RESUMO
Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.
Assuntos
Memória Imunológica/imunologia , Células Matadoras Naturais/imunologia , Transcriptoma/imunologia , Útero/imunologia , Animais , Linhagem Celular Tumoral , Decídua/imunologia , Decídua/metabolismo , Feminino , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Gravidez , Útero/citologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210-designated hypoxiamiR-and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.
Assuntos
Endotelina-2/metabolismo , Células da Granulosa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Lúteas/metabolismo , Síndrome do Ovário Policístico/metabolismo , Transdução de Sinais , Adulto , Células Cultivadas , Endotelina-2/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Valproic acid is an established structural and neurodevelopmental teratogen. Recently, we demonstrated that valproate alters the barrier function of perfused term human placentas. Here, we conducted a pilot study to evaluate the effects of subchronic valproate exposure on carrier expression in cultured placental villous explants from early human pregnancies. Placental tissue of gestational age 6-13 weeks was collected from elective pregnancy terminations in women without known epilepsy. The effects of valproate (42, 83, or 166 µg/mL) on the mRNA expression of 37 major placental carriers and related genes were evaluated by a customized gene expression array (n = 5, 5 days). Five-day exposure to valproate was associated with high variability in gene expression. However, two main gene clusters were identified, including a cluster of three major folate carriers. Exposure to low therapeutic levels of valproate (42 µg/mL) was associated with a tendency toward reduced mRNA expression of genes encoding folate and amino acid and fatty acid carriers (P = 0.065, paired analysis). Our initial findings suggest that valproate can affect the function of the human placenta during early pregnancy.
Assuntos
Proteínas de Transporte/biossíntese , Vilosidades Coriônicas/efeitos dos fármacos , Família Multigênica/efeitos dos fármacos , Teratogênicos/farmacologia , Ácido Valproico/farmacologia , Adulto , Aminoácidos/metabolismo , Proteínas de Transporte/genética , Vilosidades Coriônicas/metabolismo , Ácidos Graxos/metabolismo , Feminino , Transportadores de Ácido Fólico/biossíntese , Transportadores de Ácido Fólico/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Idade Gestacional , Humanos , Técnicas de Cultura de Órgãos , Projetos Piloto , Gravidez , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Zika virus (ZIKV) has emerged as a cause of congenital brain anomalies and a range of placenta-related abnormalities, highlighting the need to unveil the modes of maternal-fetal transmission. The most likely route of vertical ZIKV transmission is via the placenta. The earliest events of ZIKV transmission in the maternal decidua, representing the maternal uterine aspect of the chimeric placenta, have remained unexplored. Here, we show that ZIKV replicates in first-trimester human maternal-decidual tissues grown ex vivo as three-dimensional (3D) organ cultures. An efficient viral spread in the decidual tissues was demonstrated by the rapid upsurge and continued increase of tissue-associated ZIKV load and titers of infectious cell-free virus progeny, released from the infected tissues. Notably, maternal decidual tissues obtained at midgestation remained similarly susceptible to ZIKV, whereas fetus-derived chorionic villi demonstrated reduced ZIKV replication with increasing gestational age. A genome-wide transcriptome analysis revealed that ZIKV substantially upregulated the decidual tissue innate immune responses. Further comparison of the innate tissue response patterns following parallel infections with ZIKV and human cytomegalovirus (HCMV) revealed that unlike HCMV, ZIKV did not induce immune cell activation or trafficking responses in the maternal-fetal interface but rather upregulated placental apoptosis and cell death molecular functions. The data identify the maternal uterine aspect of the human placenta as a likely site of ZIKV transmission to the fetus and further reveal distinct patterns of innate tissue responses to ZIKV. Our unique experimental model and findings could further serve to study the initial stages of congenital ZIKV transmission and pathogenesis and evaluate the effect of new therapeutic interventions. IMPORTANCE: In view of the rapid spread of the current ZIKV epidemic and the severe manifestations of congenital ZIKV infection, it is crucial to learn the fundamental mechanisms of viral transmission from the mother to the fetus. Our studies of ZIKV infection in the authentic tissues of the human maternal-fetal interface unveil a route of transmission whereby virus originating from the mother could reach the fetal compartment via efficient replication within the maternal decidual aspect of the placenta, coinhabited by maternal and fetal cells. The identified distinct placental tissue innate immune responses and damage pathways could provide a mechanistic basis for some of the placental developmental abnormalities associated with ZIKV infection. The findings in the unique model of the human decidua should pave the way to future studies examining the interaction of ZIKV with decidual immune cells and to evaluation of therapeutic interventions aimed at the earliest stages of transmission.
Assuntos
Decídua/virologia , Imunidade Inata , Placenta/virologia , Complicações Infecciosas na Gravidez , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Linhagem Celular , Vilosidades Coriônicas/virologia , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/transmissão , Infecções por Citomegalovirus/virologia , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Idade Gestacional , Humanos , Transmissão Vertical de Doenças Infecciosas , Interferons/genética , Interferons/metabolismo , Gravidez , Transdução de Sinais , Infecção por Zika virus/metabolismo , Infecção por Zika virus/transmissãoRESUMO
Endothelin-2 (EDN2), expressed at a narrow window during the periovulatory period, critically affects ovulation and corpus luteum (CL) formation. LH (acting mainly via cAMP) and hypoxia are implicated in CL formation; therefore, we aimed to elucidate how these signals regulate EDN2 using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in EDN2 expression. Hypoxia (either mimetic compound-CoCl2, or low O2) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and EDN2 Hypoxia-induced miR-210 was suppressed in HIF1A-silenced SVOG cells, suggesting that miR-210 is HIF1A dependent. Elevated miR-210 levels in hypoxia or by miR-210 overexpression, increased EDN2 Conversely, miR-210 inhibition reduced EDN2 levels, even in the presence of CoCl2, indicating the importance of miR-210 in the hypoxic induction of EDN2 A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene-GPD1L, was established as a miR-210 target in both cell types. It was decreased by miR-210-mimic and was increased by miR-inhibitor. Furthermore, reducing GPD1L by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by GPD1L siRNA resulted in elevated HIF1A protein and EDN2 levels, implying a vital role for GPD1L in the hypoxic induction of EDN2 Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated HIF1A, EDN2 and miR-210 while inhibiting GPD1L Furthermore, HIF1A silencing greatly reduced forskolin's ability to elevate EDN2 and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced EDN2 by human granulosa-lutein cells.
Assuntos
Endotelina-2/metabolismo , Regulação da Expressão Gênica , Glicerolfosfato Desidrogenase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Lúteas/metabolismo , MicroRNAs/genética , Adulto , Hipóxia Celular , Células Cultivadas , Endotelina-2/genética , Feminino , Glicerolfosfato Desidrogenase/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células Lúteas/citologia , Ovulação , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl2) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1A mRNA but not its protein. The successful knockdown of HIF1A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl2. We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50âµM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl2-induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL.
Assuntos
AMP Cíclico/metabolismo , Endotelina-2/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Hormônio Luteinizante/farmacologia , Animais , Western Blotting , Bovinos , Células Cultivadas , Endotelina-2/genética , Feminino , Células da Granulosa/citologia , Humanos , Hipóxia/tratamento farmacológico , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
microRNAs constitute a large family of approximately 21-nucleotide-long, noncoding RNAs. They emerged more than 20 years ago as key posttranscriptional regulators of gene expression. The regulatory role of these small RNA molecules has recently begun to be explored in the human reproductive system. microRNAs have been shown to play an important role in control of reproductive functions, especially in the processes of oocyte maturation, folliculogenesis, corpus luteum function, implantation, and early embryonic development. Knockout of Dicer, the cytoplasmic enzyme that cleaves the pre-miRNA to its mature form, results in postimplantation embryonic lethality in several animal models, attributing to these small RNA vital functions in reproduction and development. Another intriguing characteristic of microRNAs is their presence in body fluids in a remarkably stable form that is protected from endogenous RNase activity. In this chapter we will describe the current knowledge on microRNAs, specifically relating to human gonadal cells. We will focus on their role in the ovarian physiologic process and ovulation dysfunction, regulation of spermatogenesis and male fertility, and putative involvement in human normal and aberrant trophoblast differentiation and invasion through the process of placentation.
Assuntos
MicroRNAs/genética , Ovário/metabolismo , Ovulação/genética , Reprodução/genética , Espermatogênese/genética , Animais , RNA Helicases DEAD-box/genética , Feminino , Fertilidade/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Ovário/citologia , Ovário/crescimento & desenvolvimento , Síndrome do Ovário Policístico/genética , Ribonuclease III/genética , Transdução de Sinais/genéticaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle disorder that has been associated with a contraction of 3.3-kb repeats on chromosome 4q35. FSHD is characterized by a wide clinical inter- and intrafamilial variability, ranging from wheelchair-bound patients to asymptomatic carriers. Our study is unique in comparing the gene expression profiles from related affected, asymptomatic carrier, and control individuals. Our results suggest that the expression of genes on chromosome 4q is altered in affected and asymptomatic individuals. Remarkably, the changes seen in asymptomatic samples are largely in products of genes encoding several chemokines, whereas the changes seen in affected samples are largely in genes governing the synthesis of GPI-linked proteins and histone acetylation. Besides this, the affected patient and related asymptomatic carrier share the 4qA161 haplotype. Thus, these polymorphisms by themselves do not explain the pathogenicity of the contracted allele. Interestingly, our results also suggest that the miRNAs might mediate the regulatory network in FSHD. Together, our results support the previous evidence that FSHD may be caused by transcriptional dysregulation of multiple genes, in cis and in trans, and suggest some factors potentially important for FSHD pathogenesis. The study of the gene expression profiles from asymptomatic carriers and related affected patients is a unique approach to try to enhance our understanding of the missing link between the contraction in D4Z4 repeats and muscle disease, while minimizing the effects of differences resulting from genetic background.
Assuntos
Regulação da Expressão Gênica/genética , Heterozigoto , Distrofia Muscular Facioescapuloumeral/genética , Transcrição Gênica/genética , Estudos de Casos e Controles , Cromossomos Humanos Par 4/genética , Perfilação da Expressão Gênica , Humanos , Polimorfismo Genético/genéticaRESUMO
Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. To elucidate the pathological mechanisms leading from the mutated GNE to the HIBM phenotype, we attempted to identify and characterize early occurring downstream events by analyzing the genomic expression patterns of muscle specimens from 10 HIBM patients carrying the M712T Persian Jewish founder mutation and presenting mild histological changes, compared with 10 healthy matched control individuals, using GeneChip expression microarrays. When analyzing the expression profile data sets by the intersection of three statistic methods (Student's t-test, TNoM and Info score), we found that the HIBM-specific transcriptome consists of 374 differentially expressed genes. The specificity of the HIBM transcriptome was assessed by the minimal transcript overlap found between HIBM and the transcriptome of nine additional muscle disorders including adult onset limb girdle myopathies, inflammatory myopathies and early onset conditions. A strikingly high proportion (18.6%) of the overall differentially expressed mRNAs of known function were found to encode for proteins implicated in various mitochondrial processes, revealing mitochondria pathways dysregulation. Mitochondrial morphological analysis by video-rate confocal microscopy showed a high degree of mitochondrial branching in cells of HIBM patients. The subtle involvement of mitochondrial processes identified in HIBM reveals an unexpected facet of HIBM pathophysiology which could at least partially explain the slow evolution of this disorder and give new insights in the disease mechanism.
Assuntos
Mitocôndrias/genética , Mitocôndrias/metabolismo , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The last 20 years have witnessed major advances in the understanding of muscle diseases and significant inroads are being made to treat muscular dystrophy. However, no curative therapy is currently available for any of the muscular dystrophies, despite the immense progress made using several approaches and only palliative and symptomatic treatment is available for patients. The discovery of miRNAs as new and important regulators of gene expression is expected to broaden our biological understanding of the regulatory mechanism in muscle by adding another dimension of regulation to the diversity and complexity of gene-regulatory networks. As important regulators of muscle development, unravelling the regulatory circuits involved may be challenging, given that a single miRNA can regulate the expression of many mRNA targets. Although the identification of the regulatory targets of miRNAs in muscle is a challenge, it will be critical for placing them in genetic pathways and biological contexts. Therefore, combining informatics, biochemical and genetic approaches will not only expected to reveal the elucidation of the miRNA regulatory network in skeletal muscle and to bring a better knowledge on muscle tissue regulation but will also raise new opportunities for therapeutic intervention in muscular dystrophies by identifying candidate miRNAs as potential targets for clinical application.
Assuntos
MicroRNAs/metabolismo , Músculo Esquelético , Doenças Musculares/genética , Doenças Musculares/fisiopatologia , Animais , Biomarcadores/metabolismo , Terapia Genética , Humanos , MicroRNAs/genética , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/terapia , Células-Tronco/fisiologiaRESUMO
Attention deficit hyperactivity disorder (ADHD) is a heterogeneous highly heritable disorder which has recently been described to be comorbid in obese subjects. This study investigated phenotype/genotype associations in a consanguineous family with genetic obesity due to the melanocortin-4-receptor (MC4R) (C271R) mutation. MC4R deficiency disrupts hunger/satiety regulation resulting in abnormal eating behaviors. To date, the behavioral/psychiatric characteristics of MC4R deficiency have not been described except for a possible association with Binge Eating Disorder. Twenty-nine subjects of a family known to carry the MC4R (C271R) mutation, were genotyped for the mutation and underwent extensive evaluations in search for physical/psychiatric phenotype characteristics. Subjects originated from proband nuclear families with morbid obese children (BMI percentile > 97%). All probands were homozygous for the MC4R (C271R) mutation. ADHD prevalence was higher than expected only in the groups carrying the homozygous or heterozygous mutation (P = 0.00057, 0.0028, respectively). An obvious difference was observed between the homozygous group and the rest of the family in terms of obesity: homozygous subjects had childhood morbid obesity whereas heterozygous subjects included lean, normal weight and later onset obese subjects. A significant difference was found in ADHD prevalence between the homozygous MC4R (C271R) group (80%) and the rest of the family (22%) (P = 0.033) and a significant trend was found between ADHD prevalence and the number of MC4R (C271R) alleles (P = 0.0267). We conclude that in our sample, the MC4R (C271R) mutation causing obesity, is in association with ADHD. Identifying specific subgroups in which the comorbidity of obesity and ADHD occur may contribute to the understanding of the underlying molecular mechanisms.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Expressão Gênica , Obesidade/genética , Receptor Tipo 4 de Melanocortina/deficiência , Adolescente , Alelos , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Transtorno do Deficit de Atenção com Hiperatividade/epidemiologia , Criança , Feminino , Predisposição Genética para Doença , Genótipo , Heterozigoto , Homozigoto , Humanos , Entrevistas como Assunto , Masculino , Núcleo Familiar , Pais , Linhagem , Prevalência , Receptor Tipo 4 de Melanocortina/genética , IrmãosRESUMO
OBJECTIVE: To study the role of micro-RNA (miRNA)-200b and miRNA-429 in human ovulation and to measure their expression levels in ovulatory and anovulatory patients. DESIGN: Micro-RNA-200b and miRNA-429 expression analysis in human serum and granulosa cells at different phases of the ovulation cycle in normal cycling women and women undergoing assisted reproductive technology cycles. SETTING: University-affiliated hospital and academic research laboratory. PATIENT(S): Forty women (7 normally ovulating, 15 normally ovulating with pure male infertility factor, and 18 with polycystic ovary syndrome) were included in this study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression profile of circulating miRNAs and granulosa cells was assessed by means of real-time quantitative reverse transcription-polymerase chain reaction analysis. RESULT(S): We identified miRNA-200b and miRNA-429 in the sera of all women tested. These miRNA expression levels were elevated during the early follicular phase of the cycle compared with serum levels during the early luteal phase. Anovulatory women, diagnosed with polycystic ovary syndrome, expressed significantly higher levels of miRNA-200b and miRNA-429 compared with spontaneously ovulating women. Ovulation induction with exogenous gonadotropins during an IVF cycle reduced these levels to the levels measured in normal ovulating women. CONCLUSION(S): Our findings suggest an involvement of miRNA-200b and miRNA-429 in the pituitary regulation of human ovulation. Although it is unclear whether this altered miRNA expression profile is a cause or a result of anovulation, the levels of these molecules in the serum of anovulatory women may serve as serum biomarkers for the ovulation process.
Assuntos
Anovulação/sangue , Infertilidade Feminina/sangue , MicroRNAs/sangue , Ovulação , Síndrome do Ovário Policístico/sangue , Adulto , Anovulação/genética , Anovulação/fisiopatologia , Anovulação/terapia , Estudos de Casos e Controles , Feminino , Fármacos para a Fertilidade Feminina/administração & dosagem , Fertilização in vitro , Marcadores Genéticos , Gonadotropinas/administração & dosagem , Células da Granulosa/química , Hospitais Universitários , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/fisiopatologia , Infertilidade Feminina/terapia , Masculino , Ciclo Menstrual , MicroRNAs/genética , Ovulação/efeitos dos fármacos , Ovulação/genética , Indução da Ovulação , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/fisiopatologia , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Adulto JovemRESUMO
Hereditary Inclusion Body Myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult onset and a typical muscle pathology. We have recently identified the gene encoding for a bifunctional enzyme, UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase (GNE), as the mutated gene in the prototype form of the disease presenting quadriceps sparing, particularly common in Middle Eastern Jews. Interestingly, we have identified the homozygous M712T Middle Eastern Jewish mutation also in two unrelated Middle Eastern Moslem families. We have also evaluated the involvement of GNE in several families from worldwide non-Jewish ethnic origins presenting symptoms similar to the Middle Eastern HIBM prototype. A total of 14 GNE mutations were identified (one nonsense and 13 missense), of which six are novel: an homozygous missense mutation in a consanguineous family from Italy and in a non consanguineous family from USA, and distinct compound heterozygotes in families from Germany, Italy, Ireland, Bahamas, USA and East India. This study brings to 17 the number of reported GNE mutations in quadriceps sparing myopathy, occurring either in the epimerase or the kinase domain of the enzyme. The mechanism leading to this unique phenotype still remains to be elucidated.
Assuntos
Carboidratos Epimerases/genética , Proteínas de Escherichia coli , Mutação , Miosite de Corpos de Inclusão/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Adolescente , Adulto , Análise Mutacional de DNA , Humanos , Pessoa de Meia-Idade , Miosite de Corpos de Inclusão/diagnóstico , Miosite de Corpos de Inclusão/etnologiaRESUMO
A novel human transcript, C9orf19, mapped to the genomic region involved in hereditary inclusion body myopathy (IBM2) at chromosome 9p12-p13, has been cloned and characterized. A single cDNA clone consisting of the full-length 1.9 kb transcript has been isolated from a human placenta cDNA library and further analyzed. Genomic characterization of the C9orf19 gene identified five exons extending over 27.2 kb of genomic DNA, located 12 kb centromeric to the tumor suppressor RECK gene. C9orf19 mRNA is expressed in a wide range of adult tissues as a single transcript, most abundantly in lung and peripheral blood leukocytes. The predicted protein contains the SCP-like extracellular protein signature classified to IPR001283, a family of evolutionary related proteins with extracellular domains, which includes the human glioma pathogenesis-related protein (GliPR), the human testis specific glycoprotein (TPX-1), and several other extracellular proteins from rodents (SCP), insects venom allergens (Ag5, Ag3), plants pathogenesis proteins (PR-1) and yeast hypothetical proteins. Homology searches with the deduced 154 amino acid protein sequence of C9orf19 revealed highly similar proteins in mouse, drosophila, nematode and yeast. Mutation analysis of C9orf19 in IBM2 patients excluded it as the disease causing gene and revealed four single nucleotide polymorphisms within and in the vicinity of the gene, which will certainly be useful tools to study its potential role in several human diseases mapped to chromosome 9p12-p13. Parallel to this study, the gene termed GNE, approximately 50 kb centromeric to C9orf19, was shown to be the disease causing gene in IBM2.
Assuntos
Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Sequência Conservada/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Feminino , Perfilação da Expressão Gênica , Genes/genética , Humanos , Íntrons , Masculino , Proteínas de Membrana , Dados de Sequência Molecular , Mutação , Polimorfismo Genético , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis. In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine. Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain. B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-acetylglucosamine 2-epimerase activity. Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids.
Assuntos
Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Substituição de Aminoácidos , Animais , Linfócitos B/enzimologia , Linhagem Celular , Membrana Celular/metabolismo , Citosol/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Citometria de Fluxo , Homozigoto , Humanos , Miosite de Corpos de Inclusão/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/citologia , Células Tumorais CultivadasRESUMO
MicroRNAs have recently begun to be explored in ovarian cells, uncovering their influence on fundamental ovarian features like steroidogenesis, ovulation, and curpos luteum development and function. We provide a review of the current knowledge on microRNAs, specifically relating to human ovarian cell function and microRNAs' role in the ovarian physiologic process and ovulation dysfunction. Because of microRNAs' known stability in body fluids, we enlighten also their potential use in the clinical setting as novel biomarkers for ovarian function. Future research of the role of microRNAs in physiologic and dysfunctional ovulation may offer new diagnostic and treatment strategies for infertility and other ovarian disorders.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/metabolismo , Ovário/metabolismo , Ovulação , Transdução de Sinais , Animais , Células do Cúmulo/metabolismo , RNA Helicases DEAD-box/metabolismo , Feminino , Fertilidade , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/metabolismo , Infertilidade Feminina/fisiopatologia , Infertilidade Feminina/terapia , Células Lúteas/metabolismo , Oócitos/metabolismo , Doenças Ovarianas/genética , Doenças Ovarianas/metabolismo , Doenças Ovarianas/fisiopatologia , Doenças Ovarianas/terapia , Ovário/fisiopatologia , Ovulação/genética , Gravidez , Ribonuclease III/metabolismo , Transdução de Sinais/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.
Assuntos
Proteínas de Transporte/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Proteínas do Tecido Nervoso/genética , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked myopathy resulting from the production of a nonfunctional dystrophin protein. MicroRNA (miRNA) are small 21- to 24-nucleotide RNA that can regulate both individual genes and entire cell signaling pathways. Previously, we identified several mRNA, both muscle-enriched and inflammation-induced, that are dysregulated in the skeletal muscles of DMD patients. One particularly muscle-enriched miRNA, miR-486, is significantly downregulated in dystrophin-deficient mouse and human skeletal muscles. miR-486 is embedded within the ANKYRIN1(ANK1) gene locus, which is transcribed as either a long (erythroid-enriched) or a short (heart muscle- and skeletal muscle-enriched) isoform, depending on the cell and tissue types. RESULTS: Inhibition of miR-486 in normal muscle myoblasts results in inhibited migration and failure to repair a wound in primary myoblast cell cultures. Conversely, overexpression of miR-486 in primary myoblast cell cultures results in increased proliferation with no changes in cellular apoptosis. Using bioinformatics and miRNA reporter assays, we have identified platelet-derived growth factor receptor ß, along with several other downstream targets of the phosphatase and tensin homolog deleted on chromosome 10/AKT (PTEN/AKT) pathway, as being modulated by miR-486. The generation of muscle-specific transgenic mice that overexpress miR-486 revealed that miR-486 alters the cell cycle kinetics of regenerated myofibers in vivo, as these mice had impaired muscle regeneration. CONCLUSIONS: These studies demonstrate a link for miR-486 as a regulator of the PTEN/AKT pathway in dystrophin-deficient muscle and an important factor in the regulation of DMD muscle pathology.
RESUMO
The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans [Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis]. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA-miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.