RESUMO
MicroRNAs (miRNAs) are key players in human hepatocellular carcinoma (HCC) tumorigenesis. Therefore, small molecules targeting components of miRNA biogenesis may provide new therapeutic means for HCC treatment. By a high-throughput screening and structural simplification, we identified a small molecule, CIB-3b, which suppresses the growth and metastasis of HCC in vitro and in vivo by modulating expression profiles of miRNAome and proteome in HCC cells. Mechanistically, CIB-3b physically binds to transactivation response (TAR) RNA-binding protein 2 (TRBP) and disrupts the TRBP-Dicer interaction, thereby altering the activity of Dicer and mature miRNA production. Structure-activity relationship study via the synthesis of 45 CIB-3b derivatives showed that some compounds exhibited a similar inhibitory effect on miRNA biogenesis to CIB-3b. These results support TRBP as a potential therapeutic target in HCC and warrant further development of CIB-3b along with its analogues as a novel therapeutic strategy for the treatment of HCC.
Assuntos
Carcinoma Hepatocelular , RNA Helicases DEAD-box , Neoplasias Hepáticas , MicroRNAs , Coativadores de Receptor Nuclear , Ribonuclease III , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular , RNA Helicases DEAD-box/antagonistas & inibidores , Humanos , Neoplasias Hepáticas/tratamento farmacológico , MicroRNAs/metabolismo , Coativadores de Receptor Nuclear/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/antagonistas & inibidoresRESUMO
Lowering the activity of the Insulin/IGF-1 Signaling (IIS) cascade results in elevated stress resistance, enhanced protein homeostasis (proteostasis) and extended lifespan of worms, flies and mice. In the nematode Caenorhabditis elegans (C. elegans), the longevity phenotype that stems from IIS reduction is entirely dependent upon the activities of a subset of transcription factors including the Forkhead factor DAF-16/FOXO (DAF-16), Heat Shock Factor-1 (HSF-1), SKiNhead/Nrf (SKN-1) and ParaQuat Methylviologen responsive (PQM-1). While DAF-16 determines lifespan exclusively during early adulthood and governs proteostasis in early adulthood and midlife, HSF-1 executes these functions foremost during development. Despite the central roles of SKN-1 as a regulator of lifespan and proteostasis, the temporal requirements of this transcription factor were unknown. Here we employed conditional knockdown techniques and discovered that in C. elegans, SKN-1 is primarily important for longevity and proteostasis during late larval development through early adulthood. Our findings indicate that events that occur during late larval developmental through early adulthood affect lifespan and proteostasis and suggest that subsequent to HSF-1, SKN-1 sets the conditions, partially overlapping temporally with DAF-16, that enable IIS reduction to promote longevity and proteostasis. Our findings raise the intriguing possibility that HSF-1, SKN-1 and DAF-16 function in a coordinated and sequential manner to promote healthy aging.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Longevidade , Proteostase/fisiologia , Fatores de Transcrição/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Peptídeos/farmacologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
Sequence specific recognition and functional inhibition of biomedically relevant double-helical RNAs is highly desirable but remains a formidable problem. The present study demonstrates that electroporation of a triplex-forming peptide nucleic acid (PNA), modified with 2-aminopyridine (M) nucleobases, inhibited maturation of endogenous microRNA-197 in SH-SY5Y cells, while having little effect on maturation of microRNA-155 or -27a. In vitro RNA binding and Dicer inhibition assays suggested that the observed biological activity was most likely due to a sequence-specific PNA-RNA triplex formation that inhibited the activity of endonucleases responsible for microRNA maturation. The present study is the first example of modulation of activity of endogenous noncoding RNA using M-modified triplex-forming PNA.
Assuntos
MicroRNAs/metabolismo , Ácidos Nucleicos Peptídicos/metabolismo , Linhagem Celular Tumoral , Humanos , Sequências Repetidas Invertidas , MicroRNAs/química , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , Ribonuclease III/antagonistas & inibidoresRESUMO
Guanine (G)-rich single-stranded nucleic acids can adopt G-quadruplex structures. Accumulating evidence indicates that G-quadruplexes serve important regulatory roles in fundamental biological processes such as DNA replication, transcription, and translation, while aberrant G-quadruplex formation is linked to genome instability and cancer. Understanding the biological functions played by G-quadruplexes requires detailed knowledge of their protein interactome. Here, we report that both RNA and DNA G-quadruplexes are bound by human Dicer in vitro. Using in vitro binding assays, mutation studies, and computational modeling we demonstrate that G-quadruplexes can interact with the Platform-PAZ-Connector helix cassette of Dicer, the region responsible for anchoring microRNA precursors (pre-miRNAs). Consequently, we show that G-quadruplexes efficiently and stably inhibit the cleavage of pre-miRNA by Dicer. Our data highlight the potential of human Dicer for binding of G-quadruplexes and allow us to propose a G-quadruplex-driven sequestration mechanism of Dicer regulation.
Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Quadruplex G , MicroRNAs/metabolismo , RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , RNA Helicases DEAD-box/metabolismo , DNA/química , DNA/genética , Inibidores Enzimáticos/química , Humanos , MicroRNAs/genética , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease III/metabolismoRESUMO
MicroRNAs are promising biological markers for prenatal diagnosis. They regulate placental development and are present in maternal plasma. Maternal metabolic diseases are major risk factors for placental deterioration. We analysed the influence of a maternal insulin-dependent diabetes mellitus on microRNA expression in maternal plasma and in blastocysts employing an in vivo rabbit diabetic pregnancy model and an in vitro embryo culture in hyperglycaemic and hypoinsulinaemic medium. Maternal diabetes led to a marked downregulation of Dicer protein in embryoblast cells and Drosha protein in trophoblast cells. MiR-27b, miR-141 and miR-191 were decreased in trophoblast cells and in maternal plasma of diabetic rabbits. In vitro studies indicate, that maternal hyperglycaemia and hypoinsulinaemia partially contribute to the downregulation of trophoblastic microRNAs. As the altered microRNA expression was detectable in maternal plasma, too, the plasma microRNA signature could serve as an early biological marker for the prediction of trophoblast function during a diabetic pregnancy.
Assuntos
Diabetes Mellitus Experimental/genética , Regulação para Baixo/genética , MicroRNAs/genética , Ribonuclease III/antagonistas & inibidores , Trofoblastos/metabolismo , Animais , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/sangue , Regulação para Baixo/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Feminino , Glucose/farmacologia , Insulina/farmacologia , MicroRNAs/sangue , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ribonuclease III/genética , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Trofoblastos/efeitos dos fármacosRESUMO
Colorectal cancer (CRC) is a major health problem due to its high mortality rate. The incidence of CRC is increasing in young individuals. Oxaliplatin (OXA) is an approved third-generation drug and is used for first-line chemotherapy in CRC. Although current standard chemotherapy improves the overall survival of CRC patients, an increasing number of reports of OXA resistance in CRC therapy indicates that resistance has become an urgent problem in clinical applications. Dicer is a critical enzyme involved in miRNA maturation. The expression of Dicer has been reported to be involved in the resistance to various drugs in cancer. In the present study, we aimed to investigate the role of Dicer in OXA resistance in CRC. We found that OXA treatment inhibited Dicer expression through decreasing the protein stability. OXA-induced Dicer protein degradation occurred through both proteasomal and lysosomal proteolysis, while the CHIP E3 ligase was involved in OXA-mediated Dicer ubiquitination and degradation. We established stable OXA-resistant clones from CRC cells, and observed that the CHIP E3 ligase was decreased, along with the increased Dicer expression in OXA-resistant cells. Knockdown of Dicer resensitized CRC cells to OXA treatment. In this study, we have revealed the role of miRNA biogenesis factors in OXA resistance in CRC cells.
Assuntos
Antineoplásicos/farmacologia , RNA Helicases DEAD-box/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Oxaliplatina/farmacologia , Ribonuclease III/genética , Ubiquitina-Proteína Ligases/genética , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/metabolismo , Células HCT116 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Bone marrow mesenchymal stem cells (BMMSCs) facilitate the growth of multiple myeloma (MM) cells, but the underlying mechanisms remain unclear. This study demonstrates that the senescence of MM-MSCs significantly increased, as evidenced by a decrease in proliferation and increase in the number of cells positive for senescence-associated ß-galactosidase activity. Senescent MM-MSCs displayed decreased differentiation potential and increased tumor-supporting capacity. Dicer1 knockdown in the MSCs of healthy controls promoted cellular senescence and tumor-supporting capacity, while decreasing the differentiation capacity. Dicer1 overexpression in MM-MSCs reversed the effects on differentiation and reduced cellular senescence. In addition, decreased expression of the microRNA-17 family was identified as a favorable element responsible for increasing senescence, with the expression of p21 increased in Dicer1 knockdown cells. Furthermore, we observed decreased expression of miR-93 and miR-20a in MM-MSCs, while upregulation of miR-93/miR-20a decreased cellular senescence, as evidenced by the increased p21 expression. Importantly, we found that myeloma cells could induce the senescence of MSCs from healthy controls, as observed from the decreased expression of Dicer1 and miR-93/miR-20a and increased expression of p21. Overall, MM cells downregulate Dicer1 in MSCs, which leads to senescence; in turn, senescent MSCs promote MM cell growth, which most likely contributes to disease progression.
Assuntos
RNA Helicases DEAD-box/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/genética , Plasmócitos/metabolismo , Ribonuclease III/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Estudos de Casos e Controles , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células , Senescência Celular , Técnicas de Cocultura , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/metabolismo , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Estadiamento de Neoplasias , Plasmócitos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Transdução de Sinais , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions. Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy. Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR). TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA. To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results: We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells. Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection. To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγnull mice (hu-NSG) were treated with the aptamer-siRNA conjugates. Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice. Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1.
Assuntos
Aptâmeros de Nucleotídeos/genética , RNA Helicases DEAD-box/genética , Regulação Viral da Expressão Gênica , Inativação Gênica , Infecções por HIV/terapia , HIV-1/genética , RNA Viral/genética , Ribonuclease III/genética , Animais , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/metabolismo , Modelos Animais de Doenças , Terapia Genética/métodos , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV , HIV-1/crescimento & desenvolvimento , HIV-1/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Conformação de Ácido Nucleico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/antagonistas & inibidores , RNA Viral/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Transcrição GênicaRESUMO
Laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated gene 5 (MDA5) cooperatively detect viral RNA in the cytoplasm of Cardiovirus-infected cells and activate innate immune responses. Here, we evaluated whether the double-stranded RNA-binding protein PACT plays a role in this anti-viral response to further elucidate the mechanism. Immunoprecipitation experiments demonstrated that PACT interacts with LGP2 and that this interaction is enhanced by encephalomyocarditis virus (EMCV) infection. In vitro interaction analyses using purified recombinant proteins confirmed that the single-stranded Theiler's murine encephalitis virus genome enhanced the interaction between LGP2 and PACT. Small interfering RNA knockdown experiments further indicated that PACT is required for Cardiovirus-triggered interferon responses. To support this functional interaction with LGP2, overexpressed PACT was shown to enhance EMCV-triggered interferon promoter activity only when LGP2 and MDA5 were co-expressed but not when MDA5 is expressed alone. Together, our findings indicate a possible role of PACT in regulating the Cardiovirus-triggered immune responses mediated by MDA5 and LGP2, which opens the door to novel therapeutic strategies in interferon-related autoimmune diseases and cancer.
Assuntos
Infecções por Cardiovirus/imunologia , Vírus da Encefalomiocardite , Helicase IFIH1 Induzida por Interferon/imunologia , RNA Helicases/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/virologia , Linhagem Celular , Chlorocebus aethiops , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/imunologia , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/imunologia , Técnicas de Silenciamento de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/genética , Helicase IFIH1 Induzida por Interferon/genética , Interferon beta/genética , Camundongos , Regiões Promotoras Genéticas , RNA Helicases/genética , RNA Interferente Pequeno/genética , RNA Viral/genética , RNA Viral/imunologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/imunologia , Células VeroRESUMO
The insecticide dichlorodiphenyltrichloroethane (DDT) is a nonmutagenic xenobiotic compound able to exert estrogen-like effects resulting in activation of estrogen receptor-α (ERα) followed by changed expression of its downstream target genes. In addition, studies performed over recent years suggest that DDT may also influence expression of microRNAs. However, an impact of DDT on expression of ER, microRNAs, and related target genes has not been fully elucidated. Here, using real-time PCR, we assessed changes in expression of key genes involved in hormonal carcinogenesis as well as potentially related regulatory oncogenic/tumor suppressor microRNAs and their target genes in the uterus and ovaries of female Wistar rats during single and chronic multiple-dose DDT exposure. We found that applying DDT results in altered expression of microRNAs-221, -222, -205, -126a, and -429, their target genes (Pten, Dicer1), as well as genes involved in hormonal carcinogenesis (Esr1, Pgr, Ccnd1, Cyp19a1). Notably, Cyp19a1 expression seems to be also regulated by microRNAs-221, -222, and -205. The data suggest that epigenetic effects induced by DDT as a potential carcinogen may be based on at least two mechanisms: (i) activation of ERα followed by altered expression of the target genes encoding receptor Pgr and Ccnd1 as well as impaired expression of Cyp19a1, affecting, thereby, cell hormone balance; and (ii) changed expression of microRNAs resulting in impaired expression of related target genes including reduced level of Cyp19a1 mRNA.
Assuntos
Carcinogênese/genética , DDT/toxicidade , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Ovário/efeitos dos fármacos , Útero/efeitos dos fármacos , Animais , Aromatase/genética , Aromatase/metabolismo , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Ovário/metabolismo , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/metabolismo , Útero/metabolismoRESUMO
DICER1 plays a central role in the biogenesis of microRNAs and it is important for normal development. Altered microRNA expression and DICER1 dysregulation have been described in several types of tumors, including thyroid carcinomas. Recently, our group identified a new somatic mutation (c.5438A>G; E1813G) within DICER1 gene of an unknown function. Herein, we show that DICER1 is overexpressed, at mRNA level, in a significant-relative number of papillary (70%) and anaplastic (42%) thyroid carcinoma samples, whereas is drastically downregulated in all the analyzed human thyroid carcinoma cell lines (TPC-1, BCPAP, FRO and 8505c) in comparison with normal thyroid tissue samples. Conversely, DICER1 is downregulated, at protein level, in PTC in comparison with normal thyroid tissues. Our data also reveals that DICER1 overexpression positively regulates thyroid cell proliferation, whereas its silencing impairs thyroid cell differentiation. The expression of DICER1 gene mutation (c.5438A>G; E1813G) negatively affects the microRNA machinery and cell proliferation as well as upregulates DICER1 protein levels of thyroid cells but has no impact on thyroid differentiation. In conclusion, DICER1 protein is downregulated in papillary thyroid carcinomas and affects thyroid proliferation and differentiation, while DICER1 gene mutation (c.5438A>G; E1813G) compromises the DICER1 wild-type-mediated microRNA processing and cell proliferation.
Assuntos
Diferenciação Celular , Proliferação de Células , RNA Helicases DEAD-box/metabolismo , Ribonuclease III/metabolismo , Neoplasias da Glândula Tireoide/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Regulação para Baixo , Humanos , MicroRNAs/metabolismo , Mutagênese Sítio-Dirigida , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismoRESUMO
MicroRNAs (miRNAs) help orchestrate cellular growth and survival through post-transcriptional mechanisms. The dysregulation of miRNA biogenesis can lead to cellular growth defects and chemotherapeutic resistance and plays a direct role in the development of many chronic diseases. Among these RNAs, miR-21 is consistently overexpressed in most human cancers, leading to the down-regulation of key tumor-suppressing and pro-apoptotic factors, suggesting that inhibition of miR-21 biogenesis could reverse these negative effects. However, targeted inhibition of miR-21 using small molecules has had limited success. To overcome difficulties in targeting RNA secondary structure with small molecules, we developed a class of cyclic ß-hairpin peptidomimetics which bind to RNA stem-loop structures, such as miRNA precursors, with potent affinity and specificity. We screened an existing cyclic peptide library and discovered a lead structure which binds to pre-miR21 with KD = 200 nM and prefers it over other pre-miRNAs. The NMR structure of the complex shows that the peptide recognizes the Dicer cleavage site and alters processing of the precursor to the mature miRNA in vitro and in cultured cells. The structure provides a rationale for the peptide binding activity and clear guidance for further improvements in affinity and targeting.
Assuntos
Anticarcinógenos/química , MicroRNAs/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Peptidomiméticos/farmacologia , Animais , Linhagem Celular , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/metabolismo , Humanos , Ligantes , MicroRNAs/metabolismo , Biblioteca de Peptídeos , Peptídeos Cíclicos/metabolismo , Peptidomiméticos/metabolismo , Ligação Proteica , Processamento Pós-Transcricional do RNA , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/metabolismo , Especificidade por SubstratoRESUMO
The discovery of microRNAs (miRNAs) has opened an entire new avenue for drug development. These short (15-22 nucleotides) noncoding RNAs, which function in RNA silencing and posttranscriptional regulation of gene expression, have been shown to critically affect numerous pathways in both development and disease progression. Current miRNA drug development focuses on either reintroducing the miRNA into cells through the use of a miRNA mimic or inhibiting its function via use of a synthetic antagomir. Although these methods have shown some success as therapeutics, they face challenges particularly with regard to cellular uptake and for use as systemic reagents. We recently presented a novel mechanism of inhibiting miR-544 by directed inhibition of miRNA biogenesis. We found that inhibition of DICER processing of miR-544 through the use of a small molecule abolished miR-544 function in regulating adaptation of breast cancer cells to hypoxic stress. Herein, we describe a protocol that utilizes bioinformatics to first identify lead small molecules that bind to DICER cleavage sites in pre-miRNAs and then employ an efficient, high-throughput fluorescent-based screening system to determine the inhibitory potential of the lead compounds and their derivatives.
Assuntos
Neoplasias da Mama/tratamento farmacológico , RNA Helicases DEAD-box/genética , Ensaios de Triagem em Larga Escala , MicroRNAs/genética , Ribonuclease III/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Neoplasias da Mama/genética , Biologia Computacional/métodos , RNA Helicases DEAD-box/antagonistas & inibidores , Descoberta de Drogas/métodos , Feminino , Regulação da Expressão Gênica , Humanos , MicroRNAs/antagonistas & inibidores , Ribonuclease III/antagonistas & inibidoresRESUMO
Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteína 1 de Ligação a Y-Box/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Genoma Humano , Genômica , Glioblastoma/enzimologia , Glioblastoma/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/antagonistas & inibidores , Análise de Sequência de RNARESUMO
The highly conserved, multifunctional YB-1 is a powerful breast cancer prognostic indicator. We report on a pervasive role for YB-1 in which it associates with thousands of nonpolyadenylated short RNAs (shyRNAs) that are further processed into small RNAs (smyRNAs). Many of these RNAs have previously been identified as functional noncoding RNAs (http://www.johnlab.org/YB1). We identified a novel, abundant, 3'-modified short RNA antisense to Dicer1 (Shad1) that colocalizes with YB-1 to P-bodies and stress granules. The expression of Shad1 was shown to correlate with that of YB-1 and whose inhibition leads to an increase in cell proliferation. Additionally, Shad1 influences the expression of additional prognostic markers of cancer progression such as DLX2 and IGFBP2. We propose that the examination of these noncoding RNAs could lead to better understanding of prostate cancer progression.
Assuntos
Corpo Celular/metabolismo , Neoplasias da Próstata/genética , RNA não Traduzido/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Animais , Células COS , Proliferação de Células , Chlorocebus aethiops , RNA Helicases DEAD-box/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA não Traduzido/genética , Ribonuclease III/antagonistas & inibidores , Análise de Sequência de RNA , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Dicer and Argonaute2 (Ago2) are critical components responsible not only for RNA interference but also for microRNA synthesis. The present study investigated the roles of Dicer and Ago2 in prostate cancer (Pca). First, the expression levels of Dicer and Ago2 in Pca tissues were determined by immunohistochemistry (IHC) and compared with pathological features. Next, RNA interference was used to down-regulate the expression levels of Dicer and Ago2 in the Pca cell lines LNCaP, PC-3, and DU145, and effects on proliferation, apoptosis, and cell cycle were detected using the CCK-8 assay and flow cytometry, respectively. We found that Dicer and Ago2 expression levels in Pca tissues were higher than those in adjacent benign tissues and correlated with lower Gleason patterns, with the exception of Dicer expression in localized Pca. In vitro, silencing Dicer or Ago2 inhibited cell proliferation and induced apoptosis in LNCaP, PC-3, and DU145, as well as arrested the cell cycle at the G2/M phase in androgen-dependent LNCaP, or at S phase in the androgen-independent PC-3 and DU145. Altogether these findings suggest that Dicer and Ago2 play important roles in proliferation, apoptosis, and the cell cycle in Pca and might serve as both promising biomarkers for Pca progression and potential therapeutic targets.
Assuntos
Apoptose , Proteínas Argonautas/metabolismo , Biomarcadores Tumorais/metabolismo , Proliferação de Células , RNA Helicases DEAD-box/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/patologia , Ribonuclease III/metabolismo , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Western Blotting , Ciclo Celular , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Células Tumorais CultivadasRESUMO
Ocular neovascularization is a vision-threatening complication of ischemic retinopathy that develops in various ocular disorders, such as retinopathy of prematurity (ROP) and diabetic retinopathy. Both Ang-2 and VEGF are implicated in this pathogenesis. However, their inter-regulation still remains elusive. Competitive endogenous RNAs (ceRNAs) are messenger RNA (mRNA) molecules that affect each other expression through the competition for the shared miRNA. Herein, we assessed whether the expression of Ang-2 and VEGF is interdependent through the sequestration of common miRNAs. Bioinformatics prediction and 3'-UTR luciferase assay revealed that Ang-2 and VEGF is commonly targeted by miR-351. Silencing either Ang-2 or VEGF increases the availabilities of shared miR-351, therefore reduces the activity of Luc-Ang-2 3'-UTR. The interdependence of VEGF and Ang-2 is miRNA- and 3'-UTR dependent, as silencing Dicer abolishes the interdependence. We also found that miR-351 dependency of VEGF-Ang-2 crosstalk occurs in retinal endothelial cells and rat retinas. miR-351 over-expression significantly reduces the level of VEGF and Ang-2 expression in vivo and in vitro. Overall, miRNA-dependent crosstalk between Ang-2 and VEGF plays a role in hypoxia-induced microvascular response. miRNA-based therapy can affect the expression of Ang-2 and VEGF, which represents a therapeutic potential for the treatment of vascular disease.
Assuntos
Angiopoietina-2/genética , Células Endoteliais/metabolismo , MicroRNAs/genética , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Regiões 3' não Traduzidas , Angiopoietina-2/metabolismo , Animais , Hipóxia Celular , Linhagem Celular , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células Endoteliais/patologia , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Macaca mulatta , Camundongos , MicroRNAs/metabolismo , Neovascularização Patológica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Retina/patologia , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoAssuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , RNA Helicases DEAD-box/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Pirazinas/farmacologia , Ribonuclease III/metabolismo , Western Blotting , Bortezomib , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Humanos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mutação/genética , RNA Interferente Pequeno/genética , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
STUDY QUESTION: Does Let-7a have a functional role in modulating dicer expression to activate dormant mouse blastocysts for implantation? SUMMARY ANSWER: Let-7a post-transcriptionally regulates dicer expression altering microRNA expression to affect the implantation competency of the activated blastocysts. WHAT IS KNOWN ALREADY: The Let-7a microRNA is up-regulated during blastocyst dormancy and its forced-expression suppresses embryo implantation in vitro and in vivo. Dicer is a Let-7 target, which processes pre-microRNA to mature microRNA. STUDY DESIGN, SIZE, DURATION: The effects on the expression of Let-7a and dicer in dormant blastocysts during the first 12 h after estradiol-induced activation, and the relationship between Let-7a and dicer in preimplantation embryos were determined. The effects on the microRNA expression and embryo implantation in vivo in dicer-knockdown mouse 5-8 cell embryos and dormant blastocysts at 1 h post estradiol activation were also studied. PARTICIPANTS/MATERIALS, SETTING, METHODS: ICR female mice at 6 weeks of age were ovariectomized on Day 4 of pregnancy to generate the delayed implantation model. Mouse 5-8 cell embryos and/or dormant blastocysts at 1 h after estradiol injection were electroporated with dicer siRNA and Let-7a precursor or Let-7a inhibitor. At 48 h post electroporation, the Let-7a expression, dicer transcripts and proteins in the embryos were determined using qPCR and immunostaining/western blotting, respectively. All experiments were repeated at least three times. MAIN RESULTS AND THE ROLE OF CHANCE: Estradiol injection down-regulated Let-7a and up-regulated dicer in the dormant blastocysts during the first 12 h post-activation. Dicer knockdown at 1 h post-activation of blastocysts suppressed EGFR expression, attenuated EGF binding and compromised implantation of the transferred embryos. Let-7a transcriptionally regulated dicer by binding to the 3'-UTR of dicer in trophoblast cells. Dicer knockdown in blastocysts suppressed mature Let-7a expression and compromised implantation. LIMITATIONS, REASONS FOR CAUTION: Gain- and loss-of-function approaches were used by analyzing transient expressions of transfected microRNA modulators or genes. The consequence of the Let-7a-dicer interaction on pregnancy remains to be determined. The study used the mouse as a model and the applicability of the observed phenomena in humans warrants further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the Let-7a-dicer interaction leads to differential microRNA expression in dormant blastocysts after estradiol activation. Because the expression pattern of Let-7a in human blastocysts is similar to that in mouse blastocysts, our observation that the Let-7a-dicer interaction has a role in regulating the implantation potential of the mouse blastocysts could be applicable to humans. STUDY FUNDING/COMPETING INTEREST(S): This project is supported partly by a research grant from the Research Grant Council to W.S.B.Y. The authors have no competing interests to declare.
Assuntos
RNA Helicases DEAD-box/fisiologia , Implantação do Embrião/genética , MicroRNAs/fisiologia , Ribonuclease III/fisiologia , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Linhagem Celular , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Transferência Embrionária , Fator de Crescimento Epidérmico/metabolismo , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos ICR , MicroRNAs/genética , MicroRNAs/metabolismo , Gravidez , Interferência de RNA , Ribonuclease III/antagonistas & inibidores , Ribonuclease III/genética , Fatores de TempoRESUMO
RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH (Aspartic acid, Glumatic Acid, Alanine, Histidine)-box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3'-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non-quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3'-untranslated region.