RESUMO
BACKGROUND: Breast cancer is the most frequently diagnosed malignancy among women. However, the role of microRNA (miRNA) expression in breast cancer progression is not fully understood. In this study we examined predictive interactions between differentially expressed miRNAs and mRNAs in breast cancer cell lines representative of the common molecular subtypes. Integrative bioinformatics analysis identified miR-193 and miR-210 as potential regulatory biomarkers of mRNA in breast cancer. Several recent studies have investigated these miRNAs in a broad range of tumors, but the mechanism of their involvement in cancer progression has not previously been investigated. METHODS: The miRNA-mRNA interactions in breast cancer cell lines were identified by parallel expression analysis and miRNA target prediction programs. The expression profiles of mRNA and miRNAs from luminal (MCF-7, MCF-7/AZ and T47D), HER2 (BT20 and SK-BR3) and triple negative subtypes (Hs578T e MDA-MB-231) could be clearly separated by unsupervised analysis using HB4A cell line as a control. Breast cancer miRNA data from TCGA patients were grouped according to molecular subtypes and then used to validate these findings. Expression of miR-193 and miR-210 was investigated by miRNA transient silencing assays using the MCF7, BT20 and MDA-MB-231 cell lines. Functional studies included, xCELLigence system, ApoTox-Glo triplex assay, flow cytometry and transwell inserts were performed to determine cell proliferation, cytotoxicity, apoptosis, migration and invasion, respectively. RESULTS: The most evident effects were associated with cell proliferation after miR-210 silencing in triple negative subtype cell line MDA-MB-231. Using in silico prediction algorithms, TNFRSF10 was identified as one of the potential regulated downstream targets for both miRNAs. The TNFRSF10C and TNFRSF10D mRNA expression inversely correlated with the expression levels of miR-193 and miR210 in breast cell lines and breast cancer patients, respectively. Other potential regulated genes whose expression also inversely correlated with both miRNAs were CCND1, a known mediator on invasion and metastasis, and the tumor suppressor gene RUNX3. CONCLUSIONS: In summary, our findings identify miR-193 and miR-210 as potential regulatory miRNA in different molecular subtypes of breast cancer and suggest that miR-210 may have a specific role in MDA-MB-231 proliferation. Our results highlight important new downstream regulated targets that may serve as promising therapeutic pathways for aggressive breast cancers.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/metabolismo , Biomarcadores Tumorais/análise , Mama/patologia , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Biologia Computacional , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Ciclina D1/genética , Feminino , Proteínas Ligadas por GPI/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/análise , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Membro 10c de Receptores do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/genéticaRESUMO
The rhythm of factors involved in luteal regression is crucial in determining the physiological duration of the oestrous cycle. Given the role of tumour necrosis factor (TNF)-α in luteal function and circadian regulation and that most of the effects of TNF-α are mediated by p55 TNF receptor (TNFRp55), the aims of the present study were to analyse the following during the luteal regression phase in the ovary of mice: (1) whether the pattern of expression of progesterone (P4) and the enzymes involved in the synthesis and degradation of P4 is circadian and endogenous (the rhythm persists in constant conditions, (i.e., constant darkness) with a period of about 24 hours); (2) circadian oscillations in clock gene expression; (3) whether there are daily variations in the expression of key genes involved in apoptosis and antioxidant mechanisms; and (4) the consequences of TNFRp55 deficiency. P4 was found to oscillate circadianally following endogenous rhythms of clock factors. Of note, TNFRp55 deficiency modified the circadian oscillation in P4 concentrations and its enzymes involved in the synthesis and degradation of P4, probably as a consequence of changes in the circadian oscillations of brain and muscle ARNT-Like protein 1 (Bmal1) and Cryptochrome 1 (Cry1). Furthermore, TNFRp55 deficiency modified the circadian rhythms of apoptosis genes, as well as antioxidant enzymes and peroxidation levels in the ovary in dioestrus. The findings of the present study strengthen the hypothesis that dysregulation of TNF-α signalling may be a potential cause for altered circadian and menstrual cycling in some gynaecological diseases.
Assuntos
Ritmo Circadiano/fisiologia , Corpo Lúteo/metabolismo , Expressão Gênica , Luteólise/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Apoptose/fisiologia , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Ciclo Estral/genética , Ciclo Estral/metabolismo , Feminino , Peroxidação de Lipídeos/fisiologia , Luteólise/genética , Camundongos , Camundongos Knockout , Progesterona/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/genética , Ácido Úrico/sangueRESUMO
Dendritic cells (DCs) play critical functions in the initiation of immune responses. Understanding their role in reactive arthritis (ReA) will help delineate the pathogenesis of this arthropathy. In early studies, we detected IL-12/23p40 deregulation in Yersinia entercolitica (Ye)-induced ReA in TNFRp55-deficient (TNFRp55-/-) mice. In this study, we assessed the contribution of DCs in this overproduction. First, greater levels of IL-12/23p40, IFN-γand IL-17A were confirmed in supernatants of lipopolysaccharide (LPS)-stimulated TNFRp55-/-splenocytes obtained on arthritis onset (day 14 after Ye infection). Later, DCs were identified as a precise source of IL-12/23p40 since increased frequency of splenic IL-12/23p40+DCs was detected in TNFRp55-/- mice. After robust in vivo amplification of DCs by injection of Fms-like tyrosine kinase 3-Ligand (Flt3L)-transfected BL16 melanoma, DCs were purified. These cells recapitulated the higher production of IL-12/23p40 under TNFRp55deficiency. In agreement with these results, TNFRp55-/- DCs promoted Th1 and Th17 programs by co-culture with WT CD4+lymphocytes. A mechanistic study demonstrated that JNK and p38 MAPK pathways are involved in IL-12/23p40 overproduction in purified TNFRp55-/- DCs as well as in the JAWS II cell line. This deregulation was once again attributed to TNFRp55 deficiency since CAY10500, a specific inhibitor of this pathway, compromised TNF-mediated IL-12/23p40 control in LPS-stimulated WT DCs. Simultaneously, this inhibition reduced IL-10 production, suggesting its role mediating IL-12/23p40 regulation by TNFRp55 pathway. These results provide experimental data on the existence of a TNFRp55-mediated anti-inflammatory circuit in DCs. Moreover, these cells may be considered as a novel target in the treatment of ReA.
Assuntos
Artrite Reativa/imunologia , Células Dendríticas/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Células Th1/citologia , Células Th17/citologia , Receptores Chamariz do Fator de Necrose Tumoral/genética , Yersiniose/complicações , Yersinia enterocolitica/imunologia , Animais , Artrite Reativa/patologia , Linhagem Celular , Polaridade Celular , Técnicas de Cocultura , Modelos Animais de Doenças , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Knockout , Proibitinas , Baço/imunologia , Yersiniose/imunologiaRESUMO
Endometriosis is an inflammatory disease depending on estradiol, with TNF-α being one of the most representative cytokines involved in its pathogenesis. TNF-α acts through its bond to the TNFRp55 and TNFRp75 membrane receptors. The aim of this study was to analyze the effect of the TNFRp55 deficiency on the development of ectopic endometriotic-like lesions. Endometriosis was induced surgically in mice of the C57BL/6 strain, wild type (WT) and TNFRp55-/- (KO). After four weeks, the peritoneal fluid was collected and the lesions were counted, measured with a caliper, removed, weighed, fixed or kept at -80°C. We evaluated the cell proliferation by proliferating cell nuclear antigen (PCNA) immunohistochemistry and apoptosis by TUNEL technique in the ectopic lesions. MMP-2 and MMP-9 activities (factors involved in invasiveness) were measured by zymography in the peritoneal fluid; estradiol and progesterone levels were measured by radioimmunoassay in the lesions and in the peritoneal fluid. We found that in KO animals the mean number of lesions established per mouse, the lesion volume, weight and cell proliferation increased and apoptosis decreased. In addition, the activity of MMP-2 and the estradiol level increased, whereas the progesterone level was not significantly modified. In conclusion, the deficiency of TNFRp55 promoted the establishment and development of endometriosis through an increase in the lesion size and high levels of estradiol which correlate with an increase in the MMP-2 activity. This is evidence of the possible association of the deregulation of the TNFRp55 expression and the survival of the endometriotic tissue in ectopic sites.
Assuntos
Endometriose/metabolismo , Endométrio/crescimento & desenvolvimento , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Chamariz do Fator de Necrose Tumoral/deficiência , Animais , Proliferação de Células , Modelos Animais de Doenças , Endometriose/genética , Endometriose/patologia , Endometriose/fisiopatologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/genéticaRESUMO
Spindle-shaped stromal cells, like carcinoma-associated fibroblasts and mesenchymal stem cells, influence tumor behavior and can serve as parameters in the clinical diagnosis, therapy, and prognosis of early breast cancer. Therefore, the aim of this study is to explore the clinicopathological significance of tumor necrosis factor-related apoptosis-induced ligand (TRAIL) receptors (Rs) 2 and 4 (TRAIL-R2 and R4), and interleukin-6 R (IL-6R) in spindle-shaped stromal cells, not associated with the vasculature, as prognostic determinants of early breast cancer patients. Receptors are able to trigger the migratory activity, among other functions, of these stromal cells. We conducted immunohistochemical analysis for the expression of these receptors in spindle-shaped stromal cells, not associated with the vasculature, of primary tumors from early invasive breast cancer patients, and analyzed their association with clinicopathological characteristics. Here, we demonstrate that the elevated levels of TRAIL-R2, TRAIL-R4, and IL-6R in these stromal cells were significantly associated with a higher risk of metastatic occurrence (p = 0.034, 0.026, and 0.006; respectively). Moreover, high expression of TRAIL-R4 was associated with shorter disease-free survival and metastasis-free survival (p = 0.013 and 0.019; respectively). Also, high expression of IL-6R was associated with shorter disease-free survival, metastasis-free survival, and overall survival (p = 0.003, 0.001, and 0.003; respectively). Multivariate analysis showed that IL-6R expression was an independent prognostic factor for disease-free survival and metastasis-free survival (p = 0.035). This study is the first to demonstrate that high levels of IL-6R expression in spindle-shaped stromal cells, not associated with the vasculature, could be used to identify early breast cancer patients with poor outcomes.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptores de Interleucina-6/metabolismo , Células Estromais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Receptores de Interleucina-6/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Células Estromais/patologia , Carga Tumoral , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/metabolismoRESUMO
OBJECTIVE: To assess the expression of TRAIL-R3 and the methylation of a CpG island within the TRAIL-R3 promoter both in cystadenoma tumors and primary and metastatic epithelial ovarian carcinoma (EOC). METHODS: RNA was obtained from women with normal ovarian (NO) tissues (n=18), ovarian serous cystadenoma tumors (n=11) and EOC (n=16) using Trizol. Quantitative PCR (qRT-PCR) was performed to quantify the relative levels of TRAIL-R3. The methylation frequency of the CpG island in the TRAIL-R3 promoter was assessed using the methylation-specific PCR (MSP) assay after DNA bisulfite conversion. The differences between the groups were evaluated using the chi-square, Student's t, ANOVA, Mann-Whitney U, Wilcoxon or Kruskal-Wallis tests as indicated. The survival rates were calculated using the Kaplan-Meier method. RESULTS: Cystadenoma and metastatic EOC tumors expressed significantly more TRAIL-R3 mRNA than primary EOC tumors. Methylation of the TRAIL-R3 promoter was absent in NO tissues, while hemimethylation of the TRAIL-R3 promoter was frequently found in the neoplasia samples with 45.4% of the cystadenoma tumors, 8.3% of the primary EOC samples and 11.1% of the metastatic EOC samples showing at least partial methylation (p=0.018). Neither the expression of TRAIL-R3 nor alterations in the methylation profile were associated to cumulative progression-free survival or the overall survival in EOC patients. CONCLUSIONS: Primary EOC is associated to a lower TRAIL-R3 expression, which leads to a better understanding of the complex disease and highlighting potential therapeutic targets. Promoter DNA methylation was not related to this finding, suggesting the presence of other mechanisms to transcriptional control.
Assuntos
Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/biossíntese , Receptores Chamariz do Fator de Necrose Tumoral/genética , Apoptose/fisiologia , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/patologia , Metilação de DNA , Intervalo Livre de Doença , Epigenômica , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Estudos Prospectivos , Membro 10c de Receptores do Fator de Necrose TumoralRESUMO
While cytokines are major regulators of macrophage activation following host-pathogen interactions, they also act to limit inflammation to avoid tissue damage. In previous studies we reported the development of progressive Yersinia enterocolitica-induced reactive arthritis (ReA) in mice lacking the tumor necrosis factor receptor p55 (TNFRp55). In this work, we analyzed the response of TNFRp55â»/â» macrophages to Y. enterocolitica antigens. We found higher concentration of nitric oxide (NO) in TNFRp55â»/â» compared to wild-type macrophages in response to heat-killed Yersinia (HKY) and Yersinia outer membranes (OM). Moreover, Toll-like receptor (TLR)4 expression was increased in OM-stimulated TNFRp55â»/â» versus wild-type (WT) macrophages. Accordingly, NO production was inhibited in TLR4-deficient macrophages following stimulation with OM, suggesting that LPS may function as a major OM component implicated in these responses. Thus, augmented NO production together with enhanced expression of inducible nitric oxide synthase (iNOS) and higher IL-6 production, may provide a pro-inflammatory setting in Yersinia LPS-stimulated TNFRp55â»/â» macrophages. Augmented synthesis of NO and IL-6 was prevented by treatment with Polymyxin B, or by exposure to a specific NF-κB p65 oligonucleotide antisense, indicating the involvement of TLR4-mediated NF-κB activation in the unleashed pro-inflammatory response triggered by TNFRp55 deficiency. Thus, TNFRp55 modulates macrophage functions in response to Yersinia LPS stimulation through mechanisms involving NO, IL-6 and NF-κB pathways, suggesting an essential regulatory role of TNF via TNFRp55 signaling.
Assuntos
Interleucina-6/metabolismo , Macrófagos Peritoneais/metabolismo , Óxido Nítrico/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Yersinia enterocolitica/imunologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Inflamação , Interleucina-6/genética , Interleucina-6/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Oligodesoxirribonucleotídeos Antissenso/genética , Polimixina B/farmacologia , Proibitinas , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/imunologiaRESUMO
BACKGROUND: Kinin B1 receptors are inducible molecules up-regulated after inflammatory stimuli. This study evaluated the relevance of kinin B1 receptors in a mouse depression behavior model. METHODS: Mice were exposed to a 5-min swimming session, and 30 min later they were injected with E. coli lipopolysaccharide (LPS). Depression-like behavior was assessed by determining immobility time in a tail suspension test. Different brain structures were collected for molecular and immunohistochemical studies. Anhedonia was assessed by means of a sucrose intake test. RESULTS: Our protocol elicited an increase in depression-like behavior in CF1 mice, as assessed by the tail-suspension test, at 24 h. This behavior was significantly reduced by treatment with the selective B1 receptor antagonists R-715 and SSR240612. Administration of SSR240612 also prevented an increase in number of activated microglial cells in mouse hippocampus, but did not affect a reduction in expression of mRNA for brain-derived neurotrophic factor. The increased immobility time following LPS treatment was preceded by an enhancement of hippocampal and cortical B1 receptor mRNA expression (which were maximal at 1 h), and a marked production of TNFα in serum, brain and cerebrospinal fluid (between 1 and 6 h). The depression-like behavior was virtually abolished in TNFα p55 receptor-knockout mice, and increased B1 receptor mRNA expression was completely absent in this mouse strain. Furthermore, treatment with SSR240612 was also effective in preventing anhedonia in LPS-treated mice, as assessed using a sucrose preference test. CONCLUSION: Our data show, for the first time, involvement of kinin B1 receptors in depressive behavioral responses, in a process likely associated with microglial activation and TNFα production. Thus, selective and orally active B1 receptor antagonists might well represent promising pharmacological tools for depression therapy.
Assuntos
Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Depressão/fisiopatologia , Lipopolissacarídeos/farmacologia , Receptor B1 da Bradicinina/metabolismo , Estresse Psicológico , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Humanos , Cininas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/metabolismo , Receptor B1 da Bradicinina/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/genética , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.