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1.
Am J Obstet Gynecol ; 229(1): 53.e1-53.e8, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36596438

RESUMO

BACKGROUND: In utero repair of open neural tube defects using an open hysterotomy approach (hereafter referred to as "open") has been shown to reduce the need for ventriculoperitoneal shunting and to improve motor outcomes for affected infants. Laparotomy-assisted fetoscopic repair (hereafter referred to as "hybrid") is an alternative approach that may confer similar neurologic benefits while reducing the incidence of hysterotomy-related complications. OBJECTIVE: This study aimed to analyze procedure-related maternal and fetal complications of in utero repair using the Clavien-Dindo classification, and to compare the outcomes of the hybrid and open approaches. STUDY DESIGN: This was a retrospective cohort study conducted in a single center between September 2011 and July 2021. All patients who met the Management of Myelomeningocele Study criteria and who underwent either hybrid or open fetal surgery were included. Maternal complications were classified using a unique adaptation of the Clavien-Dindo scoring system, allowing the development of a comprehensive complication index score specific to fetal surgery. Primary fetal outcome was defined as gestational age at delivery and summarized according to the World Health Organization definitions of preterm delivery. RESULTS: There were 146 fetuses with open neural tube defects who were eligible for, and underwent, in utero repair during the study period. Of these, 102 underwent hybrid fetoscopic repair and 44 underwent open hysterotomy repair. Gestational age at the time of surgery was higher in the hybrid group than in the open group (25.1 vs 24.8 weeks; P=.004). Maternal body mass index was lower in the hybrid than in the open group (25.4 vs 27.1 kg/m2; P=.02). The duration of hybrid fetoscopic surgery was significantly longer in the hybrid than in the open group (250 vs 164 minutes; P<.001). There was a significantly lower Clavien-Dindo Grade III complication rate (4.9% vs 43.2%; P<.001) and a significantly lower overall comprehensive maternal complication index (8.7 vs 22.6; P=.021) in the hybrid group than in the open group. Gestational age at delivery was significantly higher in the hybrid group than in the open group (38.1 vs 35.8 weeks; P<.001), and this finding persisted when gestational age at delivery was analyzed using the World Health Organization definitions of preterm delivery. CONCLUSION: Use of our adaptation of the standardized Clavien-Dindo classification to assess the maternal complications associated with in utero open neural tube defect repair provides a new method for objectively assessing different fetal surgical approaches. It also provides a much-needed standardized tool to allow objective comparisons between methods, which can be used when counseling patients. The hybrid open neural tube defect repair was associated with lower rates of maternal adverse events , and later gestational age at delivery compared with the open approach.


Assuntos
Meningomielocele , Defeitos do Tubo Neural , Nascimento Prematuro , Gravidez , Recém-Nascido , Lactente , Feminino , Humanos , Nascimento Prematuro/etiologia , Estudos Retrospectivos , Feto/cirurgia , Meningomielocele/cirurgia , Fetoscopia/métodos , Idade Gestacional , Defeitos do Tubo Neural/cirurgia
2.
Clin Cancer Res ; 27(4): 1139-1149, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33208342

RESUMO

PURPOSE: miRNA-155 is an oncogenic miRNA highly expressed in B-cell malignancies, particularly in the non-germinal center B-cell or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL. EXPERIMENTAL DESIGN: Preclinical studies included the delivery of cobomarsen to highly miR-155-expressing ABC-DLBCL cell lines to assess any phenotypic changes, as well as intravenous injections of cobomarsen in NSG mice carrying ABC-DLBCL xenografts, to study tumor growth and pharmacodynamics of the compound over time. To begin to test its safety and therapeutic efficacy, a patient was recruited who underwent five cycles of cobomarsen treatment. RESULTS: Cobomarsen decreased cell proliferation and induced apoptosis in ABC-DLBCL cell lines. Intravenous administration of cobomarsen in a xenograft NSG mouse model of ABC-DLBCL reduced tumor volume, triggered apoptosis, and derepressed direct miR-155 target genes. Finally, the compound reduced and stabilized tumor growth without any toxic effects for the patient. CONCLUSIONS: Our findings support the potential therapeutic application of cobomarsen in ABC-DLBCL and other types of lymphoma with elevated miR-155 expression.


Assuntos
Linfoma Difuso de Grandes Células B/tratamento farmacológico , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , MicroRNAs/metabolismo , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Invest Dermatol ; 139(5): 1073-1081, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30472058

RESUMO

MicroRNA-29 (miR-29) negatively regulates fibrosis and is downregulated in multiple fibrotic organs and tissues, including in the skin. miR-29 mimics prevent pulmonary fibrosis in mouse models but have not previously been tested in the skin. This study aimed to identify pharmacodynamic biomarkers of miR-29 in mouse skin, to translate those biomarkers across multiple species, and to assess the pharmacodynamic activity of a miR-29b mimic (remlarsen) in a clinical trial. miR-29 biomarkers were selected based on gene function and mRNA expression using quantitative reverse transcriptase polymerase chain reaction. Those biomarkers comprised multiple collagens and other miR-29 direct and indirect targets and were conserved across species; remlarsen regulated their expression in mouse, rat, and rabbit skin wounds and in human skin fibroblasts in culture, while a miR-29 inhibitor reciprocally regulated their expression. Biomarker expression translated to clinical proof-of-mechanism; in a double-blinded, placebo-randomized, within-subject controlled clinical trial of single and multiple ascending doses of remlarsen in normal healthy volunteers, remlarsen repressed collagen expression and the development of fibroplasia in incisional skin wounds. These results suggest that remlarsen may be an effective therapeutic to prevent formation of a fibrotic scar (hypertrophic scar or keloid) or to prevent cutaneous fibrosis, such as scleroderma.


Assuntos
Matriz Extracelular/metabolismo , MicroRNAs/genética , Dermatopatias/patologia , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Fibrose/genética , Fibrose/patologia , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , MicroRNAs/farmacologia , Estudos Prospectivos , Dermatopatias/tratamento farmacológico , Dermatopatias/genética , Resultado do Tratamento
4.
Br J Haematol ; 183(3): 428-444, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30125933

RESUMO

miR-155, a microRNA associated with poor prognosis in lymphoma and leukaemia, has been implicated in the progression of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). In this study, we developed and tested cobomarsen (MRG-106), a locked nucleic acid-modified oligonucleotide inhibitor of miR-155. In MF and human lymphotropic virus type 1 (HTLV-1+) CTCL cell lines in vitro, inhibition of miR-155 with cobomarsen de-repressed direct miR-155 targets, decreased expression of multiple gene pathways associated with cell survival, reduced survival signalling, decreased cell proliferation and activated apoptosis. We identified a set of genes that are significantly regulated by cobomarsen, including direct and downstream targets of miR-155. Using clinical biopsies from MF patients, we demonstrated that expression of these pharmacodynamic biomarkers is dysregulated in MF and associated with miR-155 expression level and MF lesion severity. Further, we demonstrated that miR-155 simultaneously regulates multiple parallel survival pathways (including JAK/STAT, MAPK/ERK and PI3K/AKT) previously associated with the pathogenesis of MF, and that these survival pathways are inhibited by cobomarsen in vitro. A first-in-human phase 1 clinical trial of cobomarsen in patients with CTCL is currently underway, in which the panel of proposed biomarkers will be leveraged to assess pharmacodynamic response to cobomarsen therapy.


Assuntos
Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Linfoma Cutâneo de Células T , MicroRNAs/antagonistas & inibidores , Oligonucleotídeos/farmacologia , RNA Neoplásico/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaios Clínicos Fase I como Assunto , Intervalo Livre de Doença , Feminino , Infecções por HTLV-I/tratamento farmacológico , Infecções por HTLV-I/metabolismo , Infecções por HTLV-I/mortalidade , Infecções por HTLV-I/patologia , Humanos , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/mortalidade , Linfoma Cutâneo de Células T/patologia , Masculino , MicroRNAs/metabolismo , RNA Neoplásico/metabolismo , Taxa de Sobrevida
5.
Wound Repair Regen ; 26(4): 311-323, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30118158

RESUMO

There is a strong unmet need for new therapeutics to accelerate wound healing across both chronic and acute indications. It is well established that local tissue hypoxia, vascular insufficiency, and/or insufficient angiogenesis contribute to inadequate wound repair in the context of diabetic foot ulcers as well as to other chronic wounds such as venous stasis and pressure ulcers. microRNA-92a-3p (miR-92a) is a potent antiangiogenic miRNA whose inhibition has led to increases in angiogenesis in multiple organ systems, resulting in an improvement in function following myocardial infarction, limb ischemia, vascular injury, and bone fracture. Due to their pro-angiogenic effects, miR-92a inhibitors offer potential therapeutics to accelerate the healing process in cutaneous wounds as well. This study investigated the effect of a development stage locked nucleic acid-modified miR-92a inhibitor, MRG-110, in excisional wounds in db/db mice and in normal pigs. In both acute and chronic wounds, MRG-110 increased granulation tissue formation as assessed by histology, angiogenesis as assessed by immunohistochemistry and tissue perfusion, and wound healing as measured by time to closure and percent closure over time. The effects of MRG-110 were greater than those that were observed with the positive controls rhVEGF-165 and rhPDGF-BB, and MRG-110 was at least additive with rhPDGF-BB when co-administered in db/db mouse wounds. MRG-110 was found to up-regulate expression of the pro-angiogenic miR-92a target gene integrin alpha 5 in vitro in both human vascular endothelial cells and primary human skin fibroblasts and in vivo in mouse skin, demonstrating its on-target effects in vitro and in vivo. Additional safety endpoints were assessed in both the mouse and pig studies with no safety concerns noted. These studies suggest that MRG-110 has the potential to accelerate both chronic and acute wound healing and these data provide support for future clinical trials of MRG-110.


Assuntos
Indutores da Angiogênese/farmacologia , Pé Diabético/complicações , MicroRNAs/antagonistas & inibidores , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/complicações , Ferimentos e Lesões/tratamento farmacológico , Animais , Células Endoteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Tecido de Granulação/patologia , Humanos , Masculino , Camundongos , Modelos Animais , Neovascularização Patológica/patologia , Oligonucleotídeos Antissenso/metabolismo , Transdução de Sinais , Suínos
6.
Mol Ther ; 25(3): 694-704, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28202391

RESUMO

MicroRNAs (miRNAs) are important regulators of biology and disease. Recent animal efficacy studies validate the therapeutic benefit of miRNA modulation and underscore the therapeutic value of miRNA-targeting oligonucleotides. However, whether disease conditions (stress) influence the pharmacological effects of an anti-miR is currently unknown. To study the effect of disease on target regulation after anti-miR treatment, we injected animals with anti-miR-208a, a synthetic oligonucleotide that inhibits the cardiomyocyte-specific miR-208a. Our data indicate that the presence of stress increases the number of regulated miR-208a targets, and that higher stress levels correlate with stronger target derepression. Additionally, the type of stress also influences which targets are regulated upon miR-208a inhibition. Studies in a large animal model indicate a similar stress-dependent anti-miR effect. Subsequent in vitro studies suggest that the influence of stress on anti-miR efficacy depends at least in part on increased cellular anti-miR uptake. These data indicate that the pharmacological effect of anti-miRs is stronger under disease conditions, and that both the type and severity of disease determine the therapeutic outcome. These facts will be important for assessing the therapeutic dose and predicting the therapeutic outcome when applying anti-miRs in a clinical setting.


Assuntos
Antagomirs/genética , MicroRNAs/genética , Miócitos Cardíacos/metabolismo , Estresse Fisiológico/genética , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Interferência de RNA , Ratos , Suínos
7.
Regul Toxicol Pharmacol ; 66(2): 167-76, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23557984

RESUMO

Gene expression can be modulated in plants to produce desired traits through agricultural biotechnology. Currently, biotechnology-derived crops are compared to their conventional counterparts, with safety assessments conducted on the genetic modification and the intended and unintended differences. This review proposes that this comparative safety assessment paradigm is appropriate for plants modified to express mediators of RNA-mediated gene regulation, including RNA interference (RNAi), a gene suppression mechanism that naturally occurs in plants and animals. The molecular mediators of RNAi, including long double-stranded RNAs (dsRNA), small interfering RNAs (siRNA), and microRNAs (miRNA), occur naturally in foods; therefore, there is an extensive history of safe consumption. Systemic exposure following consumption of plants containing dsRNAs that mediate RNAi is limited in higher organisms by extensive degradation of ingested nucleic acids and by biological barriers to uptake and efficacy of exogenous nucleic acids. A number of mammalian RNAi studies support the concept that a large margin of safety will exist for any small fraction of RNAs that might be absorbed following consumption of foods from biotechnology-derived plants that employ RNA-mediated gene regulation. Food and feed derived from these crops utilizing RNA-based mechanisms is therefore expected to be as safe as food and feed derived through conventional plant breeding.


Assuntos
Ração Animal , Inocuidade dos Alimentos , Alimentos Geneticamente Modificados , Plantas Geneticamente Modificadas , Animais , Biotecnologia , Regulação da Expressão Gênica , Humanos , RNA de Plantas/genética
8.
Inflamm Allergy Drug Targets ; 12(2): 88-98, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23517647

RESUMO

Chronic respiratory diseases are a significant health problem requiring novel approaches to both complement existing therapies and provide breakthrough medicines. Recent clinical advances in understanding the behavior of inhaled oligonucleotides provide the impetus for application of this technology to microRNA therapeutics. MicroRNAs are evolutionarily conserved small regulatory RNA molecules involved in tuning gene networks controlling biological and pathological processes. Deletion or overexpression of microRNAs results in phenotypic changes in animal models of disease such as cancer, fibrosis, diabetes, and inflammation. Inhibition of microRNAs in preclinical models of asthma, cystic fibrosis, and idiopathic pulmonary fibrosis has shown therapeutic promise. In animals, inhibitors of microRNAs directly delivered to the airway at doses suitable for nebulizers or hand-held inhalers up-regulate expression of cohorts of genes containing complementary "seed" sequences for specific and directed microRNA binding within their mRNA untranslated regions. These observations suggest the opportunity to exploit intervention in microRNA biology to create new therapies for chronic pulmonary disorders.


Assuntos
Pneumopatias/genética , Pneumopatias/terapia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Animais , Doença Crônica , Humanos , Pneumopatias/metabolismo , MicroRNAs/metabolismo
9.
Respir Res ; 13: 92, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-23061798

RESUMO

BACKGROUND: Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system. METHODS: Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools. RESULTS: An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts. CONCLUSIONS: These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.


Assuntos
Quimiocina CCL11/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Camundongos , Fator 2 Relacionado a NF-E2/genética , RNA Interferente Pequeno/genética
10.
Nucleic Acid Ther ; 22(4): 213-25, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22913594

RESUMO

MicroRNAs are endogenous small non-coding RNAs that regulate gene expression by interfering with translation or stability of target transcripts. The importance of microRNAs for maintaining biological functions is illustrated by the fact that microRNAs are exploited in nature to regulate phenotypes, and by the diverse disease phenotypes that result when microRNAs are mutated or improperly expressed. Disease-associated microRNAs might therefore represent a new class of therapeutic targets. With the recent demonstration that inhibition of miR-122 reduces viral load in hepatitis C patients, microRNA modulators are no longer merely theoretical, but rather, have become strong candidate therapeutics. The complexity of microRNA biology offers a novel mechanism of action for therapeutic intervention but also poses unique challenges for the development of therapeutic modulators as drugs.


Assuntos
MicroRNAs/genética , MicroRNAs/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Descoberta de Drogas , Humanos , MicroRNAs/farmacologia , Mimetismo Molecular , Terapia de Alvo Molecular , Especificidade da Espécie
11.
J Biomol Screen ; 17(10): 1316-28, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22786893

RESUMO

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).


Assuntos
Ensaios de Triagem em Larga Escala , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Apoptose/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Reparo do DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases , Interferência de RNA/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos
12.
PLoS One ; 6(3): e14758, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21412408

RESUMO

Mice lacking the p27(Kip1) Cdk inhibitor (Cdkn1b) exhibit increased susceptibility to lymphomas from the Maloney murine leukemia virus (M-MuLV), and exhibit a high frequency of viral integrations at Xpcl1 (Kis2), a locus on the X-chromosome. Xpcl1 encodes miR-106a~363, a cluster of microRNAs that are expressed in response to adjacent retroviral integrations. We report the first large-scale profile of microRNA expression in MuLV-induced lymphomas, in combination with microarray gene expression analysis. The source material was T-cell lymphomas induced by M-MuLV in p27(Kip1) knockout mice and normal thymus. Surprisingly, the overall levels of miRNA expression were equivalent in lymphomas and normal thymus. Nonetheless, the expression of specific microRNAs was altered in tumors. The miR-106a~363 miRNA were over-expressed in lymphomas, particularly those with viral integrations at the Xpcl1 locus. In contrast, p27(Kip1) deletion itself was associated with a different pattern of microRNA expression. Gene expression was dramatically altered in lymphomas, yet paralleled data from T-cell lymphomas induced by other mechanisms. Genes with altered expression in association with the p27(Kip1) null genotype were of similar functional classes to those associated with Xpcl1 integration, but with the opposite pattern of expression. Thus, the effect of p27(Kip1) deletion may be to oppose an anti-oncogenic effect of Xpcl1 rather than enhancing its oncogenic functions. A subset of miR-106a~363 target genes was consistently reduced in lymphomas with Xpcl1 integrations, particularly genes with cell cycle and immune functions. We identify four predicted target genes of miR-106a~363 miRNA, including N-Myc (Mycn), and the TGF-beta receptor (Tgfbr2) using 3'UTR reporter assays. Still, bioinformatic miRNA target predictions were poor predictors of altered gene expression in lymphomas with Xpcl1 integration. Confirmation of miR-106a~363 gene targeting relevant to the tumor phenotype requires in vivo validation, because only a subset of predicted targets are consistently reduced in tumors that overexpress miR-106a~363.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/deficiência , Regulação Neoplásica da Expressão Gênica , Linfoma/genética , MicroRNAs/genética , Animais , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Perfilação da Expressão Gênica , Genótipo , Camundongos , MicroRNAs/metabolismo , Vírus da Leucemia Murina de Moloney/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timo/metabolismo , Integração Viral/genética
13.
BMC Biol ; 8: 53, 2010 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-20529236

RESUMO

Small interfering RNAs can trigger unintended, microRNA-like off-target effects, but the impact of these effects on functional studies has been controversial. A recent study in BMC Genomics shows that microRNA-like effects can predominate among the 'hits' of functional genomics screens.


Assuntos
Perfilação da Expressão Gênica/métodos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Apoptose/genética , Modelos Genéticos , RNA Interferente Pequeno/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
14.
Nat Rev Drug Discov ; 9(1): 57-67, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20043028

RESUMO

Small interfering RNAs (siRNAs) are widely used to study gene function owing to the ease with which they silence target genes, and there is considerable interest in their potential for therapeutic applications. In a remarkably short time since their discovery, siRNAs have entered human clinical trials in various disease areas. However, rapid acceptance of the use of siRNAs has been accompanied by recognition of several hurdles for the technology, including a lack of specificity. Off-target activity can complicate the interpretation of phenotypic effects in gene-silencing experiments and can potentially lead to unwanted toxicities. Here, we describe the types of off-target effects of siRNAs and methods to mitigate them, to help enable effective application of this exciting technology.


Assuntos
Inativação Gênica , Marcação de Genes , RNA Interferente Pequeno/uso terapêutico , Animais , Ensaios Clínicos como Assunto , Terapia Genética/métodos , Humanos , MicroRNAs , RNA Interferente Pequeno/efeitos adversos
15.
EMBO J ; 28(20): 3157-70, 2009 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-19745813

RESUMO

Myc proteins are known to have an important function in stem cell maintenance. As Myc has been shown earlier to regulate microRNAs (miRNAs) involved in proliferation, we sought to determine whether c-Myc also affects embryonic stem (ES) cell maintenance and differentiation through miRNAs. Using a quantitative primer-extension PCR assay we identified miRNAs, including, miR-141, miR-200, and miR-429 whose expression is regulated by c-Myc in ES cells, but not in the differentiated and tumourigenic derivatives of ES cells. Chromatin immunoprecipitation analyses indicate that in ES cells c-Myc binds proximal to genomic regions encoding the induced miRNAs. We used expression profiling and seed homology to identify genes specifically downregulated both by these miRNAs and by c-Myc. We further show that the introduction of c-Myc-induced miRNAs into murine ES cells significantly attenuates the downregulation of pluripotency markers on induction of differentiation after withdrawal of the ES cell maintenance factor LIF. In contrast, knockdown of the endogenous miRNAs accelerate differentiation. Our data show that in ES cells c-Myc acts, in part, through a subset of miRNAs to attenuate differentiation.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Western Blotting , Diferenciação Celular/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Imuno-Histoquímica , Camundongos , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo
16.
RNA ; 15(2): 308-15, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19144911

RESUMO

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand "seed region," similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.


Assuntos
Regiões 3' não Traduzidas/genética , Apolipoproteínas B/genética , Inativação Gênica , MicroRNAs/metabolismo , RNA Interferente Pequeno/metabolismo , Regiões 3' não Traduzidas/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , RNA Interferente Pequeno/genética , Seleção Genética , Especificidade da Espécie , Transcrição Gênica
17.
Cancer Res ; 68(24): 10105-12, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19074876

RESUMO

Cell cycle arrest in response to DNA damage is an important antitumorigenic mechanism. MicroRNAs (miRNAs) were recently shown to play key regulatory roles in cell cycle progression. For example, miR-34a is induced in response to p53 activation and mediates G(1) arrest by down-regulating multiple cell cycle-related transcripts. Here we show that genotoxic stress promotes the p53-dependent up-regulation of the homologous miRNAs miR-192 and miR-215. Like miR-34a, activation of miR-192/215 induces cell cycle arrest, suggesting that multiple miRNA families operate in the p53 network. Furthermore, we define a downstream gene expression signature for miR-192/215 expression, which includes a number of transcripts that regulate G(1) and G(2) checkpoints. Of these transcripts, 18 transcripts are direct targets of miR-192/215, and the observed cell cycle arrest likely results from a cooperative effect among the modulations of these genes by the miRNAs. Our results showing a role for miR-192/215 in cell proliferation combined with recent observations that these miRNAs are underexpressed in primary cancers support the idea that miR-192 and miR-215 function as tumor suppressors.


Assuntos
Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Divisão Celular/genética , Dano ao DNA , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Fase G1/genética , Fase G2/genética , Perfilação da Expressão Gênica , Inativação Gênica , Genes p53 , Células HCT116 , Humanos , MicroRNAs/biossíntese , Neoplasias/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transfecção , Proteína Supressora de Tumor p53/biossíntese , Regulação para Cima
18.
Mol Cell Biol ; 28(7): 2167-74, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18212054

RESUMO

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.


Assuntos
Ciclo Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Genes cdc , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , Interferência de RNA , RNA Neoplásico/fisiologia , Mama/citologia , Linhagem Celular Transformada/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Doxorrubicina/toxicidade , Feminino , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética
19.
Cancer Inform ; 6: 147-64, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19259408

RESUMO

We identified gene expression signatures predicting responsiveness to a Kinesin-5 (KIF11) inhibitor (Kinesin-5i) in cultured colon tumor cell lines. Genes predicting resistance to Kinesin-5i were enriched for those from chromosome 20q, a region of frequent amplification in a number of tumor types. siRNAs targeting genes in this chromosomal region identified AURKA, TPX2 and MYBL2 as genes whose disruption enhances response to Kinesin-5i. Taken together, our results show functional interaction between these genes, and suggest that their overexpression is involved in resistance to Kinesin-5i. Furthermore, our results suggest that patients whose tumors overexpress AURKA due to amplification of 20q will more likely resist treatment with Kinesin-5 inhibitor, and that inactivation of AURKA may sensitize these patients to treatment.

20.
Nat Cell Biol ; 9(12): 1401-12, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17994010

RESUMO

Cell division is fundamental for all organisms. Here we report a genome-scale RNA-mediated interference screen in HeLa cells designed to identify human genes that are important for cell division. We have used a library of endoribonuclease-prepared short interfering RNAs for gene silencing and have used DNA content analysis to identify genes that induced cell cycle arrest or altered ploidy on silencing. Validation and secondary assays were performed to generate a nine-parameter loss-of-function phenoprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by ascribing novel functions to genes in mitosis and cytokinesis. In particular, we identify two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin.


Assuntos
Divisão Celular/fisiologia , Genoma Humano , Interferência de RNA , Perfilação da Expressão Gênica , Células HeLa , Humanos , RNA Interferente Pequeno/metabolismo
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