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2.
Mol Cancer Res ; 14(6): 548-62, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27311960

RESUMO

UNLABELLED: Malignant melanoma is the most aggressive form of skin cancer; therefore, it is crucial to disclose its underlying molecular mechanisms. MicroRNAs (miRNAs) are small endogenous noncoding RNAs able to posttranscriptionally downregulate the expression of direct target genes. Using a melanoma progression model, miR-146a was identified as a key double-acting player in melanoma malignancy. In fact, miR-146a is able to enhance tumor growth, while it suppresses dissemination. It was determined that miR-146a coordinated melanoma cell growth by its direct targets lunatic fringe (LFNG) and NUMB, which operate on the NOTCH/PTEN/Akt pathway; while inhibition of metastasis formation was linked to decreased expression of ITGAV and ROCK1. Relevantly, miR-146a expression correlated with melanoma recurrence and was enriched in both patient-derived melanoma and cutaneous metastasis specimens, while its direct targets were depleted. However, miR-146a levels drop in circulating tumor cells (CTCs), suggesting the necessity for miR-146a expression to fluctuate during tumor progression in order to favor tumor growth and allow dissemination. This study reconciles the contradictory biologic functions of miR-146a in melanoma progression and unravels distinct molecular mechanisms that need to be considered for therapeutic interventions. IMPLICATIONS: miR-146a controls melanoma progression in a dual way, promoting growth and inhibiting dissemination; however, it is poorly expressed in CTCs, resulting in overall tumor spreading and distant-site colonization. Mol Cancer Res; 14(6); 548-62. ©2016 AACR.


Assuntos
Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , Idoso , Diferenciação Celular/genética , Movimento Celular/genética , Progressão da Doença , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica
3.
Cancer Res ; 76(17): 5151-62, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27328731

RESUMO

miR-214 and miR-148b have been proposed to antagonize the effects of each other in enabling or blocking metastasis, respectively. In this study, we provide evidence deepening their role and interrelationship in the process of metastatic dissemination. Depleting miR-214 or elevating miR-148b blocked the dissemination of melanoma or breast cancer cells, an effect that could be accentuated by dual alteration. Mechanistic investigations indicated that dual alteration suppressed passage of malignant cells through the blood vessel endothelium by reducing expression of the cell adhesion molecules ITGA5 and ALCAM. Notably, transendothelial migration in vitro and extravasation in vivo impaired by singly alternating miR-214 or miR-148b could be overridden by overexpression of ITGA5 or ALCAM in the same tumor cells. In clinical specimens of primary breast cancer or metastatic melanoma, we found a positive correlation between miR-214 and ITGA5 or ALCAM along with an inverse correlation of miR-214 and miR-148b in the same specimens. Our findings define an antagonistic relationship of miR-214 and miR-148b in determining the dissemination of cancer cells via tumor-endothelial cell interactions, with possible implications for microRNA-mediated therapeutic interventions aimed at blocking cancer extravasation. Cancer Res; 76(17); 5151-62. ©2016 AACR.


Assuntos
Neoplasias da Mama/patologia , Melanoma/patologia , MicroRNAs/genética , Invasividade Neoplásica/genética , Animais , Antígenos CD/biossíntese , Neoplasias da Mama/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular Neuronais/biossíntese , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Células Endoteliais/patologia , Feminino , Proteínas Fetais/biossíntese , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Immunoblotting , Melanoma/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
4.
Oncotarget ; 6(4): 2315-30, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25537513

RESUMO

Basal-like breast cancer is an aggressive tumor subtype with a poor response to conventional therapies. Tumor formation and relapse are sustained by a cell subset of Breast Cancer Stem Cells (BrCSCs). Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation. Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal. It induces the expression of a functional estrogen receptor (ER) and renders basal-like BrCSCs responsive to hormonal therapy. The key role played by miR-100 in breast cancer free-survival is confirmed by the analysis of a cohort of patients' tumors, which shows that low expression of miR-100 is a negative prognostic factor and is associated with gene signatures of high grade undifferentiated tumors. Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.


Assuntos
Neoplasias da Mama/genética , Diferenciação Celular/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Transplante Heterólogo
5.
PLoS One ; 9(1): e84859, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24400121

RESUMO

MicroRNAs are single-stranded non-coding RNAs that simultaneously down-modulate the expression of multiple genes post-transcriptionally by binding to the 3'UTRs of target mRNAs. Here we used computational methods to predict microRNAs relevant in breast cancer progression. Specifically, we applied different microRNA target prediction algorithms to various groups of differentially expressed protein-coding genes obtained from four breast cancer datasets. Six potential candidates were identified, among them miR-223, previously described to be highly expressed in the tumor microenvironment and known to be actively transferred into breast cancer cells. To investigate the function of miR-223 in tumorigenesis and to define its molecular mechanism, we overexpressed miR-223 in breast cancer cells in a transient or stable manner. Alternatively we overexpressed miR-223 in mouse embryonic fibroblasts or HEK293 cells and used their conditioned medium to treat tumor cells. With both approaches, we obtained elevated levels of miR-223 in tumor cells and observed decreased migration, increased cell death in anoikis conditions and augmented sensitivity to chemotherapy but no effect on adhesion and proliferation. The analysis of miR-223 predicted targets revealed enrichment in cell death and survival-related genes and in pathways frequently altered in breast cancer. Among these genes, we showed that protein levels for STAT5A, ITGA3 and NRAS were modulated by miR-223. In addition, we proved that STAT5A is a direct miR-223 target and highlighted a possible correlation between miR-223 and STAT5A in migration and chemotherapy response. Our investigation revealed that a computational analysis of cancer gene expression datasets can be a relevant tool to identify microRNAs involved in cancer progression and that miR-223 has a prominent role in breast malignancy that could potentially be exploited therapeutically.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , MicroRNAs/genética , Regiões 3' não Traduzidas , Anoikis/genética , Antineoplásicos/farmacologia , Pareamento de Bases , Sequência de Bases , Neoplasias da Mama/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Biologia Computacional , Bases de Dados Factuais , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/química , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Fator de Transcrição STAT5/química , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
PLoS One ; 8(11): e73798, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24223694

RESUMO

MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication.


Assuntos
Albuminúria/urina , Diabetes Mellitus Tipo 1/urina , Nefropatias Diabéticas/urina , Exossomos/metabolismo , MicroRNAs/urina , Animais , Células Cultivadas , Expressão Gênica , Glucose/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Pessoa de Meia-Idade , Regulação para Cima
7.
Cancer Res ; 73(13): 4098-111, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23667173

RESUMO

Malignant melanoma is one of the most aggressive human cancers, but the mechanisms governing its metastatic dissemination are not fully understood. Upregulation of miR-214 and ALCAM and the loss of TFAP2 expression have been implicated in this process, with TFAP2 a direct target of miR-214. Here, we link miR-214 and ALCAM as well as identify a core role for miR-214 in organizing melanoma metastasis. miR-214 upregulated ALCAM, acting transcriptionally through TFAP2 and also posttranscriptionally through miR-148b (itself controlled by TFAP2), both negative regulators of ALCAM. We also identified several miR-214-mediated prometastatic functions directly promoted by ALCAM. Silencing ALCAM in miR-214-overexpressing melanoma cells reduced cell migration and invasion without affecting growth or anoikis in vitro, and it also impaired extravasation and metastasis formation in vivo. Conversely, cell migration and extravasation was reduced in miR-214-overexpressing cells by upregulation of either miR-148b or TFAP2. These findings were consistent with patterns of expression of miR-214, ALCAM, and miR-148b in human melanoma specimens. Overall, our results define a pathway involving miR-214, miR-148b, TFAP2, and ALCAM that is critical for establishing distant metastases in melanoma.


Assuntos
Antígenos CD/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas Fetais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , MicroRNAs/genética , MicroRNAs/fisiologia , Fator de Transcrição AP-2/genética , Animais , Antígenos CD/metabolismo , Sequência de Bases , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Feminino , Proteínas Fetais/metabolismo , Humanos , Neoplasias Pulmonares/secundário , Melanoma/secundário , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Invasividade Neoplásica , Transplante de Neoplasias , Interferência de RNA , Fator de Transcrição AP-2/metabolismo , Transcrição Genética , Regulação para Cima
8.
FASEB J ; 27(3): 1223-35, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23233531

RESUMO

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, down-regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.


Assuntos
Neoplasias da Mama/metabolismo , Integrina alfa5/biossíntese , Fator Estimulador de Colônias de Macrófagos/biossíntese , MicroRNAs/metabolismo , Proteína Oncogênica p21(ras)/biossíntese , Fosfatidilinositol 3-Quinases/biossíntese , RNA Neoplásico/metabolismo , Quinases Associadas a rho/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Progressão da Doença , Feminino , Humanos , Integrina alfa5/genética , Fator Estimulador de Colônias de Macrófagos/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteína Oncogênica p21(ras)/genética , Fosfatidilinositol 3-Quinases/genética , RNA Neoplásico/genética , Quinases Associadas a rho/genética
9.
Horm Cancer ; 3(3): 65-78, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22274890

RESUMO

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17ß-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.


Assuntos
Neoplasias da Mama/genética , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Adulto , Idoso , Sítios de Ligação/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Análise por Conglomerados , Receptor alfa de Estrogênio/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Elementos de Resposta
10.
Breast Cancer Res Treat ; 131(1): 49-63, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21336598

RESUMO

The purpose of this article is to determine the tumorigenic potential of estradiol treatment (E2) when combined with either progesterone (P4) or medroxyprogesterone acetate (MPA) in normal luminal human breast cells (HBE) and in human breast cancer cells (T47-D, MCF-7). Proliferation profiles were evaluated, along with the gene transactivation activity between the progesterone and glucocorticoid receptors (PR, GR) in HBE, T47-D, and MCF-7 cells treated by E2 + P4 or E2 + MPA. High throughput transcriptome analysis was performed on RNA from HBE cells treated by E2, E2 + MPA and E2 + P4. GR content was analyzed in normal breast cells as well. In HBE cells, E2 + P4 treatment was antiproliferative and promoted cellular differentiation. In contrast, E2 + MPA displayed mitogenic, antiapoptotic effects in HBE cells and did not influence cellular differentiation. The effect of P4 and MPA on cell proliferation was, however, variable in breast cancer cells. In cells containing GR or/and PR, MPA decreased proliferation whereas P4 antiproliferative effect needed the presence of PR. In HBE cells, the regulation of genes by E2 + P4, and E2 + MPA was significantly different, particularly in cell proliferation and cell death gene families. Further analysis revealed a modulation of the glucocorticoid receptor gene expression pathway by E2 + MPA. Predominant MPA glucocorticoid activity in normal and breast cancer cells was demonstrated using a glucocorticoid antagonist and the down-regulation of the GR by RNA interference. In normal luminal breast cells and in breast cancer cells, P4 and MPA combined with E2 treatment have opposing mitogenic effects due to GR. The consequences of MPA glucocorticoid potencies as well as the importance of GR in breast tissue merit a reappraisal.


Assuntos
Estradiol/farmacologia , Acetato de Medroxiprogesterona/farmacologia , Progesterona/farmacologia , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Adolescente , Adulto , Mama , Neoplasias da Mama , Proteínas de Transporte/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Terapia de Reposição Hormonal/métodos , Humanos , Norpregnadienos/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Receptores de Estrogênio/metabolismo , Esteroides , Transcriptoma , Adulto Jovem
11.
EMBO J ; 30(10): 1990-2007, 2011 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-21468029

RESUMO

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non-coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR-214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well-established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR-214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR-214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.


Assuntos
Regulação da Expressão Gênica , Melanoma/patologia , Melanoma/secundário , MicroRNAs/metabolismo , Metástase Neoplásica/patologia , Fator de Transcrição AP-2/biossíntese , Animais , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Integrinas/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/patologia , Camundongos , MicroRNAs/genética
12.
Int J Cancer ; 123(6): 1327-38, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18561318

RESUMO

Gene expression profiles were studied by microarray analysis in 2 sets of archival breast cancer tissues from patients with distinct clinical outcome. Seventy-seven differentially expressed genes were identified when comparing 30 cases with relapse and 30 cases without relapse within 72 months from surgery. These genes had a specific ontological distribution and some of them have been linked to breast cancer in previous studies: AIB1, the two keratin genes KRT5 and KRT15, RAF1, WIF1 and MSH6. Seven out of 77 differentially expressed genes were selected and analyzed by qRT-PCR in 127 cases of breast cancer. The expression levels of 6 upregulated genes (CKMT1B, DDX21, PRKDC, PTPN1, SLPI, YWHAE) showed a significant association to both disease-free and overall survival. Multivariate analysis using the significant factors (i.e., estrogen receptor and lymph node status) as covariates confirmed the association with survival. There was no correlation between the expression level of these genes and other clinical parameters. In contrast, SERPINA3, the only downregulated gene examined, was not associated with survival, but correlated with steroid receptor status. An indirect validation of our genes was provided by calculating their association with survival in 3 publicly available microarray datasets. CKMT1B expression was an independent prognostic marker in all 3 datasets, whereas other genes confirmed their association with disease-free survival in at least 1 dataset. This work provides a novel set of genes that could be used as independent prognostic markers and potential drug targets for breast cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Perfilação da Expressão Gênica , Expressão Gênica , Recidiva Local de Neoplasia/genética , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
FASEB J ; 22(8): 2702-14, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18443366

RESUMO

The events occurring during tumor formation and progression display similarities to some of the steps in embryonic morphogenesis. The family of AP-2 proteins consists of five different transcription factors (alpha, beta, gamma, delta, and epsilon) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP-2alpha and AP-2gamma proteins play an essential role in tumorigenesis. Down-modulation of AP-2 expression in tumor cells by RNA interference (RNAi) led to enhanced tumor growth and reduced chemotherapy-induced cell death, as well as migration and invasion. Most of these biological modulations were rescued by AP-2 overexpression. We observed that increased xenotransplant growth was mostly due to highly enhanced proliferation of the tumor cells together with reduced innate immune cell recruitment. Moreover, we showed that migration impairment was mediated, at least in part, by secreted factors. To identify the genetic programs involved in tumorigenesis, we performed whole genome microarray analysis of AP-2alpha knockdown cells and observed that AP-2alpha regulates specific genes involved in cell cycle, cell death, adhesion, and migration. In particular, we showed that ESDN, EREG, and CXCL2 play a major role in AP-2 controlled migration, as ablation of any of these genes severely altered migration.


Assuntos
Neoplasias/genética , Fator de Transcrição AP-2/genética , Animais , Sequência de Bases , Neoplasias da Mama/etiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Morte Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CXCL2/genética , Fator de Crescimento Epidérmico/genética , Epirregulina , Feminino , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Interferente Pequeno/genética , Fator de Transcrição AP-2/antagonistas & inibidores , Fator de Transcrição AP-2/metabolismo , Transplante Heterólogo
14.
Lab Invest ; 88(4): 430-40, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18305565

RESUMO

Microarray-based gene expression profiling is well suited for parallel quantitative analysis of large numbers of RNAs, but its application to cancer biopsies, particularly formalin-fixed, paraffin-embedded (FFPE) archived tissues, is limited by the poor quality of the RNA recovered. This represents a serious drawback, as FFPE tumor tissue banks are available with clinical and prognostic annotations, which could be exploited for molecular profiling studies, provided that reliable analytical technologies are found. We applied and evaluated here a microarray-based cDNA-mediated annealing, selection, extension and ligation (DASL) assay for analysis of 502 mRNAs in highly degraded total RNA extracted from cultured cells or FFPE breast cancer (MT) biopsies. The study included quantitative and qualitative comparison of data obtained by analysis of the same RNAs with genome-wide oligonucleotide microarrays vs DASL arrays and, by DASL, before and after extensive in vitro RNA fragmentation. The DASL-based expression profiling assay applied to RNA extracted from MCF-7 cells, before or after 24 h stimulation with a mitogenic dose of 17beta-estradiol, consistently allowed to detect hormone-induced gene expression changes following extensive RNA degradation in vitro. Comparable results where obtained with tumor RNA extracted from FFPE MT biopsies (6 to 19 years old). The method proved itself sensitive, reproducible and accurate, when compared to results obtained by microarray analysis of RNA extracted from snap-frozen tissue of the same tumor.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Perfilação da Expressão Gênica/métodos , RNA Neoplásico/análise , Biópsia , Linhagem Celular Tumoral , Feminino , Formaldeído , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Inclusão em Parafina , Reprodutibilidade dos Testes
15.
Maturitas ; 57(1): 50-5, 2007 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-17395409

RESUMO

Estrogen exerts a primary regulatory role on a wide variety of physiological processes in different tissues and organs. Agonistic ad antagonistic compounds are widely used in human health and, therefore, a deep understanding of their mechanisms of action at the molecular level is mandatory. The effect of 17beta-estradiol and three antiestrogenic drugs, comprising two selective estrogen receptor modulator (SERM, 4-OH-tamoxifen, Raloxifene) and the pure antiestrogen ICI 182,780, on genome-wide gene expression levels was evaluated in breast carcinoma cell lines by DNA microarray analysis. Different clusters of genes, showing specific coregulation patterns, were found. First, several groups of genes displaying temporal-specific up- or down-regulation were characterized. Second, clusters of genes responding to different antiestrogenic drugs in either antagonstic or agonistic fashion, were found. Genes responding specifically to antiestrogens, but not to estrogen, were also identified. In addition, each individual compound exhibited a very specific gene regulation. Bioinformatic analysis was applied to the regulatory sequences of different groups of genes and confirmed that specific pathways and secondary responses are activated at each temporal point and in response to different compounds. Our results underline the complexity of genomic responses to estrogen in breast cancer cells and strongly suggest that the molecular characterization of estrogen agonists and antagonists used in human therapy should be carefully studied.


Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Cloridrato de Raloxifeno/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Neoplasias da Mama/patologia , Carcinoma/patologia , Regulação para Baixo , Feminino , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Tamoxifeno/farmacologia , Células Tumorais Cultivadas , Regulação para Cima
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