RESUMEN
While the transcriptional machinery has been extensively dissected at the molecular level, little is known about regulation of chromosomal organization in the three-dimensional space of the nucleus to achieve integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories requiring nuclear actin/myosin-I transport machinery, dynein light chain 1 (DLC1), and a specific subset of transcriptional coactivators and chromatin remodeling complexes. We establish a requirement for the histone lysine demethylase, LSD1, in directing specific interchromosomal interaction loci to distinct interchromatin granules, long thought to be "storage" sites for splicing machinery, and demonstrate that these three-dimensional motor-dependent interactions are required to achieve enhanced transcription of specific estrogen-receptor target genes. These findings reveal roles for the modulation of nuclear architecture in orchestrating regulated gene-expression programs in the mammalian nucleus.
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Cromatina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Redes Reguladoras de Genes , Proteínas Motoras Moleculares/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Actinas/metabolismo , Línea Celular Tumoral , Núcleo Celular , Células Cultivadas , Histona Demetilasas , Humanos , Cuerpos de Inclusión Intranucleares/metabolismo , Transcripción GenéticaRESUMEN
Microglia, being the resident immune cells of the central nervous system, play an important role in maintaining tissue homeostasis and contributes towards brain development under normal conditions. However, when there is a neuronal injury or other insult, depending on the type and magnitude of stimuli, microglia will be activated to secrete either proinflammatory factors that enhance cytotoxicity or anti-inflammatory neuroprotective factors that assist in wound healing and tissue repair. Excessive microglial activation damages the surrounding healthy neural tissue, and the factors secreted by the dead or dying neurons in turn exacerbate the chronic activation of microglia, causing progressive loss of neurons. It is the case observed in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. This review gives a detailed account of the microglia-mediated neuroinflammation in various neurodegenerative diseases. Hence, resolving chronic inflammation mediated by microglia bears great promise as a novel treatment strategy to reduce neuronal damage and to foster a permissive environment for further regeneration effort.
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Inflamación/metabolismo , Microglía/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Animales , Humanos , Inflamación/patología , Microglía/patología , Enfermedades Neurodegenerativas/patologíaRESUMEN
RATIONALE: Pathogenic variations in the lamin gene (LMNA) cause familial dilated cardiomyopathy (DCM). LMNA insufficiency caused by LMNA pathogenic variants is believed to be the basic mechanism underpinning LMNA-related DCM. OBJECTIVE: To assess whether silencing of cardiac Lmna causes DCM and investigate the role of Yin Yang 1 (Yy1) in suppressing Lmna DCM. METHODS AND RESULTS: We developed a Lmna DCM mouse model induced by cardiac-specific Lmna short hairpin RNA. Silencing of cardiac Lmna induced DCM with associated cardiac fibrosis and inflammation. We demonstrated that upregulation of Yy1 suppressed Lmna DCM and cardiac fibrosis by inducing Bmp7 expression and preventing upregulation of Ctgf. Knockdown of upregulated Bmp7 attenuated the suppressive effect of Yy1 on DCM and cardiac fibrosis. However, upregulation of Bmp7 alone was not sufficient to suppress DCM and cardiac fibrosis. Importantly, upregulation of Bmp7 together with Ctgf silencing significantly suppressed DCM and cardiac fibrosis. Mechanistically, upregulation of Yy1 regulated Bmp7 and Ctgf reporter activities and modulated Bmp7 and Ctgf gene expression in cardiomyocytes. Downregulation of Ctgf inhibited TGF-ß (transforming growth factor-ß)/Smad signaling in DCM hearts. Regulation of both Bmp7 and Ctgf further suppressed TGFß/Smad signaling. In addition, co-modulation of Bmp7 and Ctgf reduced CD3+ T cell numbers in DCM hearts. CONCLUSIONS: Our findings demonstrate that upregulation of Yy1 or co-modulation of Bmp7 and Ctgf offer novel therapeutic strategies for the treatment of DCM caused by LMNA insufficiency.
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Proteína Morfogenética Ósea 7/biosíntesis , Cardiomiopatías/metabolismo , Cardiomiopatías/prevención & control , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Factor de Transcripción YY1/biosíntesis , Animales , Proteína Morfogenética Ósea 7/genética , Cardiomiopatías/genética , Factor de Crecimiento del Tejido Conjuntivo/genética , Endotelio Vascular/metabolismo , Fibrosis/genética , Fibrosis/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción YY1/genéticaRESUMEN
PIWI homologs constitute a subclass of the Argonaute family. Traditionally, they have been shown to associate with a specific class of small RNAs, piRNAs, to suppress transposable elements and protect genomic integrity in germ cells. Recent studies imply that PIWI proteins may also exert important biological functions in somatic contexts, including the brain. However, their exact role in neural development remains unknown. Hence we investigated whether PIWI proteins are involved in neuronal differentiation. By using an established cell model for studying neurogenesis, NTera2/D1 (NT2) cells, we found that a particular PIWI homolog, PIWIL4 was increasingly upregulated throughout the course of all-trans retinoic acid (RA)-mediated neuronal differentiation. During this process, PIWIL4 knockdown led to partial recovery of embryonic stem cell markers, while suppressing RA-induced expression of neuronal markers. Consistently, PIWIL4 overexpression further elevated their expression levels. Furthermore, co-immunoprecipitation revealed an RA-induced interaction between PIWIL4 and the H3K27me3 demethylase UTX. Chromatin immunoprecipitation showed that this interaction could be essential for the removal of H3K27me3 from the promoters of RA-inducible genes. By a similar mechanism, PIWIL4 knockdown also suppressed the expression of PTN and NLGN3, two important neuronal factors secreted to regulate glioma activity. We further noted that the conditioned medium collected from PIWIL4-silenced NT2 cells significantly reduced the proliferation of glioma cells. Thus, our data suggest a novel somatic role of PIWIL4 in modulating the expression of neuronal genes that can be further characterized to promote neuronal differentiation and to modulate the activity of glioma cells.
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Diferenciación Celular/genética , Células Madre de Carcinoma Embrionario/metabolismo , Células Madre de Carcinoma Embrionario/patología , Neuronas/metabolismo , Proteínas de Unión al ARN/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioma/genética , Glioma/metabolismo , Glioma/patología , Histona Demetilasas/metabolismo , Histonas/metabolismo , Humanos , Neuronas/citología , Unión Proteica , Proteínas de Unión al ARN/metabolismo , TranscriptomaRESUMEN
Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers.
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Empalme Alternativo/fisiología , Factor de Crecimiento Epidérmico/fisiología , Sistema de Señalización de MAP Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/genética , Células HEK293 , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Autophagy is an evolutionarily conserved cellular degradative process in which intracellular components (cellular proteins and organelles) are engulfed in autophagosomes which then fuse with lysosomes to form autolysosome for degradation. Autophagy is closely implicated in various physio-pathological processes and human diseases. Among them, the roles of autophagy in cancer have been extensively studied. Increasing evidence has demonstrated that inhibiting autophagy is a novel and promising approach in cancer therapy, based on the notion that autophagy is a pro-survival mechanism in cancer cells under therapeutic stress, and induction of autophagy is associated with chemoresistance of cancer cells to chemotherapeutic agents. Thus, suppression of autophagy would sensitize resistance tumor cells to cancer therapeutic agents, thereby supporting the clinical application of autophagy inhibitors. In recent years, significant progress has been achieved in developing autophagy inhibitors and testing their therapeutical potential, either as standalone or as adjuvant therapeutic agents, in cell and animal models, and more importantly in clinical trials. In this review, we will discuss some of these recent advances in development of novel small molecules autophagy inhibitors and their mechanisms of action, together with their applications in clinical trials.
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Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto , Descubrimiento de Drogas/métodos , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
A planar hexanuclear cobalt ring was clamped by two bivacant α1-[PW10O37](9-) with the assistance of the pyridazine bridges to form a novel sandwiched Co(II)-polyoxometalate cluster compound, [Na(H2O)6][Co3(OH) (pydz)4(H2O)7][Co6(PW10O37)2(pydz)4(H2O)6]·43H2O (1; pydz = pyridazine).This cluster was identified by X-ray single-crystal diffraction, elemental analysis, Fourier transform IR and UV-visible spectroscopies, and cyclic voltammetry (CV). Structural analysis reveals that 1 comprises a hexahydrated sodium, a trinuclear [Co3(OH) (pydz)4(H2O)7](5+) cationic cluster, and an anionic [Co6(PW10O37)2(pydz)4(H2O)6](6-) sandwiched cluster, thus giving an intrinsical intercluster compound. The isolation of such cluster was dependent on the in situ transformation of trivacant [α-P2W15O56](12-) to α1-[PW10O37](9-) under the hydrothermal condition. The CV shows reversible multielectron waves from the redox of W(VI) in 1. Cluster 1 exhibits remarkable electrocatalytic activity toward the reduction of nitrite. Magnetism studies indicated a weak anti-ferromagnetic exchange interaction between Co(II) ions within 1.
RESUMEN
In the past several decades, intensive research in this field has uncovered a surprising number of regulatory factors and their associated enzymatic properties to reveal the network of complexes that function in activation and repression of the transcriptional programs mediated by nuclear receptors (NR). These factors and their associated complexes have been extensively characterized both biochemically and functionally [34, 87, 94]. Several principles have emerged: (1) It is widely recognized that ligand-dependent cofactor complexes mediating repression and activation exhibit ligand-dependent exchange. (2) These complexes mediate modifications of chromatin structure consequent to their binding at regulatory elements, particularly at promoter and enhancer Enhancer sites. (3) The concept about the rapid exchange of coregulatory complexes at regulatory sites has been suggested [88]. Key questions in the NR field have included: (a) What are the cofactors and exchange complexes used to mediate the ligand and signaling network-dependent switches in gene regulation programs; (b) Do long non-coding RNAs (lncRNAs) serve as regulatory "factors" for ligand-dependent gene programs, and do enhancers actually regulate transcription units encoding enhancer Enhancer non-coding RNAs (eRNAs) Enhancer RNA that might have functional significance; (c) What is the relationship between DNA damage repair machinery and transcriptional machinery? (d) Do Retinoic Acid Receptors (RAR) also regulate Pol III-dependent, non-coding repeat transcriptional units in stem cells? and (e) How have new technologies such as deep sequencing altered our ability to investigate transcriptional regulatory mechanisms utilized by NRs?
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Cromatina/química , Regulación de la Expresión Génica , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide/metabolismo , Transcripción Genética , Cromatina/metabolismo , Humanos , Ligandos , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Receptores de Ácido Retinoico/genética , Elementos de Respuesta , Receptores X Retinoide/genética , Transducción de SeñalRESUMEN
Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated potent clinical efficacy in the treatment of hematopoietic malignancies. However, the application of CAR-T in solid tumors has been limited due in part to the expression of inhibitory molecules in the tumor microenvironment, leading to T-cell exhaustion. To overcome this limitation, we have developed a synthetic T-cell receptor (TCR) that targets programmed death-ligand 1 (PD-L1), a molecule that is widely expressed in various solid tumors and plays a pivotal role in T-cell exhaustion. Our novel TCR platform is based on antibody-based binding domain, which is typically a single-chain variable fragment (scFv), fused to the γδ TCRs (TCRγδ). We have utilized the T-cell receptor alpha constant (TRAC) locus editing approach to express cell surface scFv of anti-PD-L1, which is fused to the constant region of the TCRγ or TCRδ chain in activated T cells derived from peripheral blood mononuclear cells (PBMCs). Our results indicate that these reconfigured receptors, both γ-TCRγδ and δ-TCRγδ, have the capability to transduce signals, produce inflammatory cytokines, degranulate and exert tumor killing activity upon engagement with PD-L1 antigen in vitro. Additionally, we have also shown that γ-TCRγδ exerted superior efficacy than δ-TCRγδ in in vivo xenograft model.
RESUMEN
piRNAs (PIWI-interacting RNAs) are a class of small non-coding RNAs (ncRNAs) abundantly expressed in germline cells and involved in suppressing the transposon activity. Interestingly, recent studies have found piRNA expression in the central nervous system (CNS), yet the underlying biological significance remains largely unknown. In this study, we investigated the expression and function of piRNAs during the retinoic acid (RA)-mediated neuronal differentiation in NT2 cells, a human embryonal carcinoma cell line. We identified a cohort of differentially expressed piRNAs by microarray. Two piRNAs, DQ582359 and DQ596268, were increasingly upregulated during the RA-induced differentiation and involved in regulating the expression of neuronal markers, MAP2 and TUBB3. Furthermore, these piRNAs were found to associate with cold-shock domain (CSD)-containing RNA binding proteins, DIS3, DIS3L2, and YB-1. Markedly, overexpression of these piRNAs further enhanced the protein levels of MAP2 and TUBB3, potentially by downregulating DIS3, DIS3L2, and YB-1. Hence, our study has identified a novel somatic function of piRNAs in regulating neuronal gene expression. The interaction of piRNA with some CSD-containing proteins can be further explored to enhance neuronal differentiation to treat neurodegenerative diseases.
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Proteínas y Péptidos de Choque por Frío , Proteínas de Unión al ARN , Proteínas Argonautas/metabolismo , Diferenciación Celular/genética , Proteínas y Péptidos de Choque por Frío/metabolismo , Expresión Génica , Humanos , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic have captured the world's attention and remains an ongoing issue due to concerns of balancing protection against emerging variants of concern while also generating long-lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid encoding the spike protein in a lipid nanoparticle formulation (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In an effort to improve immunity, a clinically focused lymphatic drug delivery device (MuVaxx) was engineered to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) and tested to measure adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine-producing CD8 T cells, neutralizing antibodies against the Washington (wild type) strain and South African (Beta) variants, and LN-resident spike-specific memory B cells. Remarkably, a 4-fold-elevated T cell response was observed in MuVaxx-administered vaccination compared to that of IM-administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic-mediated delivery using MuVaxx for SARS-CoV-2 and VoC vaccines.
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Vacunas contra la COVID-19 , COVID-19 , Animales , Ratones , Vacunas contra la COVID-19/inmunología , Inmunidad Celular , Ratones Endogámicos C57BL , SARS-CoV-2/genéticaRESUMEN
The emerging SARS-CoV-2 variants of concern (VOCs) exhibit enhanced transmission and immune escape, reducing the effectiveness of currently approved mRNA vaccines. To achieve wider coverage of VOCs, we first constructed a cohort of mRNAs harboring a furin cleavage mutation in the spike (S) protein of predominant VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). The mutation abolished the cleavage between the S1 and S2 subunits. Systematic evaluation in vaccinated mice discovered that individual VOC mRNAs elicited strong neutralizing activity in a VOC-specific manner. In particular, the neutralizing antibodies (nAb) produced by immunization with Beta-Furin and Washington (WA)-Furin mRNAs showed potent cross-reactivity with other VOCs. However, neither mRNA elicited strong neutralizing activity against the Omicron variant. Hence, we further developed an Omicron-specific mRNA vaccine that restored protection against the original Omicron variant and some sublineages. Finally, to broaden the protection spectrum of the new Omicron mRNA vaccine, we engineered an mRNA-based chimeric immunogen by introducing the receptor-binding domain of Delta variant into the entire S antigen of Omicron. The resultant chimeric mRNA induced potent and broadly nAbs against Omicron and Delta, which paves the way to developing new vaccine candidates to target emerging variants in the future.
RESUMEN
Although the role of liganded nuclear receptors in mediating coactivator/corepressor exchange is well-established, little is known about the potential regulation of chromosomal organization in the 3-dimensional space of the nucleus in achieving integrated transcriptional responses to diverse signaling events. Here, we report that ligand induces rapid interchromosomal interactions among specific subsets of estrogen receptor alpha-bound transcription units, with a dramatic reorganization of nuclear territories, which depends on the actions of nuclear actin/myosin-I machinery and dynein light chain 1. The histone lysine demethylase, LSD1, is required for these ligand-induced interactive loci to associate with distinct interchromatin granules, long thought to serve as "storage" sites for the splicing machinery, some critical transcription elongation factors, and various chromatin remodeling complexes. We demonstrate that this 2-step nuclear rearrangement is essential for achieving enhanced, coordinated transcription of nuclear receptor target genes.
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Células Epiteliales/fisiología , Redes Reguladoras de Genes/fisiología , Oxidorreductasas N-Desmetilantes/genética , Receptores Citoplasmáticos y Nucleares/genética , Transcripción Genética/fisiología , Neoplasias de la Mama , Línea Celular Tumoral , Núcleo Celular/fisiología , Cromatina/fisiología , Células Epiteliales/citología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Regulación de la Expresión Génica/fisiología , Histona Demetilasas , Humanos , Hibridación Fluorescente in Situ , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oxidorreductasas N-Desmetilantes/química , Oxidorreductasas N-Desmetilantes/metabolismo , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Factor Trefoil-1 , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Cisplatin and other platinum-based compounds are frequently used to treat breast cancer, but their utility is severely compromised by drug resistance. Many genes dictating drug responsiveness are subject to pre-mRNA alternative splicing which is regulated by key kinases such as the serine-arginine protein kinase 1 (SRPK1). However, its contribution to drug resistance remains controversial. In this study, we have identified that Tip60-mediated acetylation of SRPK1 is closely associated with chemotherapy sensitivity. In breast cancer cells, cisplatin induced SRPK1 acetylation but in the corresponding resistant cells, it reduced acetylation yet increased phosphorylation and kinase activity of SRPK1, favouring the splicing of some anti-apoptotic variants. Significantly, the cisplatin-resistant cells could be re-sensitized by enhancing SRPK1 acetylation or inhibiting its kinase activity. Hence, our study reveals a key role of SRPK1 in the development of cisplatin resistance in breast cancer cells and suggests a potential therapeutic avenue for overcoming chemotherapy resistance.
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Empalme Alternativo , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Resistencia a Medicamentos/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Acetilación , Neoplasias de la Mama , Humanos , Células MCF-7RESUMEN
Y-box binding protein-1 (YB-1) facilitates cancer chemoresistance through the upregulation of ATP-binding cassette (ABC) transporters associated with multidrug resistance, which is one of the primary obstacles in cancer treatment. Since aberrant Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling is also implicated in chemoresistance in numerous human malignancies, the interaction between YB-1 and JAK/STAT signaling was explored underlying the chemoresistance of NUGC3 gastric cancer cells. It was demonstrated that YB-1 translocated into the nuclei of NUGC3 cells exposed to doxorubicin hydrochloride, suggesting its important role in chemoresistance. Consistently, knockdown of YB-1 significantly decreased the chemoresistance of cells to doxorubicin hydrochloride and epirubicin hydrochloride, as evidenced by a decrease in cell viability. Notably, JAK inhibitor AG490 treatment further decreased the cell viability caused by YB-1 inhibition and doxorubicin hydrochloride. It was also observed that YB-1 transcriptionally regulated the ABCC3 transporter, whereas STAT3 modulated ABCC2 transporter levels. These findings suggest that YB-1 and STAT3 act together to facilitate chemoresistance via modulating the expression of different ABC transporters in NUGC3 cells. Notably, siYB-1 did not exhibit any significant effect on STAT3 expression. Similarly, siSTAT3 failed to alter YB-1 expression, suggesting that the two may not regulate each other in a mutual manner. However, double knockdown of YB-1 and STAT3 led to a synergistic inhibition of cell invasion in NUGC3 cells. Nonetheless, the combined treatment of YB-1 antagonists with STAT3 inhibitors may serve as an effective therapy in gastric cancer.
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Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Antineoplásicos , Factor de Transcripción STAT3/metabolismo , Neoplasias Gástricas/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Transporte de Proteínas/efectos de los fármacos , Factor de Transcripción STAT3/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Tirfostinos/farmacología , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
A majority of breast cancer cases are positive for the estrogen receptor (ER), which means that they can respond to the estrogen hormone to achieve growth. Hence, the ER signaling pathway has been extensively targeted in pharmaceutical research and development in order to suppress tumor growth. However, prevalent hormone therapy and targeted therapy often become ineffective as cancer cells ultimately develop resistance, suggesting that there could be unidentified signaling molecules and events that regulate breast cancer growth. Notably, recent studies have uncovered that Piwilike (Piwil) proteins, which were initially found in germline cells, are expressed in a wide spectrum of human cancers, including breast cancers. Although Piwil proteins have been well established to silence retrotransposons and to promote heterochromatin formation in germline cells, their somatic functions in cancer cells remain largely unknown. In the present study, we profiled the expression of four Piwi homologs in an ERpositive breast cancer cell line, MCF7, and found that only Piwil4 was upregulated by 17ßestradiol treatment. Notably, Piwil4 upregulation was not observed in an ERpositive but nontumorigenic breast cancer cell line, MCF12A. In addition, the induced expression of Piwil4 was dependent on estrogen/ERα signaling. To explore the biological significance of Piwil4 in breast cancer growth, we knocked down Piwil4 with multiple siRNAs and observed the suppressed expression of some canonical targets of ER. The knockdown of Piwil4 expression also decreased the migration and invasion capabilities of MCF7 cells. Furthermore, the lossoffunction of Piwil4 reduced the motility of MCF7 cells in woundhealing assays, which could be associated to decreased expression of vimentin and Ncadherin. Collectively, these findings revealed that Piwil4 is a novel regulator of ER signaling that could be targeted to inhibit breast cancer growth and migration.
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Proteínas Argonautas/genética , Neoplasias de la Mama/genética , Estradiol/farmacología , Receptores de Estrógenos/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Células MCF-7 , Proteínas de Unión al ARN , Transducción de Señal/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
Reactive oxygen species (ROS) play important roles in fundamental cellular processes such as proliferation and survival. Here we investigated the effect of oxidative stress on stem cell maintenance and neuronal differentiation in a human embryonic stem cell (hESC) model, Ntera2 (NT2). CM-H2DCFDA and DHE assays confirmed that the oxidizing agent paraquat could induce a high level of ROS in NT2 cells. Quantitative PCR, Western blotting and immunocytochemistry showed that paraquat-induced oxidative stress suppressed the expression of stemness markers, including NANOG, OCT4 and TDGF1, whereas it enhanced the spontaneous expression of neuronal differentiation markers such as PAX6, NEUROD1, HOXA1, NCAM, GFRA1 and TUJ1. The treated cells even exhibited a strikingly different morphology from control cells, extending out long neurite-like processes. The neurogenic effect of ROS on stem cell behaviour was confirmed by the observations that the expression of neuronal markers in the paraquat-treated cells was suppressed by an antioxidant while further enhanced by knocking down Nrf2, a key transcription factor associated with antioxidant signaling. Lastly, paraquat dose-dependently activated the neurogenic MAPK-ERK1/2, which can be reversed by the MEK1/2 inhibitor SL327. Our study suggests that excessive intracellular ROS can trigger the exit from stem cell state and promote the neuronal differentiation of hESCs, and that MAPK-ERK1/2 signaling may play a proactive role in the ROS-induced neuronal differentiation of hESCs.
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The last few decades have witnessed a tremendous advancement in understanding the genetic basis of major human diseases such as cancer. Intriguingly, there is also an evergrowing body of evidence that suggest the critical role of epigenetic regulation in pathogenesis. In contrast to genetic mechanisms often associated with changes in DNA sequence, epigenetics generally refers to the regulation of gene expression featuring alterations in histone modification, DNA methylation, chromatin conformation and non-coding RNAs, with the first two categories being the best-characterized so far. A growing list of epigenetic factors, including writers, readers and erasers have been identified, and huge differences in genome-wide epigenetic modifications, so-called epigenome, have been reported between normal tissues and cancer. Significantly, since the epigenetic regulation is largely dependent on enzymes, they are generally reversible and thus more amenable to pharmaceutical intervention. Hence, it is believed that a comprehensive understanding of how alterations in epigenome lead to tumorigenesis, progression and drug resistance is of primary importance to develop epigenetic therapies and to ensure long-term efficacy of cancer treatment.