RESUMO
DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.
Assuntos
Cílios , RNA Helicases DEAD-box , Modelos Animais de Doenças , Neoplasias Hepáticas , Ribonuclease III , Animais , Ribonuclease III/genética , Ribonuclease III/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/deficiência , Cílios/patologia , Cílios/metabolismo , Camundongos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Mutação , Fígado/patologia , Fígado/metabolismo , Ductos Biliares/patologiaAssuntos
Biomarcadores Tumorais , RNA Helicases DEAD-box/deficiência , Resistencia a Medicamentos Antineoplásicos/genética , Linfoma Difuso de Grandes Células B/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ciclina D1/genética , Progressão da Doença , Humanos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma não Hodgkin/diagnóstico , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Prognóstico , Fator de Transcrição STAT3/metabolismo , Sequenciamento do ExomaRESUMO
Insulin resistance is a major pathophysiologic defect in type 2 diabetes and obesity, while anti-inflammatory M2-like macrophages are important in maintaining normal metabolic homeostasis. Here, we show that M2 polarized bone marrow-derived macrophages (BMDMs) secrete miRNA-containing exosomes (Exos), which improve glucose tolerance and insulin sensitivity when given to obese mice. Depletion of their miRNA cargo blocks the ability of M2 BMDM Exos to enhance insulin sensitivity. We found that miR-690 is highly expressed in M2 BMDM Exos and functions as an insulin sensitizer both in vivo and in vitro. Expressing an miR-690 mimic in miRNA-depleted BMDMs generates Exos that recapitulate the effects of M2 BMDM Exos on metabolic phenotypes. Nadk is a bona fide target mRNA of miR-690, and Nadk plays a role in modulating macrophage inflammation and insulin signaling. Taken together, these data suggest miR-690 could be a new therapeutic insulin-sensitizing agent for metabolic disease.
Assuntos
Exossomos/metabolismo , Macrófagos/metabolismo , MicroRNAs/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Antagomirs/metabolismo , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Dieta Hiperlipídica , Hepatócitos/citologia , Hepatócitos/metabolismo , Insulina/metabolismo , Resistência à Insulina , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genéticaRESUMO
DICER is a key enzyme in microRNA (miRNA) biogenesis. Here we show that aerobic exercise training up-regulates DICER in adipose tissue of mice and humans. This can be mimicked by infusion of serum from exercised mice into sedentary mice and depends on AMPK-mediated signaling in both muscle and adipocytes. Adipocyte DICER is required for whole-body metabolic adaptations to aerobic exercise training, in part, by allowing controlled substrate utilization in adipose tissue, which, in turn, supports skeletal muscle function. Exercise training increases overall miRNA expression in adipose tissue, and up-regulation of miR-203-3p limits glycolysis in adipose under conditions of metabolic stress. We propose that exercise training-induced DICER-miR-203-3p up-regulation in adipocytes is a key adaptive response that coordinates signals from working muscle to promote whole-body metabolic adaptations.
Assuntos
Tecido Adiposo/metabolismo , RNA Helicases DEAD-box/metabolismo , Exercício Físico/fisiologia , Ribonuclease III/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adaptação Fisiológica/fisiologia , Adipócitos/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Feminino , Glicólise , Humanos , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Condicionamento Físico Animal , Ribonuclease III/deficiência , Ribonuclease III/genéticaRESUMO
DICER is a key rate-limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER-dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh-Dicer) was partly due to the up-regulation of miR-16-2-3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh-Dicer HTR8 cells have an anti-angiogenic effect, due to reduced angiogenic factors and increased anti-angiogenic miRNAs (including let-7d, miR-1-6-2 and miR-15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER-miR-16-2-COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER-containing MVs mediate the pro-angiogenic effect of trophoblast-derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.
Assuntos
RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Neovascularização Fisiológica , Ribonuclease III/deficiência , Ribonuclease III/genética , Trofoblastos/metabolismo , Indutores da Angiogênese/metabolismo , Linhagem Celular , Movimento Celular , Colágeno Tipo I/metabolismo , Meios de Cultivo Condicionados/metabolismo , Eletroporação , Células Endoteliais/metabolismo , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/metabolismo , Nanopartículas/química , Neovascularização Patológica , Placenta/metabolismo , Placentação , Gravidez , RNA Interferente Pequeno/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: Dietary restriction (DR) improves health and prolongs lifespan in part by upregulating type III endoribonuclease DICER in adipose tissue. In this study, we aimed to specifically test which missing dietary component was responsible for DICER upregulation. METHODS: We performed a nutrient screen in mouse preadipocytes and validated the results in vivo using different kinds of dietary interventions in wild type or genetically modified mice and worms, also testing the requirement of DICER on the effects of the diets. RESULTS: We found that sulfur amino acid restriction (i.e., methionine or cysteine) is sufficient to increase Dicer mRNA expression in preadipocytes. Consistently, while DR increases DICER expression in adipose tissue of mice, this effect is blunted by supplementation of the diet with methionine, cysteine, or casein, but not with a lipid or carbohydrate source. Accordingly, dietary methionine or protein restriction mirrors the effects of DR. These changes are associated with alterations in serum adiponectin. We also found that DICER controls and is controlled by adiponectin. In mice, DICER plays a role in methionine restriction-induced upregulation of Ucp1 in adipose tissue. In C. elegans, DR and a model of methionine restriction also promote DICER expression in the intestine (an analog of the adipose tissue) and prolong lifespan in a DICER-dependent manner. CONCLUSIONS: We propose an evolutionary conserved mechanism in which dietary sulfur amino acid restriction upregulates DICER levels in adipose tissue leading to beneficial health effects.
Assuntos
Cisteína/deficiência , RNA Helicases DEAD-box/metabolismo , Metionina/deficiência , Adipócitos/citologia , Adipócitos/metabolismo , Adiponectina/sangue , Adiponectina/metabolismo , Tecido Adiposo Bege/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Linhagem Celular , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Dieta/métodos , Dieta/veterinária , Mucosa Intestinal/metabolismo , Longevidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ribonuclease III/genética , Ribonuclease III/metabolismo , Proteína Desacopladora 1/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: This study investigated the role of microRNAs generated from adipose tissue macrophages (ATMs) during adipose tissue remodeling induced by pharmacological and nutritional stimuli. METHODS: Macrophage-specific Dicer knockout (KO) mice were used to determine the roles of microRNA generated in macrophages in adipose tissue remodeling induced by the ß3-adrenergic receptor agonist CL316,243 (CL). RNA-seq was performed to characterize microRNA and mRNA expression profiles in isolated macrophages and PDGFRα+ adipocyte stem cells (ASCs). The role of miR-10a-5p was further investigated in cell culture, and in adipose tissue remodeling induced by CL treatment and high fat feeding. RESULTS: Macrophage-specific deletion of Dicer elevated pro-inflammatory gene expression and prevented CL-induced de novo beige adipogenesis in gonadal white adipose tissue (gWAT). Co-culture of ASCs with ATMs of wild type mice promoted brown adipocyte gene expression upon differentiation, but co-culture with ATMs of Dicer KO mice did not. Bioinformatic analysis of RNA expression profiles identified miR-10a-5p as a potential regulator of inflammation and differentiation in ATMs and ASCs, respectively. CL treatment increased levels of miR-10a-5p in ATMs and ASCs in gWAT. Interestingly, CL treatment elevated levels of pre-mir-10a in ATMs but not in ASCs, suggesting possible transfer from ATMs to ASCs. Elevating miR-10a-5p levels inhibited proinflammatory gene expression in cultured RAW 264.7 macrophages and promoted the differentiation of C3H10T1/2 cells into brown adipocytes. Furthermore, treatment with a miR-10a-5p mimic in vivo rescued CL-induced beige adipogenesis in Dicer KO mice. High fat feeding reduced miR-10a-5p levels in ATMs of gWAT, and treatment of mice with a miR-10a-5p mimic suppressed pro-inflammatory responses, promoted the appearance of new white adipocytes in gWAT, and improved systemic glucose tolerance. CONCLUSIONS: These results demonstrate an important role of macrophage-generated microRNAs in adipogenic niches and identify miR-10a-5p as a key regulator that reduces adipose tissue inflammation and promotes therapeutic adipogenesis.
Assuntos
Tecido Adiposo Branco/metabolismo , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Adipócitos Marrons/metabolismo , Adipogenia , Animais , Antagomirs/metabolismo , Diferenciação Celular , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Dioxóis/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células RAW 264.7 , Receptores Adrenérgicos beta 3/química , Receptores Adrenérgicos beta 3/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.
Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Spliceossomos/efeitos dos fármacos , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Ácidos Indolacéticos/farmacologia , Clivagem do RNA/efeitos dos fármacos , RNA Helicases/deficiência , RNA Helicases/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/deficiência , Fatores de Processamento de RNA/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Studies from last two decades have established microRNAs (miRNAs) as the most influential regulator of gene expression, especially at the post-transcriptional stage. The family of small RNA molecules including miRNAs is highly conserved and expressed throughout the multicellular organism. MiRNAs regulate gene expression by binding to 3' UTR of protein-coding mRNAs and initiating either decay or movement of mRNAs to stress granules. Tissues or cells, which go through cell fate transformation like stem cells, brain cells, iPSCs, or cancer cells show very dynamic expression profile of miRNAs. Inability to pass the developmental stages of Dicer (miRNA maturation enzyme) knockout animals has confirmed that expression of mature and functional miRNAs is essential for proper development of different organs and tissues. Studies from our laboratory and elsewhere have demonstrated the role of miR-200 and miR-34 families in neural development and have shown higher expression of both families in mature and differentiated neurons. In present review, we have provided a general overview of miRNAs and focused on the role of miR-34 and miR-200, two miRNA families, which have the capability to change the phenotype and fate of a cell in different tissues and situations.
Assuntos
MicroRNAs/genética , Neurogênese/genética , Plasticidade Neuronal/fisiologia , Regiões 3' não Traduzidas , Animais , Diferenciação Celular/genética , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/fisiologia , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mamíferos , Camundongos , Células PC12 , RNA não Traduzido/classificação , RNA não Traduzido/genética , Ratos , Ribonuclease III/deficiência , Ribonuclease III/fisiologia , Terminologia como AssuntoRESUMO
RATIONALE: RNA helicases, highly conserved enzymes, are currently believed to be not only involved in RNA modulation, but also in other biological processes. We recently reported that RNA helicase DDX (DEAD-box protein)-5 is required for maintaining the homeostasis of vascular smooth muscle cells (SMCs). However, the expression and function of RNA helicase in vascular physiology and disease is unknown. OBJECTIVE: To investigate the role of RNA helicase in vascular diseases. METHODS AND RESULTS: We showed here that DDX-5 was the most abundant DEAD-box protein expressed in human and rodent artery, which mainly located in SMCs. It was demonstrated that DDX-5 levels were reduced in cytokine-stimulated SMCs and vascular lesions. DDX-5 knocking down or deficiency increased SMC proliferation and migration, whereas overexpression of DDX-5 prevented aberrant proliferation and migration of SMCs. Mechanistic studies revealed transcription factor GATA (GATA-binding protein)-6 as a novel downstream target of DDX-5, which directly interacted with GATA-6 and protected it from MDM (mouse double minute)-2-mediated degradation. Our ChIP assay identified a previously unreported binding of p27Kip1 promoter to GATA-6. DDX-5 increased the recruitment of GATA-6 to p27Kip1 promoter, which enhanced p27Kip1 expression and maintained SMC quiescence. Finally, we showed exacerbated neointima formation in DDX-5 SMC-deficient mice after femoral artery injury, whereas overexpression of DDX-5 potently inhibited vascular remodeling in balloon-injured rat carotid artery. CONCLUSIONS: These findings provide the first evidence for a role of RNA helicase DDX-5 in the protection against SMC proliferation, migration, and neointimal hyperplasia. Our data extend the fundamental role of RNA helicase beyond RNA modulation, which provides the basic information for new therapeutic strategies for vascular diseases.
Assuntos
Proliferação de Células/fisiologia , RNA Helicases DEAD-box/metabolismo , Miócitos de Músculo Liso/fisiologia , Remodelação Vascular/fisiologia , Animais , Lesões das Artérias Carótidas , Movimento Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , RNA Helicases DEAD-box/deficiência , Artéria Femoral/lesões , Artéria Femoral/metabolismo , Fator de Transcrição GATA6/metabolismo , Humanos , Camundongos , Miócitos de Músculo Liso/metabolismo , Neointima/etiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RatosRESUMO
BACKGROUND: The timing, location, and level of gene expression are crucial for normal organ development, because morphogenesis requires strict genetic control. MicroRNAs (miRNAs) are noncoding small single-stranded RNAs that play a critical role in regulating gene expression level. Although miRNAs are known to be involved in many biological events, the role of miRNAs in organogenesis is not fully understood. Mammalian eyelids fuse and separate during development and growth. In mice, failure of this process results in the eye-open at birth (EOB) phenotype. RESULTS: It has been shown that conditional deletion of mesenchymal Dicer (an essential protein for miRNA processing; Dicer fl/fl ;Wnt1Cre) leads to the EOB phenotype with full penetrance. Here, we identified that the up-regulation of Wnt signaling resulted in the EOB phenotype in Dicer mutants. Down-regulation of Fgf signaling observed in Dicer mutants was caused by an inverse relationship between Fgf and Wnt signaling. Shh and Bmp signaling were down-regulated as the secondary effects in Dicer fl/fl ;Wnt1Cre mice. Wnt, Shh, and Fgf signaling were also found to mediate the epithelial-mesenchymal interactions in eyelid development. CONCLUSIONS: miRNAs control eyelid development through Wnt. Developmental Dynamics 248:201-210, 2019. © 2019 Wiley Periodicals, Inc.
Assuntos
Pálpebras/crescimento & desenvolvimento , MicroRNAs/fisiologia , Via de Sinalização Wnt , Animais , RNA Helicases DEAD-box/deficiência , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Organogênese , Fenótipo , Ribonuclease III/deficiênciaRESUMO
The contractile and metabolic properties of adult skeletal muscle change in response to endurance exercise. The mechanisms of transcriptional regulation in exercise-induced skeletal muscle adaptation, including fiber-type switching and mitochondrial biogenesis, have been investigated intensively, whereas the role of microRNA (miRNA)-mediated posttranscriptional gene regulation is less well understood. We used tamoxifen-inducible Dicer1 knockout (iDicer KO) mice to reduce the global expression of miRNAs in adult skeletal muscle and subjected these mice to 2 wk of voluntary wheel running. Dicer mRNA expression was completely depleted in fast-twitch plantaris muscle after tamoxifen injection. However, several muscle-enriched miRNAs, including miR-1 and miR-133a, were reduced by only 30-50% in both the slow and fast muscles. The endurance exercise-induced changes that occurred for many parameters (i.e., fast-to-slow fiber-type switch and increases in succinate dehydrogenase, respiratory chain complex II, and citrate synthase activity) in wild type (WT) also occurred in the iDicer KO mice. Protein expression of myosin heavy chain IIa, peroxisome proliferator-activated receptor-γ coactivator-1α, and cytochrome c complex IV was also increased in the iDicer KO mice by the voluntary running. Furthermore, there was no significant difference in oxygen consumption rate in the isolated mitochondria between the WT and iDicer KO mice. These data indicate that muscle-enriched miRNAs were detectable even after 4 wk of tamoxifen treatment and there was no apparent specific endurance-exercise-induced muscle phenotype in the iDicer KO mice.
Assuntos
Adaptação Fisiológica/fisiologia , RNA Helicases DEAD-box/deficiência , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Resistência Física/fisiologia , Ribonuclease III/deficiência , Fatores Etários , Animais , RNA Helicases DEAD-box/genética , Masculino , Camundongos , Camundongos Knockout , Condicionamento Físico Animal/métodos , Ribonuclease III/genéticaRESUMO
DICER1 is a key enzyme responsible for the maturation of microRNAs. Recent evidences suggested that DICER1 and microRNAs expressed in epididymis were involved in the control of male fertility. However, the exact mechanism remains to be elucidated. Here, we created a mouse line by targeted disruption of Dicer1 gene in the principal cells of distal caput epididymis. Our data indicated that a set of ß-defensin genes were downregulated by DICER1 rather than by microRNAs. Moreover, DICER1 was significantly enriched in the promoter of ß-defensin gene and controlled transcription. Besides, the antibacterial ability of the adult epididymis significantly declined upon Dicer1 deletion both in vitro and in vivo. And a higher incidence of reproductive defect was observed in middle-aged Dicer1-/- males. These results suggest that DICER1 plays an important role in transcription of ß-defensin genes, which are associated with the natural antibacterial properties in a microRNA-independent manner, and further impacts the male fertility.
Assuntos
RNA Helicases DEAD-box/metabolismo , Epididimo/metabolismo , Ribonuclease III/metabolismo , beta-Defensinas/metabolismo , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Epididimo/citologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Regiões Promotoras Genéticas , Ribonuclease III/deficiência , Ribonuclease III/genética , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Transcrição Gênica , beta-Defensinas/genéticaRESUMO
The pleiotropic roles of DEAD-box helicase 3, X-linked (DDX3X), including its functions in transcriptional and translational regulation, chromosome segregation, DNA damage, and cell growth control, have highlighted the association between DDX3X and tumorigenesis. However, mRNA transcripts and protein levels of DDX3X in patient specimens have shown the controversial correlations of DDX3X with hepatocellular carcinoma (HCC) prevalence. In this study, generation of hepatocyte-specific Ddx3x-knockout mice revealed that loss of Ddx3x facilitates liver tumorigenesis. Loss of Ddx3x led to profound ductular reactions, cell apoptosis, and compensatory proliferation in female mutants at 6 weeks of age. The sustained phosphorylation of histone H2AX (γH2AX) and significant accumulation of DNA single-strand breaks and double-strand breaks in liver indicated that the replicative stress occurred in female mutants. Further chromatin immunoprecipitation analyses demonstrated that DDX3X bound to promoter regions and regulated the expression of DNA repair factors, DDB2 and XPA, to maintain genome stability. Loss of Ddx3x led to decreased levels of DNA repair factors, which contributed to an accumulation of unrepaired DNA damage, replication stress, and eventually, spontaneous liver tumors and DEN-induced HCCs in Alb-Cre/+;Ddx3xflox/flox mice. IMPLICATIONS: These data identify an important role of DDX3X in the regulation of DNA damage repair to protect against replication stress in liver and HCC development and progression.
Assuntos
RNA Helicases DEAD-box/deficiência , Dano ao DNA , Neoplasias Hepáticas Experimentais/enzimologia , Animais , Carcinogênese , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Dietilnitrosamina , Feminino , Neoplasias Hepáticas Experimentais/induzido quimicamente , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.
Assuntos
RNA Helicases DEAD-box/deficiência , Nefropatias Diabéticas/enzimologia , Túbulos Renais Proximais/enzimologia , Nefrite/enzimologia , Insuficiência Renal Crônica/enzimologia , Ribonuclease III/deficiência , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Obstrução Ureteral/enzimologia , Animais , RNA Helicases DEAD-box/genética , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Nefrite/etiologia , Nefrite/genética , Nefrite/patologia , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Ribonuclease III/genética , Transdução de Sinais , Regulação para Cima , Obstrução Ureteral/complicações , Obstrução Ureteral/genética , Obstrução Ureteral/patologiaRESUMO
BACKGROUND: The recovery of endothelial cells (ECs) after vascular injury is mainly mediated by the proliferation of resident ECs, thereby reducing neointima formation. The RNase Dicer processes microRNAs (miRNAs) and regulates EC function by controlling miRNA-mediated regulation of gene expression. This study aimed to investigate the impact of miRNA biogenesis in ECs on endothelial repair during lesion formation after vascular injury. METHODS AND RESULTS: To study the effect of Dicer on ECs during neointima formation, conditional deletion of Dicer was induced in Apoe-/- mice (EC-Dicerflox) by tamoxifen injection. Following wire-induced injury to carotid arteries of EC-Dicerflox mice, the EC recovery was impaired and the neointima formation and lesional macrophage accumulation was increased. Moreover, conditional deletion of Dicer in ECs diminished the expression of miR-126-5p in EC-Dicerflox mice. Notably, reconstitution of miR-126-5p in the injured arteries of EC-Dicerflox mice using miR-126-5p mimic, prevented the impaired endothelial recovery and increased lesion formation observed in EC-Dicerflox mice. CONCLUSIONS: Deficiency of endothelial Dicer diminished endothelial recovery and promoted neointima formation probably due to impaired miR-126-5p expression. Treatment with miR-126-5p mimics promotes endothelial recovery and thereby limits neointima formation. Thus, miR-126-5p therapy represents a potential approach to improve endothelial recovery and prevent restenosis following vascular injury.
Assuntos
Lesões das Artérias Carótidas/metabolismo , RNA Helicases DEAD-box/deficiência , Endotélio Vascular/metabolismo , MicroRNAs/biossíntese , Neointima/metabolismo , Ribonuclease III/deficiência , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , RNA Helicases DEAD-box/genética , Endotélio Vascular/lesões , Endotélio Vascular/patologia , Camundongos , Camundongos Knockout , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Neointima/genética , Neointima/patologia , Ribonuclease III/genéticaRESUMO
DExD/H-box helicase 9 (DHX9), or RNA helicase A (RHA), is an abundant multifunctional nuclear protein. Although it was previously reported to act as a cytosolic DNA sensor in plasmacytoid dendritic cells (pDCs), the role and molecular mechanisms of action of DHX9 in cells that are not pDCs during DNA virus infection are not clear. Here, a macrophage-specific knockout and a fibroblast-specific knockdown of DHX9 impaired antiviral innate immunity against DNA viruses, leading to increased virus replication. DHX9 enhanced NF-κB-mediated transactivation in the nucleus, which required its ATPase-dependent helicase (ATPase/helicase) domain, but not the cytosolic DNA-sensing domain. In addition, DNA virus infection did not induce cytoplasmic translocation of nuclear DHX9 in macrophages and fibroblasts. Nuclear DHX9 was associated with a multiprotein complex including both NF-κB p65 and RNA polymerase II (RNAPII) in chromatin containing NF-κB-binding sites. DHX9 was essential for the recruitment of RNAPII rather than NF-κB p65, to the corresponding promoters; this function also required its ATPase/helicase activity. Taken together, our results show a critical role of nuclear DHX9 (as a transcription coactivator) in the stimulation of NF-κB-mediated innate immunity against DNA virus infection, independently of DHX9's DNA-sensing function.
Assuntos
RNA Helicases DEAD-box/genética , DNA Viral/genética , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , NF-kappa B/genética , RNA Polimerase II/genética , Animais , Chlorocebus aethiops , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/imunologia , DNA Viral/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Gammaherpesvirinae/genética , Gammaherpesvirinae/crescimento & desenvolvimento , Gammaherpesvirinae/imunologia , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crescimento & desenvolvimento , Herpesvirus Humano 1/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/imunologia , Células-Tronco Embrionárias Murinas/virologia , NF-kappa B/imunologia , Células NIH 3T3 , Cultura Primária de Células , RNA Polimerase II/imunologia , Transdução de Sinais , Células Vero , Replicação ViralRESUMO
Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures1,2. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.
Assuntos
Herpesvirus Humano 1/imunologia , RNA de Cadeia Dupla/imunologia , RNA Mitocondrial/imunologia , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Exorribonucleases/deficiência , Exorribonucleases/genética , Exorribonucleases/metabolismo , Regulação da Expressão Gênica/imunologia , Células HeLa , Herpesvirus Humano 1/genética , Humanos , Interferon Tipo I/antagonistas & inibidores , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/metabolismo , Mutação , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , Análise de Célula Única , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Myelinating glial cells (MGCs), oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS), generate myelin sheaths that insulate axons. After myelination is completed in adulthood, MGC functions independent from myelin are required to support axon survival, but the underlying mechanisms are still unclear. Dicer is a key enzyme that is responsible for generating functional micro-RNAs (miRNAs). Despite the importance of Dicer in initiating myelination, the role of Dicer in mature MGCs is still unclear. Here, Dicer was specifically deleted in mature MGCs in 2-month old mice (PLP-CreERT; Dicer fl/fl) by tamoxifen administration. Progressive motor dysfunction was observed in the Dicer conditional knockout mice, which displayed hind limb ataxia at 3 months post recombination that deteriorated into paralysis within 5 months. Massive axonal degeneration/atrophy in peripheral nerves was responsible for this phenomenon, but overt demyelination was not observed in either the CNS or PNS. In contrast to the PNS, signs of axonal degeneration were not observed in the CNS of these animals. We induced a Dicer deletion in oligodendroglia at postnatal day 5 in NG2-CreERT; Dicer fl/fl mice to evaluate whether Dicer expression in OLs is essential for axonal survival. Dicer deletion in oligodendroglia did not cause motor dysfunction at the age of 7 months. Neither axonal atrophy nor demyelination was observed in the CNS. Based on our results, Dicer expression in SCs is required to maintain axon integrity in adult PNS, and Dicer is dispensable for maintaining myelin sheaths in MGCs.
Assuntos
Axônios/enzimologia , RNA Helicases DEAD-box/deficiência , Bainha de Mielina/enzimologia , Degeneração Neural/enzimologia , Ribonuclease III/deficiência , Animais , Ataxia/enzimologia , Ataxia/patologia , Atrofia , Axônios/patologia , RNA Helicases DEAD-box/genética , Progressão da Doença , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/fisiologia , Bainha de Mielina/patologia , Degeneração Neural/patologia , Nervo Óptico/enzimologia , Nervo Óptico/patologia , Paralisia/enzimologia , Paralisia/patologia , Ribonuclease III/genética , Nervo Isquiático/enzimologia , Nervo Isquiático/patologia , Medula Espinal/enzimologia , Medula Espinal/patologia , Substância Branca/enzimologia , Substância Branca/patologiaRESUMO
Recent studies have elucidated the crucial role for microRNAs in peripheral nerve myelination by ablating components of the microRNA synthesis machinery. Few studies have focused on the role of individual microRNAs. To fill this gap, we focused this study on miR-138, which was shown to be drastically reduced in Dicer1 and Dgcr8 knockout mice with hypomyelinating phenotypes and to potentially target the negative regulators of Schwann cell differentiation. Here, we show that of two miR-138 encoding loci, mir-138-1 is the predominant locus transcribed in Schwann cells. mir-138-1 is transcriptionally upregulated during myelination and downregulated upon nerve injury. EGR2 is required for mir-138-1 transcription during development, and both SOX10 and EGR2 bind to an active enhancer near the mir-138-1 locus. Based on expression analyses, we hypothesized that miR-138 facilitates the transition between undifferentiated Schwann cells and myelinating Schwann cells. However, in conditional knockouts, we could not detect significant changes in Schwann cell proliferation, cell cycle exit, or myelination. Overall, our results demonstrate that miR-138 is an Egr2-dependent microRNA but is dispensable for Schwann cell myelination.