RESUMO
In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci. Instead, Rap1 interacts with the TIP60/p400 complex and modulates its histone acetyltransferase activity. Notably, we show that deletion of Rap1 in mouse embryonic stem cells increases the fraction of two-cell-like cells. Specifically, Rap1 enhances the repressive activity of Tip60/p400 across a subset of two-cell-stage genes, including Zscan4 and the endogenous retrovirus MERVL. Preferential up-regulation of genes proximal to MERVL elements in Rap1-deficient settings implicates these endogenous retroviral elements in the derepression of proximal genes. Altogether, our study reveals an unprecedented link between Rap1 and the TIP60/p400 complex in the regulation of pluripotency.
Assuntos
Proteínas de Ligação a Telômeros , Telômero , Animais , Regulação da Expressão Gênica , Genoma , Mamíferos/genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Chromosomal translocations frequently promote carcinogenesis by producing gain-of-function fusion proteins. Recent studies have identified highly recurrent chromosomal translocations in patients with endometrial stromal sarcomas (ESSs) and ossifying fibromyxoid tumors (OFMTs), leading to an in-frame fusion of PHF1 (PCL1) to six different subunits of the NuA4/TIP60 complex. While NuA4/TIP60 is a coactivator that acetylates chromatin and loads the H2A.Z histone variant, PHF1 is part of the Polycomb repressive complex 2 (PRC2) linked to transcriptional repression of key developmental genes through methylation of histone H3 on lysine 27. In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation. The chimeric protein assembles a megacomplex harboring both NuA4/TIP60 and PRC2 activities and leads to mislocalization of chromatin marks in the genome, in particular over an entire topologically associating domain including part of the HOXD cluster. This is linked to aberrant gene expression-most notably increased expression of PRC2 target genes. Furthermore, we show that JAZF1-implicated with a PRC2 component in the most frequent translocation in ESSs, JAZF1-SUZ12-is a potent transcription activator that physically associates with NuA4/TIP60, its fusion creating outcomes similar to those of EPC1-PHF1 Importantly, the specific increased expression of PRC2 targets/HOX genes was also confirmed with ESS patient samples. Altogether, these results indicate that most chromosomal translocations linked to these sarcomas use the same molecular oncogenic mechanism through a physical merge of NuA4/TIP60 and PRC2 complexes, leading to mislocalization of histone marks and aberrant Polycomb target gene expression.
Assuntos
Neoplasias do Endométrio , Sarcoma do Estroma Endometrial , Sarcoma , Cromatina , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Histonas/metabolismo , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Sarcoma/genética , Sarcoma do Estroma Endometrial/genética , Sarcoma do Estroma Endometrial/metabolismo , Sarcoma do Estroma Endometrial/patologia , Translocação Genética/genéticaRESUMO
Endometrial stromal sarcomas (ESS) are morphologically and molecularly heterogeneous. We report novel gene fusions (EPC1::EED, EPC1::EZH2, ING3::PHF1) identified by targeted RNA sequencing in five cases. The ING3::PHF1-fusion positive ESS presented in a 58-year-old female as extrauterine mesocolonic, ovarian masses, and displayed large, monomorphic ovoid-to-epithelioid cells arranged in solid sheets. The patient remained alive with disease 13 months after surgery. The three ESS with EPC1::EED occurred in the uterine corpus in patients with a median age of 58 years (range 27-62 years). One tumor showed a uniform epithelioid nested morphology, while the other two were composed of monomorphic spindle cells in fascicles with elevated mitotic figures, focal tumor cell necrosis, and lymphovascular invasion. At a median follow-up of 20 months, two patients developed local recurrence, including one with concomitant distant metastasis, while one patient remained free of disease. All three patients were alive at the last follow-up. The EPC1::EZH2-fusion positive ESS presented in a 52-year-old female in the uterus, and displayed uniform spindled cells arranged in short fascicles, with focally elevated mitotic activity but without necrosis. The patient remained free of disease 3 months after surgery. All cases were diffusely positive for CD10; four diffusely express estrogen and progesterone receptors. Our study expands the molecular spectrum of EPC1 and PHF1-related gene fusions in ESS to include additional novel subunits of the PRC2 and/or NuA4/TIP60 complexes. These cases displayed a monomorphic epithelioid or spindled phenotype, spanning low-grade and high-grade cytomorphology, all expressing CD10 and commonly ER and PR, and are prone to local and/or distant spread.
Assuntos
Neoplasias do Endométrio , Sarcoma do Estroma Endometrial , Feminino , Humanos , Sarcoma do Estroma Endometrial/genética , Sarcoma do Estroma Endometrial/cirurgia , Sarcoma do Estroma Endometrial/patologia , Montagem e Desmontagem da Cromatina , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/cirurgia , Neoplasias do Endométrio/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fusão Gênica , Proteínas de Homeodomínio/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Endometrial stromal sarcoma encompasses a heterogeneous group of uterine mesenchymal neoplasms, which are currently divided into low-grade and high-grade subtypes. Low-grade endometrial stromal sarcoma is morphologically bland; molecularly, these tumors frequently contain JAZF1-SUZ12, JAZF1-PHF1, and EPC1-PHF1 fusions. In contrast, high-grade endometrial stromal sarcoma is characterized by morphologically undifferentiated neoplasms with high-grade nuclear features; these tumors likewise appear to be genetically diverse with YWHAE-NUTM2 and ZC3H7B-BCOR representing the most frequent gene fusions. Herein, we describe two novel EPC1 fusion genes in endometrial stromal sarcoma: EPC1-SUZ12 and EPC1-BCOR. Both tumors were characterized be an aggressive clinical course.
Assuntos
Proteínas Cromossômicas não Histona/genética , Neoplasias do Endométrio/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras/genética , Sarcoma do Estroma Endometrial/genética , Proteínas Cromossômicas não Histona/química , Neoplasias do Endométrio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/química , Proteínas Repressoras/química , Sarcoma do Estroma Endometrial/patologiaRESUMO
Enhancer of Polycomb (EPC) was first identified for its contributions to development in Drosophila and was soon-thereafter purified as a subunit of the NuA4/TIP60 acetyltransferase complex. Since then, EPC has often been left in the shadows as an essential, yet non-catalytic subunit of NuA4/TIP60; however, its deep conservation and disease association make clear that it warrants additional attention. In fact, recent studies in yeast demonstrated that its Enhancer of Polycomb, Epl1, was just as important for gene expression and acetylation as is the catalytic subunit of NuA4. Despite its conservation, studies of EPC have often remained siloed between organisms. Here, our goal is to provide a cohesive view of the current state of the EPC literature as it stands among the major model organisms in which it has been studied. EPC is involved in multiple processes, beginning with its cardinal role in regulating global and targeted histone acetylation. EPC also frequently serves as an important interaction partner in these basic cellular functions, as well as in multicellular development, such as in hematopoiesis and skeletal muscle differentiation, and in human disease. Taken together, a unifying theme from these studies highlights EPC as a critical genomic regulator.
Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação da Expressão Gênica , Genômica , Animais , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Genômica/métodos , Humanos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Biossíntese de ProteínasRESUMO
Ossifying fibromyxoid tumor (OFMT) is a rare soft tissue neoplasm of uncertain histogenesis, primarily arising in subcutaneous tissues of the extremities, head and neck, or trunk. Most cases present as well-circumscribed masses with a characteristic morphologic appearance, comprising cytologically bland ovoid cells with fibromyxoid stroma, a peripheral rim of metaplastic bone, and lobulated architecture. Nevertheless, tumors displaying unusual morphologic characteristics pose significant diagnostic challenges, requiring the detection of a pathogenic fusion for a definitive diagnosis. The majority of OFMTs exhibits PHF1 gene rearrangements. Herein, we present six cases of molecularly confirmed OFMTs with uncommon histomorphologic features, including the absence of myxoid stroma, extensive chondroid differentiation, prominent clear cell morphology, collagen entrapment, interdigitating fibrocollagenous and fibromyxoid stromal elements, and conspicuous red blood cell extravasation. One case harbored a novel fusion (EPC1::SUZ12). This study emphasizes the broad range of morphologic manifestations that can be encountered in OFMT and the crucial role of molecular testing in establishing a conclusive diagnosis in such cases.
RESUMO
Alzheimer's disease (AD) is the most common dementia in the world. Increasing evidence has shown that exosomes from hypoxic pretreated adipose-derived stem cells (ADSCs) could be an effective cognitive function therapeutic in AD-associated pathophysiology. However, their role and regulatory mechanism remain largely unknown. High-throughput sequencing was used to identify differentially expressed circRNAs from ADSCs or hypoxia pretreated ADSC exosomes. Luciferase reporter assays and RT-qPCR were used to investigate the relationships between circ-Epc1, miR-770-3p, and TREM2. APP/PS1 double transgenic AD model mice were then used to study therapeutic effects regarding circ-Epc1 in ADSC exosomes. BV2 cells were used to show the regulatory relationships between circ-Epc1, miR-770-3p, and TREM2 and to show how these interactions modulated phenotypic transformations and inflammatory cytokine expressions in microglia. The results showed that exosomes from hypoxia pretreated ADSCs had a good therapeutic effect on improving cognitive functions by decreasing neuronal damage in the hippocampus. High-throughput sequencing showed that circ-Epc1 played an important role in hypoxia-pretreated ADSC exosomes regarding their ability to improve cognitive functions. Luciferase reporter assays showed that TREM2 and miR-770-3p were downstream targets of circ-Epc1. Overexpressing miR-770-3p or downregulating TREM2 reversed the effects of circ-Epc1 on M2 microglia during lipopolysaccharide treatment. In vivo experiments showed that circ-Epc1-containing ADSC exosomes increased the therapeutic effect of exosomes by improving cognitive function, decreasing neuronal damage, and shifting hippocampal microglia from the M1 polarization to the M2 polarization stages. Taken together, the data show that hypoxic pretreatment of ADSC exosomes improved cognition by delivery of circ-Epc1 and by shifting microglial M1/M2 polarization in an AD mouse model.
Assuntos
Doença de Alzheimer , Exossomos , MicroRNAs , Adipócitos/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/terapia , Animais , Cognição , Modelos Animais de Doenças , Exossomos/metabolismo , Hipóxia/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Microglia/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Células-Tronco/metabolismoRESUMO
BACKGROUND: Comb homolog enhancer 1 (EPC1) gene is one of the important members of epigenetic inhibitor PCG family. It shows carcinogenic potential in a variety of malignant tumors, but the expression and role of EPC1 in nasopharyngeal carcinoma are unclear. The aim of this study was to explore the expression and function of enhancer of polycomb homolog 1 (EPC1) in nasopharyngeal carcinoma (NPC). METHODS: The differential expression of EPC1 in the cancer tissues and cell lines of NPC was examined by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). EPC1 expression, cell proliferation, and apoptosis were detected in NPC cell lines after EPC1 silencing, and the levels of the epithelial-mesenchymal transition (EMT)-related proteins E-cadherin and vimentin were detected in NPC cells after EPC1 silencing. The study was performed at Fujian Provincial Hospital, Fujian, China, from 2018 to 2019. RESULTS: We found that EPC1 was significantly upregulated in the cancer tissues and cell lines of NPC (P<0.001). Furthermore, knockdown of EPC1 inhibited the growth and metastasis of NPC cells. E-cadherin and vimentin were detected in NPC cells after EPC1 was knocked out. It was confirmed that inhibition of EPC1 resulted in increased E-cadherin expression (P<0.001) and decreased vimentin expression (P<0.001), suggesting that inhibition of EPC1 could inhibit the EMT in NPC cells. CONCLUSION: EPC1 expression was upregulated in NPC tissues and cell lines. Knockout of EPC1 effectively inhibited the growth of NPC cells, induced apoptosis, and inhibited invasion and metastasis. Inhibition of EPC1 could inhibit the EMT in NPC cells. All of the above findings support the viewpoint that EPC1 plays a pro-cancer role in NPC.
Assuntos
Neoplasias Nasofaríngeas , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Invasividade Neoplásica/genéticaRESUMO
MBTD1, a H4K20me reader, has recently been identified as a component of the NuA4/TIP60 acetyltransferase complex, regulating gene expression and DNA repair. NuA4/TIP60 inhibits 53BP1 binding to chromatin through recognition of the H4K20me mark by MBTD1 and acetylation of H2AK15, blocking the ubiquitination mark required for 53BP1 localization at DNA breaks. The NuA4/TIP60 non-catalytic subunit EPC1 enlists MBTD1 into the complex, but the detailed molecular mechanism remains incompletely explored. Here, we present the crystal structure of the MBTD1-EPC1 complex, revealing a hydrophobic C-terminal fragment of EPC1 engaging the MBT repeats of MBTD1 in a site distinct from the H4K20me binding site. Different cellular assays validate the physiological significance of the key residues involved in the MBTD1-EPC1 interaction. Our study provides a structural framework for understanding the mechanism by which MBTD1 recruits the NuA4/TIP60 acetyltransferase complex to influence transcription and DNA repair pathway choice.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Lisina Acetiltransferase 5/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Linhagem Celular , Dano ao DNA , Análise Mutacional de DNA , Humanos , Lisina Acetiltransferase 5/química , Modelos Moleculares , Ligação Proteica , Homologia Estrutural de ProteínaRESUMO
Long noncoding RNAs (lncRNAs), such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. However, lncRNA expression in the serum of HCC patients is still unclear. To identify candidate lncRNAs for HCC diagnosis, we purified exosomal total RNA from the serum of healthy volunteers (controls) and hepatitis, cirrhosis, and HCC patients. To assess the function of lncRNAs, small interfering RNAs and overexpression vectors were designed and cell viability and cell apoptosis assays conducted. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients. qPCR assays showed that lnc-FAM72D-3, lnc-GPR89B-15, lncZEB2-19, and lnc-EPC1-4 are differentially expressed in HCC. Furthermore, the expression level of lnc-EPC1-4 correlated with age. While the expression levels of lnc-GPR89B-15 and lnc-EPC1-4 correlated with serum alpha-fetoprotein level. lnc-FAM72D-3 knockdown decreased cell viability and promoted cell apoptosis, indicating that lnc-FAM72D-3 functions as an oncogene in HCC. In contrast, lnc-EPC1-4 overexpression inhibited cell proliferation and induced cell apoptosis, indicating that it functions as a tumor suppressor gene. Collectively, these findings show that lnc-FAM72D-3 and lnc-EPC1-4 play a novel role that might contribute to hepatocarcinogenesis and identify potential candidate biomarkers for HCC diagnosis.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Exossomos/metabolismo , Neoplasias Hepáticas/diagnóstico , RNA Longo não Codificante/sangue , Adolescente , Adulto , Apoptose/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células/genética , Detecção Precoce de Câncer/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Voluntários Saudáveis , Hepatite/sangue , Humanos , Cirrose Hepática/sangue , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Oncogenes , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA-Seq , Adulto JovemRESUMO
Three recombinant proteins of Echinococcus granulosus including two antigen B sub-units EgAgB8/1 and EgAgB8/2 and Echinococcus protoscolex calcium binding protein 1 (EPC1) were expressed in prokaryotic expression vectors. The diagnostic potential of these three recombinant proteins was evaluated in the detection of cystic echinococcosis in buffaloes in IgG-ELISA. The EgAgB8/1 and EgAgB8/2 recombinant proteins reacted fairly with the hydatid infected buffaloes with sensitivity of 75.0% and 78.6%, respectively and specificity of 75.8% while EPC1 recombinant protein showed higher sensitivity (89.3%) but lower specificity (51.5%). Cross-reactivity of these three antigens was assayed with buffalo sera naturally infected with Explanatum explanatum, Paramphistomum epiclitum, Gastrothylax spp., Fasciola gigantica and Sarcocystis spp. EgAgB8/1 and EPC1 antigens cross-reacted with all these sera while EgAgB8/2 showed no cross-reaction with Sarcocystis spp. and reacted with some of the E. explanatum infected buffalo sera. This study explores the potential of three hydatid antigens viz. EgAgB8/1, EgAgB8/2 and EPC1 for their diagnostic potential in buffaloes positive for cystic echinococcosis.
Assuntos
Antígenos de Helmintos/imunologia , Búfalos/parasitologia , Equinococose/veterinária , Echinococcus granulosus/genética , Proteínas de Helminto/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Búfalos/imunologia , Equinococose/diagnóstico , Equinococose/imunologia , Echinococcus granulosus/química , Echinococcus granulosus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Helminto/química , Imunoglobulina G/sangue , Proteínas Recombinantes/imunologia , Sensibilidade e EspecificidadeRESUMO
The immunodiagnostic tests for cystic echinococcosis (CE) are mostly serological tests based on ELISA that use hydatid cyst antigens for primary screening because of its simple preparation and availability. The challenge to develop new serological methods (as compared to those based on the hydatid cyst fluid antigens) to meet the gold standard remains. Appropriate sources of antigenic material are necessary for application to improve the efficacy of immunodiagnostic tests at a population level. In the current study, a fusion protein containing the coding sequence of antigen B2t and two sequences of EPC1 antigen with some modifications was reconstructed. Using bioinformatics tools, these sequences were joined together by applying the sequence of a rigid α-helix-forming linker to obtain an appropriate structure of a fusion protein. Synthetic recombinant fusion protein was expressed using pET28a as a vector and evaluated by indirect ELISA test for sera from patients with hepatic CE and other parasitic infections. The sensitivity of the fusion protein was lower (88.46%) than the available ELISA kit (96.15%). However, the differences in sensitivity were not statistically significant as compared to the recombinant fusion peptide with the commercial kit (p = 0.269). The specificity of the recombinant fusion protein (95.45%) was not significantly lower than the commercial kit (96.59%; p = 1.000). Moreover, surprisingly there was no difference in the cross-reactivity values of performance between the recombinant-ELISA and commercial kit. The positive and negative predictive values of the recombinant antigen were achieved as 92% and 93.33%, respectively, while for the commercial kit, they were obtained as 94.33% and 97.70%, respectively. In conclusion, as an early evaluation of these antigens the performance of our recombinant fusion protein in ELISA is relatively promising. Although, it seemed that this peptide with specific antigenic epitopes might be more appropriate for the serological evaluation of CE by use of bioinformatics tools, our findings showed that cross-reactions and a negative reaction could occur in clinical performance. This fusion protein may have utility for diagnosis in humans, but further evaluation is needed using the WHO ultrasound classification for CE.
Assuntos
Antígenos de Helmintos/imunologia , Equinococose/diagnóstico , Proteínas de Helminto/imunologia , Proteínas Recombinantes de Fusão/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Kit de Reagentes para Diagnóstico , Testes SorológicosRESUMO
Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.
Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Espermátides/crescimento & desenvolvimento , Espermatogênese , Transativadores/metabolismo , Acetilação , Animais , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Histona Acetiltransferases/genética , Lisina Acetiltransferase 5 , Masculino , Camundongos , Proteínas Repressoras/genética , Espermátides/metabolismo , Transativadores/genéticaRESUMO
BACKGROUND AND AIM: Lung cancer is one of leading malignant tumor worldwide with a high mortality rate. A new therapy target, enhancer of polycomb1 (EPC1) knocked down by short hairpin RNA (shRNA) interference technology, for lung cancer was established to investigate its effects on lung cancer in present study. METHODS: RNA interference technology was applied to down-regulate the expression of EPC1 by specific-shRNA with lentivirus vector in neoplastic human alveolar basal epithelial cells (A549 cells). The survival rate and apoptosis were respectively measured by MTT and Flow Cytometry to evaluate the effects of shRNA EPC1 on cells. Mice xenografts of HCT116 cells with shRNA EPC1 were also established to assess the effect on tumor growth. The levels of AKT and p65 were detected by western blotting. RESULTS: The down-regulation of EPC1 by specific-shRNA with lentivirus vector was significantly decreased the survival rate and apoptosis of A549 cells, and the tumors in EPC1 shRNA transfection group had a significant lower size and weight compared with the ones with control shRNA. The protein expression of p-AKT and p65 was reduced by EPC1 shRNA in both in vitro and in vivo experiments. CONCLUSION: Silencing EPC1 by shRNA technology had the inhibition effects on cell proliferation and tumor growth in lung cancer, which provided a new potential target for treatment of cancers.
Assuntos
Apoptose/genética , Proteínas Cromossômicas não Histona/biossíntese , Neoplasias Pulmonares/patologia , RNA Interferente Pequeno , Proteínas Repressoras/biossíntese , Animais , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Cystic echinococcosis is an important zoonosis caused by the Echinococcus granulosus with a substantial impact in human and animal health in endemic areas. The purpose of the present study was serodiagnosis optimizing of dog echinococcosis in order to achieve a rapid diagnostic method. METHODS: Eight dogs were challenged with protoscoleces in order to have positive echinococcosis serum and 2 two-month old puppies were used as uninfected controls. Colloidal gold was prepared by controlled reduction of a boiling solution of chloroauric acid (H [AuCl4]) with sodium citrate and labeled with recombinant EPC1. Dot immunogold filtration assay (DIGFA) was developed by coating rEPC1 labeled colloidal gold on nitrocellulose membrane. The canine sera, taken three times including, 15, 28 and 35 days post infection were tested. A total of 30 serum samples including 24 sera from 8 infected dogs and 6 sera from 2 puppies were comparatively detected with both DIGFA and ELISA. RESULTS: Gold labeled antigen, showed a dark purple dot with agglutination particles in positive sera and light purple dot without agglutination in negative sera. Among 30 serum samples, 23 were positive and 7 were negative with DIGFA and 24 were positive, 6 were negative with ELISA. CONCLUSION: DIGFA as a rapid and simple procedure could be utilized in quickly diagnosis of echinococcosis.