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
Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.
Assuntos
Endometriose/imunologia , Endometriose/metabolismo , Endométrio/inervação , Endométrio/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/deficiência , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Endométrio/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The present study was conducted to investigate whether the deficiency of tumor necrosis factor receptor p55 (TNFRp55) modulates oxidative/nitrosative stress and metallomic profile into the peritoneal cavity during the experimental endometriosis progression in mice. Female C57BL/6 mice, wild-type (WT) and TNFRp55 knockout (KO) of two months were used. Endometriosis was induced experimentally by autotransplanting three pieces of the right uterine horn to the intestinal mesentery. After four weeks, endometriotic-like lesions and peritoneal lavage fluid were collected. The obtained peritoneal fluid was analyzed for nitrite levels using the Griess method and trace elements concentrations by ICP-MS. Both endometriotic-like lesions and cells isolated from peritoneal lavage were analyzed for the following oxidative/nitrosative stress markers: inducible nitric oxide synthase (iNOS) expression by Western Blot; total antioxidant capacity (TAC), the activity of two antioxidant enzymes (CAT and GPX) and thiobarbituric acid-reactive substances (TBARS) concentration, by spectrophotometric method; and protein carbonyl content and nitrotyrosine presence by ELISA. In comparison to WT group, KO mice exhibited larger lesion volume; higher levels of nitrite, copper (Cu) and strontium (Sr) in the peritoneal fluid; increased TAC, CAT, and GPX in peritoneal lavage cells; decreased concentration of TBARS in lesions and protein carbonyl in peritoneal lavage cells. Significant positive correlations between Cu and lesion volume, Sr and lesion volume, and Cu and Sr were obtained. Our results suggest that the TNFRp55 deficiency increases antioxidant protection and promotes high Cu-Sr concentrations in the peritoneal cavity, which favors the progression of experimental endometriosis.
Assuntos
Cobre/metabolismo , Endometriose/metabolismo , Nitritos/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Estrôncio/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Animais , Antioxidantes/metabolismo , Cobre/análise , Progressão da Doença , Endometriose/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/análise , Estresse Oxidativo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Estrôncio/análise , Receptores Chamariz do Fator de Necrose Tumoral/deficiênciaRESUMO
OBJECTIVE: Circadian rhythms are generated by the suprachiasmatic nucleus of the hypothalamus and involve rhythmic expression of clock genes and proteins. This rhythmicity is transferred to peripheral tissues by neural and hormonal signals. Late pregnancy is considered a state of inflammation which impacts on peripheral tissues such as joints. Tumor necrosis factor (TNF) mediates inflammatory and circadian responses through its p55 receptor (TNFRp55). Neuroimmunoendocrine interactions in joints have not been studied completely. The purpose of this study was to analyze these interactions, investigating the circadian rhythms of progesterone (Pg) and pro- and anti-inflammatory cytokines in the joints at the end of pregnancy (gestational day 18). Moreover, the impact of TNFRp55 deficiency on these temporal oscillations was explored. METHODS: Wild-type and TNFRp55-deficient (KO) C57BL/6 mice were kept under constant darkness in order to study their endogenous circadian rhythms. The expression of the clock genes Bmal1 and Per1 at circadian time 7 was studied by reverse transcription polymerase chain reaction in the ankle joints of nonpregnant and pregnant (gestational day 18) mice. In late pregnancy, Pg and the cytokines interleukin 17 (IL-17), IL-6, and IL-10 were measured in the joints throughout a 24-h period by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: A significant increase in Bmal1 and Per1 mRNA expression was detected in the joints of pregnant KO mice. Furthermore, KO mice displayed a desynchronization of articular Pg and cytokine production. CONCLUSIONS: Our results show that TNF, via TNFRp55 signaling, modulates articular Pg and cytokine circadian rhythms in late pregnancy. These findings suggest a temporal neuroimmunoendocrine association in peripheral tissues in late pregnancy.
Assuntos
Ritmo Circadiano/fisiologia , Citocinas/metabolismo , Articulações/metabolismo , Neuroimunomodulação/fisiologia , Progesterona/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , GravidezRESUMO
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
In addition to its well-known pro-inflammatory effects, tumor necrosis factor (TNF) displays anti-inflammatory activities through mechanisms poorly understood. Previously, we reported the development of severe chronic Yersinia enterocolitica-induced reactive arthritis (ReA) in mice lacking the TNF receptor (TNFR)p55. As regulatory T (T(reg)) cells limit chronic inflammation, here we aim to investigate the expansion and function of CD4(+)CD25(+)FoxP3(+) T(reg) cells in the ReA animal model. The number of T(reg) cells as well as the FoxP3 mRNA expression and interleukin (IL)-10 levels were significantly decreased in joint regional lymph nodes (RLNs) of TNFRp55(-/-) mice vs wild-type (WT) mice at the arthritis onset. However, at chronic phase of arthritis, the number of T(reg) cell in TNFRp55(-/-) was similar to WT mice. To explore the in vivo function of T(reg) cells at this chronic phase in WT and TNFRp55-deficient mice, we adoptively transferred CD4(+) T cells from TNFRp55-deficient mice of day 21, into naïve WT or TNFRp55(-/-) mice. When knockout mice were used as recipients we observed higher delayed-type hypersensitivity (DTH) responses and joint inflammation after heat-killed Yersinia (HKY) stimulation. Accordingly, we found higher levels of IL-17, interferon (IFN)-γ, IL-6, transforming growth factor (TGF)-ß1 and IL-12/23p40 and lower IL-10 levels in RLN of paws challenged with HKY in TNFRp55(-/-) recipient mice. In addition, we found that CD4(+) T cells from TNFRp55(-/-) mice controlled antigen-specific IL-12/23(p40) production in recipient WT mice. Our results show that TNFRp55 controls the induction and function of T(reg) cells through differential regulation of cytokine production, suggesting a novel molecular target for immune intervention in ReA.
Assuntos
Artrite Reativa/imunologia , Artrite Reativa/microbiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Linfócitos T Reguladores/imunologia , Receptores Chamariz do Fator de Necrose Tumoral/metabolismo , Yersiniose/imunologia , Yersiniose/microbiologia , Yersinia/imunologia , Transferência Adotiva , Animais , Artrite Reativa/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Interleucina-10/biossíntese , Interleucina-12/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Articulações/imunologia , Articulações/patologia , Linfonodos/metabolismo , Linfonodos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Mucosa/metabolismo , Proibitinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Transdução de Sinais/imunologia , Linfócitos T Reguladores/patologia , Receptores Chamariz do Fator de Necrose Tumoral/deficiência , Yersiniose/patologiaRESUMO
The pathogenic mechanisms of bone loss caused by Brucella species have not been completely deciphered. Although T lymphocytes (LTs) are considered important to control infection, the mechanism of Brucella-induced T-cell responses to immunopathological features is not known. We present in vitro and in vivo evidence showing that Brucella abortus-induced inflammatory response leads to the activation of LTs, which further promote osteoclastogenesis. Pre-activated murine LTs treated with culture supernatant from macrophages infected with B. abortus induced bone marrow-derived monocytes (BMMs) to undergo osteoclastogenesis. Furthermore, osteoclastogenesis was mediated by CD4(+) T cells. Although B. abortus-activated T cells actively secreted the pro-osteoclastogenic cytokines RANKL and IL-17, osteoclastogenesis depended on IL-17, because osteoclast generation induced by Brucella-activated T cells was completely abrogated when these cells were cultured with BMMs from IL-17 receptor knockout mice. Neutralization experiments indicated that IL-6, generated by Brucella infection, induced the production of pro-osteoclastogenic IL-17 from LTs. By using BMMs from tumor necrosis factor receptor p55 knockout mice, we also demonstrated that IL-17 indirectly induced osteoclastogenesis through the induction of tumor necrosis factor-α from osteoclast precursors. Finally, extensive and widespread osteoclastogenesis was observed in the knee joints of mice injected with Brucella-activated T cells. Our results indicate that activated T cells, elicited by B. abortus-infected macrophages and influenced by the inflammatory milieu, promote the generation of osteoclasts, leading to bone loss.