RESUMO
In this cross-sectional study, we hypothesized that a high dietary ratio of omega-6 (n-6) to omega-3 (n-3) fatty acids could be associated with an altered gut bacterial composition and with the disease severity in patients with nonalcoholic fatty liver disease (NAFLD). A total of 101 NAFLD patients were included in the study, of which 63 underwent a liver biopsy. All 101 patients completed a 14-day food and activity record. Ebispro 2016 professional software was used to calculate individual macronutrients and micronutrients consumed. Patients were grouped into 3 quantiles (Q) according to a low (Q1: <6.1, nâ¯=â¯34), moderate (Q2: 6.1-7.8, nâ¯=â¯33), or high (Q3: >7.8, nâ¯=â¯34) dietary n-6/n-3 ratio. Stool samples were analyzed using 16S rRNA gene sequencing. Spearman correlation coefficients and principal coordinate analysis were used to detect differences in the bacterial composition of the gut microbiota. The median dietary n-6/n-3 ratio of all patients was 6.7 (range, 3.1-14.9). No significant associations between the dietary n-6/n-3 ratio and the gut microbiota composition or disease severity were observed. However, the abundance of specific bacteria such as Catenibacterium or Lactobacillus ruminis were found to be positively correlated and the abundance of Clostridium were negatively correlated with dietary n-6 fatty acid intake. The results indicate that a high dietary n-6/n-3 ratio is probably not a highly relevant factor in the pathogenesis of human NAFLD. Further studies are needed to clarify the importance of interactions between gut bacterial taxa and n-6 fatty acids in the pathophysiology of NAFLD.
Assuntos
Ácidos Graxos Ômega-3 , Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/etiologia , Microbioma Gastrointestinal/fisiologia , RNA Ribossômico 16S/genética , Estudos Transversais , Bactérias/genética , Índice de Gravidade de DoençaRESUMO
Objective: The DIRAS2 gene is associated with ADHD, but its function is largely unknown. Thus, we aimed to explore the genes and molecular pathways affected by DIRAS2. Method: Using short hairpin RNAs, we downregulated Diras2 in murine hippocampal primary cells. Gene expression was analyzed by microarray and affected pathways were identified. We used quantitative real-time polymerase chain reaction (qPCR) to confirm expression changes and analyzed enrichment of differentially expressed genes in an ADHD GWAS (genome-wide association studies) sample. Results:Diras2 knockdown altered expression of 1,612 genes, which were enriched for biological processes involved in neurodevelopment. Expression changes were confirmed for 33 out of 88 selected genes. These 33 genes showed significant enrichment in ADHD patients in a gene-set-based analysis. Conclusion: Our findings show that Diras2 affects numerous genes and thus molecular pathways that are relevant for neurodevelopmental processes. These findings may further support the hypothesis that DIRAS2 is linked to etiological processes underlying ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Estudo de Associação Genômica Ampla , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , GTP Fosfo-Hidrolases , Técnicas de Silenciamento de Genes , Hipocampo , Humanos , CamundongosRESUMO
Oncogenic RAS provides crucial survival signaling for up to half of multiple myeloma cases, but has so far remained a clinically undruggable target. RAL is a member of the RAS superfamily of small GTPases and is considered to be a potential mediator of oncogenic RAS signaling. In primary multiple myeloma, we found RAL to be overexpressed in the vast majority of samples when compared with pre-malignant monoclonal gammopathy of undetermined significance or normal plasma cells. We analyzed the functional effects of RAL abrogation in myeloma cell lines and found that RAL is a critical mediator of survival. RNAi-mediated knockdown of RAL resulted in rapid induction of tumor cell death, an effect which was independent from signaling via mitogen-activated protein kinase, but appears to be partially dependent on Akt activity. Notably, RAL activation was not correlated with the presence of activating RAS mutations and remained unaffected by knockdown of oncogenic RAS. Furthermore, transcriptome analysis yielded distinct RNA expression signatures after knockdown of either RAS or RAL. Combining RAL depletion with clinically relevant anti-myeloma agents led to enhanced rates of cell death. Our data demonstrate that RAL promotes multiple myeloma cell survival independently of oncogenic RAS and, thus, this pathway represents a potential therapeutic target in its own right.
Assuntos
GTP Fosfo-Hidrolases , Mieloma Múltiplo , Sobrevivência Celular/genética , Genes ras , Humanos , Mieloma Múltiplo/genética , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismoRESUMO
Myocardial infarction (MI) is a major cause of death worldwide. Here, we identify the macrophage MR (mineralocorticoid receptor) as a crucial pathogenic player in cardiac wound repair after MI. Seven days after left coronary artery ligation, mice with myeloid cell-restricted MR deficiency compared with WT (wild type) controls displayed improved cardiac function and remodeling associated with enhanced infarct neovascularization and scar maturation. Gene expression profiling of heart-resident and infarct macrophages revealed that MR deletion drives macrophage differentiation in the ischemic microenvironment toward a phenotype outside the M1/M2 paradigm, with regulation of multiple interrelated factors controlling wound healing and tissue repair. Mechanistic and functional data suggest that inactivation of the macrophage MR promotes myocardial infarct healing through enhanced efferocytosis of neutrophils, the suppression of free radical formation, and the modulation of fibroblast activation state. Crucially, targeted delivery of MR antagonists to macrophages, with a single administration of RU28318 or eplerenone-containing liposomes at the onset of MI, improved the healing response and protected against cardiac remodeling and functional deterioration, offering an effective and unique therapeutic strategy for cardiac repair.
Assuntos
Eplerenona/farmacologia , Coração/fisiopatologia , Infarto do Miocárdio , Miocárdio , Receptores de Mineralocorticoides , Cicatrização , Animais , Diferenciação Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Lipossomos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Receptores de Mineralocorticoides/deficiência , Receptores de Mineralocorticoides/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/fisiologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
Cell- and tissue-specific actions of glucocorticoids are mediated by the glucocorticoid receptor. Here, we demonstrate that the glucocorticoid receptor (GR) in macrophages is essential for cardiac healing after myocardial infarction. Compared with GRflox (wild-type controls), GRLysMCre mice that lacked GR in myeloid cells showed increased acute mortality as a result of cardiac rupture. Seven days after left coronary artery ligation, GRLysMCre mice exhibited worse cardiac function and adverse remodeling associated with impaired scar formation and angiogenic response to ischemic injury. Inactivation of GR altered the functional differentiation/maturation of monocyte-derived macrophages in the infarcted myocardium. Mechanistically, CD45+/CD11b+/Ly6G-/F4/80+ macrophages isolated from GRLysMCre infarcts showed deregulation of factors that control inflammation, neovascularization, collagen degradation, and scar tissue formation. Moreover, we demonstrate that cardiac fibroblasts sorted from the ischemic myocardium of GRLysMCre mice compared with cells isolated from injured GRflox hearts displayed higher matrix metalloproteinase 2 expression, and we provide evidence that the macrophage GR regulates myofibroblast differentiation in the infarct microenvironment during the early phase of wound healing. In summary, GR signaling in macrophages, playing a crucial role in tissue-repairing mechanisms, could be a potential therapeutic target during wound healing after ischemic myocardial injury.-Galuppo, P., Vettorazzi, S., Hövelmann, J., Scholz, C.-J., Tuckermann, J. P., Bauersachs, J., Fraccarollo, D. The glucocorticoid receptor in monocyte-derived macrophages is critical for cardiac infarct repair and remodeling.
Assuntos
Macrófagos/metabolismo , Monócitos/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Monócitos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Receptores de Glucocorticoides/genéticaRESUMO
In a previous study, we identified the single nucleotide polymorphism (SNP) rs4500567, located in the upstream region of tetraspanin 8 (TSPAN8), to be associated with bipolar disorder (BD). Due to its proximal position, the SNP might have an impact on promoter activity, thus on TSPAN8 gene expression. We investigated the impact of rs4500567 on TSPAN8 expression in vitro with luciferase-based promoter assays in human embryonic kidney (HEK293) and neuroblastoma cells (SH-SY5Y), and its effect on expression of downstream associated genes by microarray-based transcriptome analyses. Immunohistochemical localization studies on murine brain slices served to identify possible target regions of altered TSPAN8 expression in the brain. Promoter assays revealed decreased TSPAN8 expression in presence of the minor allele. Transcriptome analyses of TSPAN8-knockdown cells, mirroring the effects of putatively reduced TSPAN8 expression in minor allele carriers, resulted in 231 differentially expressed genes with enrichments of relevant signaling pathways for psychiatric disorders and neuronal development. Finally, we demonstrate Tspan8 abundance in mouse cerebellum and hippocampus. These findings point to a role of TSPAN8 in neuronal function or development. Considering a rather protective effect of the minor allele of rs4500567, our findings reveal a possible novel mechanism that contributes to the development of BD.
Assuntos
Transtorno Bipolar/patologia , Encéfalo/patologia , Regulação da Expressão Gênica , Neuroblastoma/patologia , Polimorfismo de Nucleotídeo Único , Tetraspaninas/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Encéfalo/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma/genética , Neuroblastoma/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Tetraspaninas/genética , Células Tumorais CultivadasRESUMO
The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.
Assuntos
Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/metabolismo , Proteínas de Ligação a Retinoblastoma/metabolismo , Neoplasias Cutâneas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/patologia , Carcinoma de Célula de Merkel/virologia , Humanos , Poliomavírus das Células de Merkel/metabolismo , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Transfecção , Ubiquitina-Proteína Ligases/genéticaRESUMO
Colon cancer is one of the most common tumors in the human population. Recent studies have shown a reduced risk for colon cancer in patients given the antidepressant fluoxetine (FLX). The exact mechanism by which FLX might protect from colon cancer remains however controversial. Here, FLX reduced the development of different colon tumor xenografts, as well as proliferation in hypoxic tumor areas within them. FLX treatment also decreased microvessel numbers in tumors. Although FLX did not increase serum and tumor glucose levels as much as the colon chemotherapy gold standard Fluorouracil did, lactate levels were significantly augmented within tumors by FLX treatment. The gene expression of the MCT4 lactate transporter was significantly downregulated. Total protein amounts from the third and fifth mitochondrial complexes were significantly decreased by FLX in tumors. Cell culture experiments revealed that FLX reduced the mitochondrial membrane potential significantly and disabled the reactive oxygen species production of the third mitochondrial complex. Furthermore, FLX arrested hypoxic colon tumor cells in the G0/G1 phase of the cell-cycle. The expression of key cell-cycle-related checkpoint proteins was enhanced in cell culture and in vivo experiments. Therefore, we suggest FLX impairs energy generation, cell cycle progression and proliferation in tumor cells, especially under condition of hypoxia. This then leads to reduced microvessel formation and tumor shrinkage in xenograft models.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Fluoxetina/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Animais , Células CACO-2 , Hipóxia Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fase G1/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of "restored" CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430).
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células/métodos , Leucócitos Mononucleares/citologia , Especificidade do Receptor de Antígeno de Linfócitos T , Contagem de Células , Células Cultivadas , Criança , Humanos , Leucócitos Mononucleares/fisiologiaRESUMO
Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , PTEN Fosfo-Hidrolase/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Citoesqueleto de Actina , Actinas/biossíntese , Benzotiazóis/farmacologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Interferência de RNA , RNA Interferente Pequeno , Serina-Treonina Quinases TOR/biossíntese , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVES: Methylphenidate (MPH) is a commonly used stimulant medication for treating attention-deficit/hyperactivity disorder (ADHD). Besides inhibiting monoamine reuptake there is evidence that MPH also influences gene expression directly. METHODS: We investigated the impact of MPH treatment on gene expression levels of lymphoblastoid cells derived from adult ADHD patients and healthy controls by hypothesis-free, genome-wide microarray analysis. Significant findings were subsequently confirmed by quantitative Real-Time PCR (qRT PCR) analysis. RESULTS: The microarray analysis from pooled samples after correction for multiple testing revealed 138 genes to be marginally significantly regulated due to MPH treatment, and one gene due to diagnosis. By qRT PCR we could confirm that GUCY1B3 expression was differential due to diagnosis. We verified chronic MPH treatment effects on the expression of ATXN1, HEY1, MAP3K8 and GLUT3 in controls as well as acute treatment effects on the expression of NAV2 and ATXN1 specifically in ADHD patients. CONCLUSIONS: Our preliminary results demonstrate MPH treatment differences in ADHD patients and healthy controls in a peripheral primary cell model. Our results need to be replicated in larger samples and also using patient-derived neuronal cell models to validate the contribution of those genes to the pathophysiology of ADHD and mode of action of MPH.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Transtorno do Deficit de Atenção com Hiperatividade/genética , Regulação da Expressão Gênica , Metilfenidato/uso terapêutico , Adulto , Ataxina-1/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Linhagem Celular , DNA Helicases , Feminino , Transportador de Glucose Tipo 3/genética , Guanilato Ciclase/genética , Humanos , MAP Quinase Quinase Quinases/genética , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Citoplasmáticos e Nucleares/genética , Guanilil Ciclase Solúvel , Adulto JovemRESUMO
A lack of reliably informative biomarkers to distinguish indolent and lethal prostate cancer is one reason this disease is overtreated. miR-221 has been suggested as a biomarker in high-risk prostate cancer, but there is insufficient evidence of its potential utility. Here we report that miR-221 is an independent predictor for cancer-related death, extending and validating earlier findings. By mechanistic investigations we showed that miR-221 regulates cell growth, invasiveness, and apoptosis in prostate cancer at least partially via STAT1/STAT3-mediated activation of the JAK/STAT signaling pathway. miR-221 directly inhibits the expression of SOCS3 and IRF2, two oncogenes that negatively regulate this signaling pathway. miR-221 expression sensitized prostate cancer cells for IFN-γ-mediated growth inhibition. Our findings suggest that miR-221 offers a novel prognostic biomarker and therapeutic target in high-risk prostate cancer.
Assuntos
Fator Regulador 2 de Interferon/genética , MicroRNAs/genética , Neoplasias da Próstata/metabolismo , Interferência de RNA , Proteínas Supressoras da Sinalização de Citocina/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 2 de Interferon/metabolismo , Interferon gama/fisiologia , Estimativa de Kaplan-Meier , Masculino , Invasividade Neoplásica , Fosforilação , Prognóstico , Modelos de Riscos Proporcionais , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Processamento de Proteína Pós-Traducional , Fatores de Transcrição STAT/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , TranscriptomaRESUMO
The treatment of high-risk prostate cancer (HRPCa) is a tremendous challenge for uro-oncologists. The identification of predictive moleculobiological markers allowing risk assessment of lymph node metastasis and systemic progression is essential in establishing effective treatment. In the current study, we investigate the prognostic potential of miR-205 in HRPCa study and validation cohorts, setting defined clinical endpoints for both. We demonstrate miR-205 to be significantly down-regulated in over 70% of the HRPCa samples analysed and that reconstitution of miR-205 causes inhibition of proliferation and invasiveness in prostate cancer (PCa) cell lines. Additionally, miR-205 is increasingly down-regulated in lymph node metastases compared to the primary tumour indicating that miR-205 plays a role in migration of PCa cells from the original location into extraprostatic tissue. Nevertheless, down-regulation of miR-205 in primary PCa was not correlated to the synchronous presence of metastasis and failed to predict the outcome for HRPCa patients. Moreover, we found a tendency for miR-205 up-regulation to correlate with an adverse outcome of PCa patients suggesting a pivotal role of miR-205 in tumourigenesis. Overall, we showed that miR-205 is involved in the development and metastasis of PCa, but failed to work as a useful clinical biomarker in HRPCa. These findings might have implications for the use of miR-205 as a prognostic or therapeutic target in HRPCa.
Assuntos
Metástase Linfática/genética , MicroRNAs/genética , Prognóstico , Neoplasias da Próstata/genética , Biomarcadores Tumorais , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Invasividade Neoplásica/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapiaRESUMO
CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology (5 transmembrane domains) and a deviating spectrum of functions can be expected.
RESUMO
BACKGROUND: The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. METHODOLOGY AND PRINCIPAL FINDINGS: We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (nâ=â98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. CONCLUSION: Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteína HMGA1a/genética , MicroRNAs/metabolismo , Recidiva Local de Neoplasia/metabolismo , Neoplasias da Próstata/metabolismo , Transcriptoma , Regiões 3' não Traduzidas , Adulto , Idoso , Sítios de Ligação , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Estudos de Coortes , Intervalo Livre de Doença , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Proteína HMGA1a/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/cirurgia , Interferência de RNA , Risco , Falha de TratamentoRESUMO
PURPOSE: Patients with high risk prostate cancer with pT3 tumor and positive surgical margins have a high risk of biochemical failure after radical prostatectomy and adjuvant androgen deprivation therapy. Predictors of cancer related death in this patient group are necessary. MATERIALS AND METHODS: We performed subset analysis of a prospective trial including 550 consecutive patients with preoperative high risk prostate cancer (prostate specific antigen greater than 20 ng/ml ± cT3/4 ± biopsy Gleason 8-10). Men who underwent radical prostatectomy and received continuous adjuvant androgen deprivation therapy for pT3a/b N0-1 positive surgical margin disease were included in the analysis, and none of the patients received neoadjuvant androgen deprivation therapy or adjuvant radiation therapy. RESULTS: Overall 173 of 550 patients (31.5%) with a median followup of 67 months met the study inclusion criteria. For these men the estimated 8-year prostate cancer specific and overall survival rates were 86.3% and 77.0%, respectively. Tumor stage and positive surgical margin at the bladder neck were independent predictors of prostate cancer specific survival and overall survival, and were used to substratify cases. Those with pT3b disease with positive surgical margins at the bladder neck had the highest risk of death (5-year cancer specific survival 60.0% and overall survival 52.3%), while pT3a disease (regardless of positive surgical margin location and lymph node invasion) and pT3b tumors with negative bladder neck margins had 8-year prostate cancer specific survival and overall survival rates of 92.0% and 84.9%, respectively. CONCLUSIONS: The results of this trial demonstrated the heterogeneity of high risk prostate cancer cases with T3 tumors and positive surgical margins. The presented substratification by tumor stage and positive surgical margin location identifies men at high risk for prostate cancer related death and might help in the design of adjuvant therapy trials.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Androgênios/sangue , Flutamida/uso terapêutico , Estadiamento de Neoplasias , Prostatectomia/métodos , Neoplasias da Próstata/cirurgia , Idoso , Biópsia , Quimioterapia Adjuvante , Endossonografia , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológico , Estudos Retrospectivos , Fatores de Risco , Fatores de Tempo , Tomografia Computadorizada por Raios XRESUMO
Introduction. To assess the role of adjuvant androgen deprivation therapy (ADT) in high-risk prostate cancer patients (PCa) after surgery. Materials and Methods. The analysis case matched 172 high-risk PCa patients with positive section margins or non-organ confined disease and negative lymph nodes to receive adjuvant ADT (group 1, n = 86) or no adjuvant ADT (group 2, n = 86). Results. Only 11.6% of the patients died, 2.3% PCa related. Estimated 5-10-year clinical progression-free survival was 96.9% (94.3%) for group 1 and 73.7% (67.0%) for group 2, respectively. Subgroup analysis identified men with T2/T3a tumors at low-risk and T3b margins positive disease at higher risk for progression. Conclusion. Patients with T2/T3a tumors are at low-risk for metastatic disease and cancer-related death and do not need adjuvant ADT. We identified men with T3b margin positive disease at highest risk for clinical progression. These patients benefit from immediate adjuvant ADT.
RESUMO
Tetraspanins affect protein trafficking and are known to influence a wide variety of physiologic processes. Recently, single nucleotide polymorphisms (SNPs) of the tetraspanin gene TSPAN8 were found among the best ranked markers of genome wide association studies on bipolar disorder (BPD) (rs1705236) and type-2 diabetes, but functional consequences remained largely unknown. In the present study, we examined 13 tagging SNPs covering the TSPAN8 gene, the intronic TSPAN8 SNP rs1705236 as well as two non-synonymous (ns) SNPs in schizophrenia (SCZ) and BPD samples. In our analysis setting, we were not able to replicate the association of rs1705236 with BPD, nor did we find an association with SCZ. In the TSPAN8 upstream transcriptional control region however, we found rs4500567 to be associated with BPD. In contrast, in SCZ the nsSNP rs3763978 was associated with disease. The significance of both associations withstood conservative Bonferroni correction. In an attempt to link the polymorphisms to functional consequences, we performed an allele-specific in silico mapping of transcription factor binding sites around rs4500567 and predicted the tolerance of the Gly73Ala exchange caused by rs3763978. The results argue for a differential promoter activity specific for the variant associated with BPD, but impaired protein functionality in SCZ. This suggests that TSPAN8 contributes to both diseases, yet with different underlying mechanisms: regulatory versus structural. Similar phenomena might also occur in other risk genes for both BPD and SCZ, providing a molecular basis for the genetic overlap of both entities.
Assuntos
Transtorno Bipolar/genética , Esquizofrenia/genética , Alelos , Antígenos de Neoplasias , Estudos de Casos e Controles , Estudo de Associação Genômica Ampla , Humanos , Entrevistas como Assunto , Glicoproteínas de Membrana , Proteínas de Membrana , Estudos Multicêntricos como Assunto , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , TetraspaninasRESUMO
Emerging evidence shows that microRNAs (miR) are involved in the pathogenesis of a variety of cancers, including prostate carcinoma (PCa). Little information is available regarding miR expression levels in lymph node metastasis of prostate cancer or the potential of miRs as prognostic markers in this disease. Therefore, we analyzed the global expression of miRs in benign, hyperplastic prostate tissue (BPH), primary PCa of a high risk group of PCa patients, and corresponding metastatic tissues by microarray analysis. Consistent with the proposal that some miRs are oncomirs, we found aberrant expression of several miRs, including the downregulation of miR-221, in PCa metastasis. Downregulation of miR-221 was negatively correlated with the expression of the proto-oncogen c-kit in primary carcinoma. In a large study cohort, the prostate-specific oncomir miR-221 was progressively downregulated in aggressive forms of PCa. Downregulation of miR-221 was associated with clinicopathological parameters, including the Gleason score and the clinical recurrence during follow up. Kaplan-Meier estimates and Cox proportional hazard models showed that miR-221 downregulation was linked to tumor progression and recurrence in a high risk prostate cancer cohort. Our results showed that progressive miR-221 downregulation hallmarks metastasis and presents a novel prognostic marker in high risk PCa. This suggests that miR-221 has potential as a diagnostic marker and therapeutic target in PCa.
Assuntos
Biomarcadores Tumorais/genética , MicroRNAs/fisiologia , Recidiva Local de Neoplasia/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Idoso , Estudos de Coortes , Biologia Computacional , Progressão da Doença , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Análise de Sequência com Séries de Oligonucleotídeos , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We showed that Tspan-1, a tetraspanin overexpressed in many human cancers, harbours oligosaccharides at all four potential N-glycosylation sites. Its most abundant form contained only mannose-rich sugar chains but two distinct glycosylation sites could also contain complex carbohydrates. Glycosylation seemed to be required for correct folding and subsequent transition through the endoplasmic reticulum.