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1.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-33804856

RESUMEN

Cancer is one of the leading causes of death worldwide. Conventional therapies, including surgery, radiation, and chemotherapy have achieved increased survival rates for many types of cancer over the past decades. However, cancer recurrence and/or metastasis to distant organs remain major challenges, resulting in a large, unmet clinical need. Oligonucleotide therapeutics, which include antisense oligonucleotides, small interfering RNAs, and aptamers, show promising clinical outcomes for disease indications such as Duchenne muscular dystrophy, familial amyloid neuropathies, and macular degeneration. While no approved oligonucleotide drug currently exists for any type of cancer, results obtained in preclinical studies and clinical trials are encouraging. Here, we provide an overview of recent developments in the field of oligonucleotide therapeutics in oncology, review current clinical trials, and discuss associated challenges.


Asunto(s)
Antagomirs/genética , Neoplasias/terapia , Oligonucleótidos Antisentido/genética , Tratamiento con ARN de Interferencia/métodos , Animales , Aptámeros de Nucleótidos/genética , Ensayos Clínicos como Asunto , Humanos , ARN Interferente Pequeño/genética
2.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-33805602

RESUMEN

Carriers of genetic material are divided into vectors of viral and non-viral origin. Viral carriers are already successfully used in experimental gene therapies, but despite advantages such as their high transfection efficiency and the wide knowledge of their practical potential, the remaining disadvantages, namely, their low capacity and complex manufacturing process, based on biological systems, are major limitations prior to their broad implementation in the clinical setting. The application of non-viral carriers in gene therapy is one of the available approaches. Poly(amidoamine) (PAMAM) dendrimers are repetitively branched, three-dimensional molecules, made of amide and amine subunits, possessing unique physiochemical properties. Surface and internal modifications improve their physicochemical properties, enabling the increase in cellular specificity and transfection efficiency and a reduction in cytotoxicity toward healthy cells. During the last 10 years of research on PAMAM dendrimers, three modification strategies have commonly been used: (1) surface modification with functional groups; (2) hybrid vector formation; (3) creation of supramolecular self-assemblies. This review describes and summarizes recent studies exploring the development of PAMAM dendrimers in anticancer gene therapies, evaluating the advantages and disadvantages of the modification approaches and the nanomedicine regulatory issues preventing their translation into the clinical setting, and highlighting important areas for further development and possible steps that seem promising in terms of development of PAMAM as a carrier of genetic material.


Asunto(s)
Dendrímeros/síntesis química , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/terapia , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/síntesis química , Dendrímeros/administración & dosificación , Regulación Gubernamental , Humanos , MicroARNs/administración & dosificación , MicroARNs/genética , MicroARNs/metabolismo , Nanomedicina/legislación & jurisprudencia , Nanomedicina/métodos , Nanopartículas/administración & dosificación , Nanopartículas/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Plásmidos/administración & dosificación , Plásmidos/química , Plásmidos/metabolismo , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Propiedades de Superficie
3.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799604

RESUMEN

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men in Western countries, and there is still an urgent need for a better understanding of PCa progression to inspire new treatment strategies. Skp2 is a substrate-recruiting component of the E3 ubiquitin ligase complex, whose activity is regulated through neddylation. Slug is a transcriptional repressor involved in the epithelial-to-mesenchymal transition, which may contribute to therapy resistance. Although Skp2 has previously been associated with a mesenchymal phenotype and prostate cancer progression, the relationship with Slug deserves further elucidation. We have previously shown that a high Gleason score (≥8) is associated with higher Skp2 and lower E-cadherin expression. In this study, significantly increased expression of Skp2, AR, and Slug, along with E-cadherin downregulation, was observed in primary prostate cancer in patients who already had lymph node metastases. Skp2 was slightly correlated with Slug and AR in the whole cohort (Rs 0.32 and 0.37, respectively), which was enhanced for both proteins in patients with high Gleason scores (Rs 0.56 and 0.53, respectively) and, in the case of Slug, also in patients with metastasis to lymph nodes (Rs 0.56). Coexpression of Skp2 and Slug was confirmed in prostate cancer tissues by multiplex immunohistochemistry and confocal microscopy. The same relationship between these two proteins was observed in three sets of prostate epithelial cell lines (PC3, DU145, and E2) and their mesenchymal counterparts. Chemical inhibition of Skp2, but not RNA interference, modestly decreased Slug protein in PC3 and its docetaxel-resistant subline PC3 DR12. Importantly, chemical inhibition of Skp2 by MLN4924 upregulated p27 and decreased Slug expression in PC3, PC3 DR12, and LAPC4 cells. Novel treatment strategies targeting Skp2 and Slug by the neddylation blockade may be promising in advanced prostate cancer, as recently documented for other aggressive solid tumors.


Asunto(s)
Proteína NEDD8/genética , Neoplasias de la Próstata/genética , Procesamiento Proteico-Postraduccional , Proteínas Quinasas Asociadas a Fase-S/genética , Factores de Transcripción de la Familia Snail/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antineoplásicos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclopentanos/farmacología , Docetaxel/farmacología , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Masculino , Proteína NEDD8/metabolismo , Clasificación del Tumor , Células PC-3 , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Pirimidinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo
5.
Respir Res ; 22(1): 99, 2021 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-33823870

RESUMEN

BACKGROUND: COVID-19 pneumonia has been associated with severe acute hypoxia, sepsis-like states, thrombosis and chronic sequelae including persisting hypoxia and fibrosis. The molecular hypoxia response pathway has been associated with such pathologies and our recent observations on anti-hypoxic and anti-inflammatory effects of whole aqueous extract of Adhatoda Vasica (AV) prompted us to explore its effects on relevant preclinical mouse models. METHODS: In this study, we tested the effect of whole aqueous extract of AV, in murine models of bleomycin induced pulmonary fibrosis, Cecum Ligation and Puncture (CLP) induced sepsis, and siRNA induced hypoxia-thrombosis phenotype. The effect on lung of AV treated naïve mice was also studied at transcriptome level. We also determined if the extract may have any effect on SARS-CoV2 replication. RESULTS: Oral administration AV extract attenuates increased airway inflammation, levels of transforming growth factor-ß1 (TGF-ß1), IL-6, HIF-1α and improves the overall survival rates of mice in the models of pulmonary fibrosis and sepsis and rescues the siRNA induced inflammation and associated blood coagulation phenotypes in mice. We observed downregulation of hypoxia, inflammation, TGF-ß1, and angiogenesis genes and upregulation of adaptive immunity-related genes in the lung transcriptome. AV treatment also reduced the viral load in Vero cells infected with SARS-CoV2. CONCLUSION: Our results provide a scientific rationale for this ayurvedic herbal medicine in ameliorating the hypoxia-hyperinflammation features and highlights the repurposing potential of AV in COVID-19-like conditions.


Asunto(s)
Antiinflamatorios/farmacología , Reposicionamiento de Medicamentos , Hipoxia/tratamiento farmacológico , Justicia , Pulmón/efectos de los fármacos , Extractos Vegetales/farmacología , Neumonía/prevención & control , Fibrosis Pulmonar/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Animales , Antiinflamatorios/aislamiento & purificación , Bleomicina , /virología , Ciego/microbiología , Ciego/cirugía , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Hipoxia/genética , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Mediadores de Inflamación/metabolismo , Justicia/química , Ligadura , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Extractos Vegetales/aislamiento & purificación , Neumonía/genética , Neumonía/metabolismo , Neumonía/microbiología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Sepsis/genética , Sepsis/metabolismo , Sepsis/microbiología , Transcriptoma
6.
Life Sci ; 274: 119313, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33667511

RESUMEN

AIMS: To design and evaluate a novel AWRK6 peptide-based long-acting GLP-1 receptor agonist (GLP-1RA) conjugated a recombinant polyethylene glycol mimetic (XTEN protein) with significant therapeutic potential on type 2 diabetes mellitus (T2DM) alone as well as Herpes simplex virus type 2 (HSV-2) infection in combination with double shRNA. MAIN METHODS: First, four AWRK6 analogs (termed XA-1 to XA-4) were designed and produced by solid phase synthesis strategy. Further surface plasmon resonance (SPR) measurement and in vitro cAMP accumulation assay were performed to detect the GLP-1R binding affinities and GLP-1R activation, respectively. The in vivo efficacy evaluation including pharmacokinetic test, oral glucose tolerance test (OGTT), hypoglycemic duration test and chronic pharmacodynamics study in rodent animals were all carefully performed. KEY FINDINGS: Four XA peptides were synthesized with purity >99%. High binding affinity as well as activation potency of XA-4 for GLP-1R were demonstrated by SPR and cell-based luciferase reporter assay, respectively. Additionally, XA-4 exhibited the long-lasting antidiabetic effects in the multiple OGTTs, hypoglycemic duration test and chronic study in mice. Furthermore, combined treatment of XA-4 and double shRNA (D-shRNA) achieved potent antiviral effects in HSV-2 infected HEK293 cells. SIGNIFICANCE: XA-4 exhibited promising pharmaceutical potential to be a therapeutic drug for treating T2D, and also held potential to against the HSV-2 infection, which is really an accidental discovery. The strategy of recombinant XTENylation can also be applied to other peptides or small molecules for the development of long-acting therapeutic drugs.


Asunto(s)
Diabetes Mellitus Experimental/terapia , Herpes Simple/terapia , Herpesvirus Humano 2/efectos de los fármacos , Obesidad/complicaciones , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , ARN Interferente Pequeño/administración & dosificación , Animales , Terapia Combinada , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/patología , Dieta Alta en Grasa/efectos adversos , Herpes Simple/genética , Herpes Simple/virología , Herpesvirus Humano 2/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Fragmentos de Péptidos/química , Péptidos/química , ARN Interferente Pequeño/genética
7.
Int J Nanomedicine ; 16: 1805-1817, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33692623

RESUMEN

Introduction: RNA interference is a promising therapy in glioma treatment. However, the application of RNA interference has been limited in glioma therapy by RNA instability and the lack of tumor targeting. Here, we report a novel DNA tetrahedron, which can effectively deliver small interfering RNA to glioma cells and induce apoptosis. Methods: siRNA, a small interfering RNA that can suppress the expression of survivin in glioma, was loaded into the DNA tetrahedron (TDN). To enhance the ability of active targeting of this nanoparticle, we modified one side of the DNA nanostructure with aptamer as1411 (As-TDN-R), which can selectively recognize the nucleolin in the cytomembrane of tumor cells. The modified nanoparticles were characterized by agarose gel electrophoresis, dynamic light scattering, and transmission electron microscopy. The serum stability was evaluated by agarose gel electrophoresis. Nucleolin was detected by Western blot and immunofluorescence, and targeted cellular uptake was examined by flow cytometry. The TUNEL assay, flow cytometry, and Western Blot were used to detect apoptosis in U87 cells. The gene silencing of survivin was examined by qPCR, Western Blot, and immunofluorescence. Results: As-TDN-R alone showed better stability towards siRNA, indicating that TDN was a good siRNA protector. Compared with TDN alone, there was increased intercellular uptake of As-TDN-R by U87 cells, evidenced by overexpressed nucleolin in glioma cell lines. TUNEL assay, flow cytometry, and Western Blot revealed increased apoptosis in the As-TDN-R group. The downregulation of survivin protein and mRNA expression levels indicated that As-TDN-R effectively silenced the target gene. Conclusion: The novel nanoparticle can serve as a good carrier for targeting siRNA delivery in glioma. Further exploration of the DNA nanostructure can greatly promote the application of DNA-based drug systems in glioma.


Asunto(s)
ADN/química , Técnicas de Transferencia de Gen , Glioma/terapia , Nanoestructuras/química , ARN Interferente Pequeño/administración & dosificación , Apoptosis/efectos de los fármacos , Aptámeros de Nucleótidos/química , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Endocitosis , Silenciador del Gen , Glioma/patología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Nanoestructuras/ultraestructura , Oligodesoxirribonucleótidos/química , Fosfoproteínas/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Survivin/metabolismo
8.
Nat Commun ; 12(1): 1436, 2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33664241

RESUMEN

Acute kidney injury (AKI) is a prevalent and lethal adverse event that severely affects cancer patients receiving chemotherapy. It is correlated with the collateral damage to renal cells caused by reactive oxygen species (ROS). Currently, ROS management is a practical strategy that can reduce the risk of chemotherapy-related AKI, but at the cost of chemotherapeutic efficacy. Herein, we report catalytic activity tunable ceria nanoparticles (CNPs) that can prevent chemotherapy-induced AKI without interference with chemotherapeutic agents. Specifically, in the renal cortex, CNPs exhibit catalytic activity that decomposes hydrogen peroxide, and subsequently regulate the ROS-involved genes by activating the Nrf2/Keap1 signaling pathway. These restore the redox homeostasis for the protection of kidney tubules. Under an acidic tumor microenvironment, CNPs become inert due to the excessive H+ that disrupts the re-exposure of active catalytic sites, allowing a buildup of chemotherapy-mediated ROS generation to kill cancer cells. As ROS-modulating agents, CNPs incorporated with context-dependent catalytic activity, hold a great potential for clinical prevention and treatment of AKI in cancer patients.


Asunto(s)
Lesión Renal Aguda/prevención & control , Antineoplásicos/efectos adversos , Cerio/farmacología , Túbulos Renales/patología , Especies Reactivas de Oxígeno/metabolismo , Células A549 , Animales , Antineoplásicos/uso terapéutico , Dominio Catalítico , Línea Celular Tumoral , Cerio/química , Femenino , Células Hep G2 , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral
9.
BMC Cancer ; 21(1): 207, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33648461

RESUMEN

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.


Asunto(s)
Adenocarcinoma/genética , Adenoma/genética , Neoplasias Colorrectales/genética , Genes Relacionados con las Neoplasias , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-ets/fisiología , Adenocarcinoma/química , Adenocarcinoma/patología , Adenoma/química , Adenoma/patología , Anciano , Biomarcadores de Tumor/genética , Brasil , División Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-ets/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Análisis de Matrices Tisulares , Transcriptoma , Ensayo de Tumor de Célula Madre
10.
J Biomed Nanotechnol ; 17(2): 322-329, 2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33785102

RESUMEN

Due to the complex physiological characteristics of tumors, chemotherapy or gene therapy alone cannot completely kill tumor cells. Therefore, combining chemotherapy with gene therapy for combination therapy is the key to solving this problem. However, there are still significant challenges in how to simultaneously deliver and rapidly release the drugs and siRNA into cancer cells. In this work, a triblock copolymer was synthesized to co-deliver siRNA and paclitaxel to tumor cells. This system has an acid-sensitive subsurface layer, which can not only load siRNA to prevent premature drug release but also has good controlled release performance. In vitro experiments showed that polymeric vectors can efficiently deliver siRNA and paclitaxel simultaneously into tumor cells for rapid release within the tumor cells. This study reveals that this novel polymeric micelle is a suitable vector for the codelivery of chemotherapeutic drugs and siRNA to cancer cells, representing an important advance in nanotechnology, nanomedicine, drug delivery, and cancer therapy.


Asunto(s)
Micelas , Neoplasias , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Concentración de Iones de Hidrógeno , Neoplasias/genética , Neoplasias/terapia , Paclitaxel , Polímeros , ARN Interferente Pequeño/genética
11.
Nat Commun ; 12(1): 1946, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782401

RESUMEN

Numerous substrates have been identified for Type I and II arginine methyltransferases (PRMTs). However, the full substrate spectrum of the only type III PRMT, PRMT7, and its connection to type I and II PRMT substrates remains unknown. Here, we use mass spectrometry to reveal features of PRMT7-regulated methylation. We find that PRMT7 predominantly methylates a glycine and arginine motif; multiple PRMT7-regulated arginine methylation sites are close to phosphorylations sites; methylation sites and proximal sequences are vulnerable to cancer mutations; and methylation is enriched in proteins associated with spliceosome and RNA-related pathways. We show that PRMT4/5/7-mediated arginine methylation regulates hnRNPA1 binding to RNA and several alternative splicing events. In breast, colorectal and prostate cancer cells, PRMT4/5/7 are upregulated and associated with high levels of hnRNPA1 arginine methylation and aberrant alternative splicing. Pharmacological inhibition of PRMT4/5/7 suppresses cancer cell growth and their co-inhibition shows synergistic effects, suggesting them as targets for cancer therapy.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias Colorrectales/genética , Ribonucleoproteína Nuclear Heterogénea A1/genética , Neoplasias de la Próstata/genética , Proteína-Arginina N-Metiltransferasas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Arginina/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Inhibidores Enzimáticos/farmacología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea A1/antagonistas & inhibidores , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Masculino , Metilación/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Empalmosomas/efectos de los fármacos , Empalmosomas/genética , Empalmosomas/metabolismo , Especificidad por Sustrato
12.
Nat Commun ; 12(1): 1940, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782411

RESUMEN

Metabolic enzymes and metabolites display non-metabolic functions in immune cell signalling that modulate immune attack ability. However, whether and how a tumour's metabolic remodelling contributes to its immune resistance remain to be clarified. Here we perform a functional screen of metabolic genes that rescue tumour cells from effector T cell cytotoxicity, and identify the embryo- and tumour-specific folate cycle enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2). Mechanistically, MTHFD2 promotes basal and IFN-γ-stimulated PD-L1 expression, which is necessary for tumourigenesis in vivo. Moreover, IFN-γ stimulates MTHFD2 through the AKT-mTORC1 pathway. Meanwhile, MTHFD2 drives the folate cycle to sustain sufficient uridine-related metabolites including UDP-GlcNAc, which promotes the global O-GlcNAcylation of proteins including cMYC, resulting in increased cMYC stability and PD-L1 transcription. Consistently, the O-GlcNAcylation level positively correlates with MTHFD2 and PD-L1 in pancreatic cancer patients. These findings uncover a non-metabolic role for MTHFD2 in cell signalling and cancer biology.


Asunto(s)
Aminohidrolasas/genética , Antígeno B7-H1/genética , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Enzimas Multifuncionales/genética , Neoplasias Pancreáticas/genética , Procesamiento Proteico-Postraduccional , Linfocitos T Citotóxicos/inmunología , Aminohidrolasas/antagonistas & inhibidores , Aminohidrolasas/inmunología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Carcinogénesis/inmunología , Carcinogénesis/patología , Línea Celular Tumoral , Embrión de Mamíferos , Fibroblastos/inmunología , Fibroblastos/patología , Ácido Fólico/inmunología , Ácido Fólico/metabolismo , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/inmunología , Metilenotetrahidrofolato Deshidrogenasa (NADP)/antagonistas & inhibidores , Metilenotetrahidrofolato Deshidrogenasa (NADP)/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Enzimas Multifuncionales/antagonistas & inhibidores , Enzimas Multifuncionales/inmunología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/inmunología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Transducción de Señal , Linfocitos T Citotóxicos/patología , Carga Tumoral , Escape del Tumor , Uridina Difosfato N-Acetilglucosamina/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Int J Mol Sci ; 22(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652577

RESUMEN

Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells' viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.


Asunto(s)
Silenciador del Gen , Técnicas de Transferencia de Gen , Virus de la Hepatitis B , Plásmidos , Proteínas Proto-Oncogénicas c-bcl-2 , ARN Interferente Pequeño , Neoplasias del Cuello Uterino , Femenino , Células HeLa , Humanos , Plásmidos/genética , Plásmidos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología
14.
Nat Commun ; 12(1): 1881, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767157

RESUMEN

To achieve the very high oncoprotein levels required to drive the malignant state cancer cells utilise the ubiquitin proteasome system to upregulate transcription factor levels. Here our analyses identify ALYREF, expressed from the most common genetic copy number variation in neuroblastoma, chromosome 17q21-ter gain as a key regulator of MYCN protein turnover. We show strong co-operativity between ALYREF and MYCN from transgenic models of neuroblastoma in vitro and in vivo. The two proteins form a nuclear coactivator complex which stimulates transcription of the ubiquitin specific peptidase 3, USP3. We show that increased USP3 levels reduce K-48- and K-63-linked ubiquitination of MYCN, thus driving up MYCN protein stability. In the MYCN-ALYREF-USP3 signal, ALYREF is required for MYCN effects on the malignant phenotype and that of USP3 on MYCN stability. This data defines a MYCN oncoprotein dependency state which provides a rationale for future pharmacological studies.


Asunto(s)
Carcinogénesis/patología , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/patología , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cromosomas Humanos Par 17/genética , Variaciones en el Número de Copia de ADN/genética , Células HEK293 , Humanos , Ratones , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Activación Transcripcional/genética , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/fisiología
15.
Nat Commun ; 12(1): 1865, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767158

RESUMEN

Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of this global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of "stemness" and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.


Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 2/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Células Cultivadas , Cromatina/metabolismo , Femenino , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño/genética , Ubiquitina Tiolesterasa/genética
16.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(3): 233-239, 2021 Mar.
Artículo en Chino | MEDLINE | ID: mdl-33766231

RESUMEN

Objective To investigate the effects of knockdown of Aurora-A gene on the proliferation and apoptosis of HepG2 human hepatocellular carcinoma cells. Methods Aurora-A short hairpin RNA (Aurora-A shRNA) was designed and Aurora-A shRNA lentiviral vector was constructed and packed, and then transfected into HepG2 cells. Aurora-A mRNA expression was detected by real-time quantitative PCR. Aurora-A protein expression and phosphorylation level were detected by Western blotting. Cell proliferation was tested by MTT assay. Cell apoptosis was analyzed by flow cytometry. Results The Aurora-A shRNA lentiviral vector was successfully constructed and Aurora-A protein phosphorylation level was significantly reduced in HepG2 cells transfected with the lentiviral vector. When Aurora-a was knocked down, the proliferation of HepG2 cells decreased and the apoptosis rate increased significantly. Conclusion Knockdown of Aurora-A can inhibit the proliferation and promote the apoptosis of HepG2 cells.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptosis/genética , Aurora Quinasa A , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Proliferación Celular , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , ARN Interferente Pequeño/genética
17.
Braz J Med Biol Res ; 54(4): e10117, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33656053

RESUMEN

The long noncoding RNA (lncRNA) H19 is involved in the pathogenesis of endometriosis by modulating the proliferation and invasion of ectopic endometrial cells in vitro, but related in vivo studies are rare. This study aimed to investigate the role of lncRNA H19 in a nude mouse model of endometriosis. Ectopic endometrial stromal cells (ecESCs) were isolated from ectopic endometrium of patients with endometriosis and infected with lentiviruses expressing short hairpin RNA (shRNA) negative control (LV-NC-shRNA) or lncRNA-H19 shRNA (LV-H19-shRNA). The ecESCs infected with LV-NC-shRNA and LV-H19-shRNA were subcutaneously implanted into forty 6- to 8-week-old female nude mice. The size and weight of the endometriotic implants were measured at 1, 2, 3, and 4 weeks after implantation and compared, and lncRNA H19 levels in endometriotic implants were evaluated using real-time polymerase chain reaction (RT-PCR). All nude mice survived the experimental period, and no significant differences in body weight were observed between the experimental group and the control group. All nude mice developed histologically confirmed subcutaneous endometriotic lesions with glandular structures and stroma after 1 week of implantation. The subcutaneous lesions in the LV-NC-shRNA group after 1, 2, 3, and 4 weeks of implantation were larger than those in the LV-H19-shRNA group, and lncRNA H19 levels in subcutaneous lesions in the LV-NC-shRNA group were significantly higher than those in the LV-H19-shRNA group. Knockdown of lncRNA H19 suppresses endometriosis in vivo. Further study is required to explore the underlying mechanism in the future.


Asunto(s)
Endometriosis , ARN Largo no Codificante , Animales , Proliferación Celular/genética , Endometriosis/genética , Endometrio , Femenino , Humanos , Ratones , Ratones Desnudos , ARN Largo no Codificante/genética , ARN Interferente Pequeño/genética
18.
Zhonghua Gan Zang Bing Za Zhi ; 29(2): 126-132, 2021 Feb 20.
Artículo en Chino | MEDLINE | ID: mdl-33685080

RESUMEN

Objective: To study the use of preS1-tp fusion protein as a novel vector to mediate the entry of small interfering RNA (siRNA) targeting the carboxy-terminal nuclear localization signal (NLS) region of hepatitis B virus (HBV) core protein into HBV-infected hepatocytes, and to further explore the HBV replication inhibition and covalently closed circular DNA synthesis. Methods: HepG2.2.15 cells expressing the human sodium taurocholate co-transporting polypeptide were established on the basis of lentivirus infection system. siRNA against HBV NLS region was designed and synthesized. PreS1-tp fusion protein expression and purification was observed to test its ability to cell entry and DNA binding. NLS siRNA were delivered into HepG2.2.15- sodium taurocholate co-transporting polypeptide cells by preS1-tp fusion protein as a vector to observe the effects of NLS siRNA on HBV replication and covalently closed circular DNA levels. Analysis of variance was used for comparison between multiple groups, and the measurement data differences between groups were analyzed by t-test. Results: HepG2.2.15-sodium taurocholate co-transporting polypeptide cell line was successfully constructed. Screened synthetic HBV NLS siRNA had significantly inhibited HBV replication. The preS1-tp fusion protein was expressed and purified on a large-scale. The fusion protein as a vector for HBV NLS siRNA had targeted delivery. The result showed that the fusion protein had effectively targeted siRNA to Hepg2.2.15-sodium taurocholate co-transporting polypeptide cell, which not only had effectively inhibited the expression of HBV mRNA, HBsAg and HBeAg, but also had significantly reduced the levels of HBV DNA and covalently closed circular DNA. Conclusion: The preS1-tp fusion protein constructed in this study uses the dual functional characteristics of preS1 binding to hepatocyte HBV receptor, and tp binding to nucleic acids, and targets HBV NLS siRNA against HBV-infected cells and block rcDNA from being transported to nucleus. siRNA plays a role in inhibiting HBV replication and covalently close circular DNA synthesis, providing a new strategy for the treatment of chronic hepatitis B caused by HBV infection, and a new research perspective for the complete elimination of HBV from the body.


Asunto(s)
Virus de la Hepatitis B , Hepatitis B , ADN Circular/genética , ADN Viral/genética , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , ARN Interferente Pequeño/genética , Replicación Viral
19.
Methods Mol Biol ; 2244: 115-132, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33555585

RESUMEN

Human fibroblasts represent the most extensively used cell type for the investigation of lytic human cytomegalovirus (HCMV) replication. However, analyzing the function of specific proteins during infection can be challenging since primary cells are difficult to transfect. An alternative approach is the use of lentiviral transduction with vectors for stable or inducible shRNA expression. This approach provides a versatile tool to study the role of host cell factors during HCMV infection. The essential steps to achieve an efficient target protein knockdown are shRNA design, cloning, generation of transgenic lentiviral particles, and, finally, transduction of the cells. However, these steps are highly dependent on the selected vector system. Here we focus on two different vector systems and describe how to successfully generate stable and inducible knockdown fibroblasts. Additionally, we demonstrate different methods to validate the knockdown of the target protein.


Asunto(s)
Citomegalovirus/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Cultivo Primario de Células/métodos , Línea Celular , Infecciones por Citomegalovirus/virología , Fibroblastos/citología , Fibroblastos/metabolismo , Vectores Genéticos/genética , Humanos , ARN Interferente Pequeño/genética , Transfección/métodos , Proteínas Virales , Replicación Viral/fisiología
20.
Nucleic Acids Res ; 49(5): 2700-2720, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33590099

RESUMEN

In animal gonads, transposable elements are actively repressed to preserve genome integrity through the PIWI-interacting RNA (piRNA) pathway. In mice, piRNAs are abundantly expressed in male germ cells, and form effector complexes with three distinct PIWIs. The depletion of individual Piwi genes causes male-specific sterility with no discernible phenotype in female mice. Unlike mice, most other mammals have four PIWI genes, some of which are expressed in the ovary. Here, purification of PIWI complexes from oocytes of the golden hamster revealed that the size of the PIWIL1-associated piRNAs changed during oocyte maturation. In contrast, PIWIL3, an ovary-specific PIWI in most mammals, associates with short piRNAs only in metaphase II oocytes, which coincides with intense phosphorylation of the protein. An improved high-quality genome assembly and annotation revealed that PIWIL1- and PIWIL3-associated piRNAs appear to share the 5'-ends of common piRNA precursors and are mostly derived from unannotated sequences with a diminished contribution from TE-derived sequences, most of which correspond to endogenous retroviruses. Our findings show the complex and dynamic nature of biogenesis of piRNAs in hamster oocytes, and together with the new genome sequence generated, serve as the foundation for developing useful models to study the piRNA pathway in mammalian oocytes.


Asunto(s)
Proteínas Argonauta/metabolismo , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonauta/genética , Femenino , Genómica , Masculino , Mesocricetus , Metafase , Fosforilación , ARN Interferente Pequeño/genética , Testículo/metabolismo
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