylated AKT in CRC cells. CONCLUSION: Our findings demonstrate that AUF1 is probably involved in the progression of CRC via the activation of the ERK1/2 and AKT pathways. AU-rich RNA-binding factor 1 could be used as a novel prognostic biomarker and a potential therapeutic target for CRC.
Asunto(s)
Neoplasias Colorrectales/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ribonucleoproteína Nuclear Heterogénea D0/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anciano , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Activación Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Ribonucleoproteína Nuclear Heterogénea D0/genética , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Fosforilación , Transducción de Señal , Carga Tumoral , Regulación hacia ArribaRESUMEN
Enterovirus 71 (EV71) poses serious threats to human health, particularly in Southeast Asia, and no drugs or vaccines are available. Previous work identified the stem loop II structure of the EV71 internal ribosomal entry site as vital to viral translation and a potential target. After screening an RNA-biased library using a peptide-displacement assay, we identify DMA-135 as a dose-dependent inhibitor of viral translation and replication with no significant toxicity in cell-based studies. Structural, biophysical, and biochemical characterization support an allosteric mechanism in which DMA-135 induces a conformational change in the RNA structure that stabilizes a ternary complex with the AUF1 protein, thus repressing translation. This mechanism is supported by pull-down experiments in cell culture. These detailed studies establish enterovirus RNA structures as promising drug targets while revealing an approach and mechanism of action that should be broadly applicable to functional RNA targeting.