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1.
J Hepatol ; 78(2): 364-375, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36848245

RESUMO

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour. METHODS: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts. RESULTS: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth. CONCLUSIONS: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA. IMPACT AND IMPLICATIONS: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Proteína Forkhead Box O1 , MicroRNAs , Humanos , Neoplasias dos Ductos Biliares/genética , Ductos Biliares , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/genética , MicroRNAs/genética , Proteína Forkhead Box O1/metabolismo
2.
J Invest Dermatol ; 141(10): 2449-2458, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33862068

RESUMO

Staphylococcal enterotoxins are believed to fuel disease activity in cutaneous T-cell lymphoma. Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sézary syndrome, the most common forms of cutaneous T-cell lymphoma. Yet, it remains incompletely characterized how staphylococcal enterotoxins fuel disease activity. In this study, we show that staphylococcal enterotoxins induce the expression of the oncogenic microRNA miR-155 in primary malignant T cells. Thus, staphylococcal enterotoxins and Staphyloccocus aureus isolates from lesional skin of patients induce miR-155 expression at least partly through the IL-2Rg‒Jak‒signal transducer and activator of transcription 5 pathway, and the effect is augmented by the presence of nonmalignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus, express Y-phosphorylated signal transducer and activator of transcription 5, and display enhanced miR-155 expression, when compared with nonlesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased Y-phosphorylated signal transducer and activator of transcription 5 and miR-155 expression in lesional skin in two patients with Sézary syndrome. In conclusion, we show that S. aureus and its enterotoxins induce enhanced expression of oncogenic miR-155, providing mechanistic insight into the role of S. aureus in cutaneous T-cell lymphoma. Our findings support that environmental stimuli such as bacteria can fuel disease progression in cutaneous T-cell lymphoma.


Assuntos
Enterotoxinas/toxicidade , Linfoma Cutâneo de Células T/etiologia , MicroRNAs/fisiologia , Fator de Transcrição STAT5/fisiologia , Neoplasias Cutâneas/etiologia , Staphylococcus aureus/patogenicidade , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Humanos
3.
Aging (Albany NY) ; 13(5): 6359-6374, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33647883

RESUMO

Despite significant evidence that the expression of several microRNAs (miRNAs) impacts disease activity in patients with ulcerative colitis (UC), it remains unknown if the more severe disease phenotype seen in pediatric onset UC can be explained by an altered miRNA expression. In this study, we assessed the relationship between miRNA expression, age, and disease severity in pediatric and adult patients with UC. Using RT-qPCR, we analyzed the expression of miR-21, miR-31, miR-126, miR-142 and miR-155 in paraffin embedded rectum biopsies from 30 pediatric and 30 adult-onset UC patients. We found that lesions from adult patients had significantly higher expression levels of miR-21 compared to pediatric patients and that the expression levels of miR-31 (all patients) and miR-155 (pediatric patients only) correlated inversely with histological assessed disease severity. Using in situ hybridization followed by image analysis, the expression level estimates of miR-21 and miR-126 correlated with histological assessed disease severity. In conclusion, we found that the expression of miRNAs depends on the age of the patient and/or the severity of the disease, suggesting that miRNAs may contribute to the regulation of inflammation in UC and could be useful biomarkers in the surveillance of disease severity.


Assuntos
Colite Ulcerativa/metabolismo , Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Índice de Gravidade de Doença , Adolescente , Adulto , Biomarcadores/metabolismo , Criança , Feminino , Humanos , Masculino , Adulto Jovem
4.
Int J Mol Sci ; 21(21)2020 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-33114313

RESUMO

Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC). These are chronic autoimmune diseases of unknown etiology affecting the gastrointestinal tract. The IBD population includes a heterogeneous group of patients with varying disease courses requiring personalized treatment protocols. The complexity of the disease often delays the diagnosis and the initiation of appropriate treatments. In a subset of patients, IBD leads to colitis-associated cancer (CAC). MicroRNAs are single-stranded regulatory noncoding RNAs of 18 to 22 nucleotides with putative roles in the pathogenesis of IBD and colorectal cancer. They have been explored as biomarkers and therapeutic targets. Both tissue-derived and circulating microRNAs have emerged as promising biomarkers in the differential diagnosis and in the prognosis of disease severity of IBD as well as predictive biomarkers in drug resistance. In addition, knowledge of the cellular localization of differentially expressed microRNAs is a prerequisite for deciphering the biological role of these important epigenetic regulators and the cellular localization may even contribute to an alternative repertoire of biomarkers. In this review, we discuss findings based on RT-qPCR, microarray profiling, next generation sequencing and in situ hybridization of microRNA biomarkers identified in the circulation and in tissue biopsies.


Assuntos
Neoplasias Associadas a Colite/genética , Marcadores Genéticos , Doenças Inflamatórias Intestinais/genética , MicroRNAs/genética , Neoplasias Associadas a Colite/etiologia , Diagnóstico Diferencial , Diagnóstico Precoce , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/complicações , Masculino
5.
J Biomed Opt ; 25(7): 1-13, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32638570

RESUMO

SIGNIFICANCE: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing. AIM: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers. APPROACH: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio. RESULTS: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images. CONCLUSIONS: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.


Assuntos
Microscopia , Imagem Óptica , Biomarcadores , Corantes Fluorescentes , Microscopia de Fluorescência por Excitação Multifotônica , Fótons
6.
Appl Immunohistochem Mol Morphol ; 28(5): 347-353, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30920963

RESUMO

Immunohistochemical (IHC) quantification of estrogen receptor-α (ER) is used for assessment of treatment regimen in breast cancer. Different ER IHC assays may produce diverging results, because of different antibody clones, protocols, and stainer platforms. Objective tissue-based techniques to assess sensitivity and specificity of IHC assays are therefore needed. We tested the usability of ER mRNA-in situ hybridization (mRNA-ISH) in comparison with assays based on clones SP1 and 6F11. We selected 56 archival specimens according to their reported ER IHC positivity, representing a wide spectrum from negative to strongly positive cases. The specimens were used to prepare 4 TMAs with 112 cores. Serial sections of each TMA were stained for ER and pan-cytokeratin (PCK) by IHC and ESR1 (ER gene) by mRNA-ISH. Digital image analysis (DIA) was used to determine ER IHC H-score. ESR1 mRNA-ISH was scored both manually and by DIA. DIA showed a nonlinear correlation between IHC and ESR1 mRNA-ISH with R-values of 0.80 and 0.78 for the ER antibody clones SP1 and 6F11, respectively. Comparison of manual mRNA-ISH scoring categories and SP1 and 6F11 IHC H-scores showed a highly significant relationship (P<0.001). In conclusion, the study showed good correlation between mRNA-ISH and IHC, suggesting that mRNA-ISH can be a valuable tool in the assessment of the sensitivity and specificity of ER IHC assays.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal/metabolismo , Receptor alfa de Estrogênio/metabolismo , RNA Mensageiro/metabolismo , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal/diagnóstico por imagem , Carcinoma Ductal/genética , Carcinoma Ductal/patologia , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Imuno-Histoquímica , Hibridização In Situ , RNA Mensageiro/genética , Estudos Retrospectivos , Sensibilidade e Especificidade , Análise Serial de Tecidos
7.
Int J Mol Sci ; 20(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999696

RESUMO

MicroRNA-21 (miR-21) is upregulated in many cancers including colon cancers and is a prognostic indicator of recurrence and poor prognosis. In colon cancers, miR-21 is highly expressed in stromal fibroblastic cells and more weakly in a subset of cancer cells, particularly budding cancer cells. Exploration of the expression of inflammatory markers in colon cancers revealed tumor necrosis factor alpha (TNF-α) mRNA expression at the invasive front of colon cancers. Surprisingly, a majority of the TNF-α mRNA expressing cells were found to be cancer cells and not inflammatory cells. Because miR-21 is positively involved in cell survival and TNF-α promotes necrosis, we found it interesting to analyze the presence of miR-21 in areas of TNF-α mRNA expression at the invasive front of colon cancers. For this purpose, we developed an automated procedure for the co-staining of miR-21, TNF-α mRNA and the cancer cell marker cytokeratin based on analysis of frozen colon cancer tissue samples (n = 4) with evident cancer cell budding. In all four cases, TNF-α mRNA was seen in a small subset of cancer cells at the invasive front. Evaluation of miR-21 and TNF-α mRNA expression was performed on digital slides obtained by confocal slide scanning microscopy. Both co-expression and lack of co-expression with miR-21 in the budding cancer cells was noted, suggesting non-correlated expression. miR-21 was more often seen in cancer cells than TNF-α mRNA. In conclusion, we report that miR-21 is not linked to expression of the pro-inflammatory cytokine TNF-α mRNA, but that miR-21 and TNF-α both take part in the cancer expansion at the invasive front of colon cancers. We hypothesize that miR-21 may protect both fibroblasts and cancer cells from cell death directed by TNF-α paracrine and autocrine activity.


Assuntos
Colo/patologia , Neoplasias Colorretais/patologia , MicroRNAs/análise , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , MicroRNAs/genética , Microscopia Confocal , RNA Mensageiro/genética
8.
Nucleic Acids Res ; 46(8): 4213-4227, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29481642

RESUMO

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.


Assuntos
Senescência Celular/genética , Dipeptidil Peptidase 4/genética , RNA Longo não Codificante/metabolismo , Dipeptidil Peptidase 4/metabolismo , Expressão Gênica , Genes ras , Genoma , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo
9.
Nucleic Acids Res ; 46(1): e4, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29059327

RESUMO

Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system.


Assuntos
Núcleo Celular/genética , Regulação da Expressão Gênica , Hibridização in Situ Fluorescente/métodos , RNA Longo não Codificante/genética , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , Sondas de DNA/genética , Amplificação de Genes , Células HeLa , Humanos , Células MCF-7 , Reprodutibilidade dos Testes
10.
Melanoma Res ; 27(5): 417-428, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28614272

RESUMO

Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours. Using array analysis, samples mainly clustered according to their biological group (benign vs. malignant) and 77 miRNAs differed significantly between nevi and melanoma samples. Increase of miR-21 and miR-142, and decrease of miR-125b, miR-211, miR-101 and miR-513c in the melanomas were verified in both cohorts using qPCR, whereas the decrease of miR-205 observed with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.


Assuntos
Melanoma/genética , MicroRNAs/genética , Neoplasias Cutâneas/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Hibridização In Situ , Melanoma/patologia , Reação em Cadeia da Polimerase , Neoplasias Cutâneas/patologia
11.
Appl Immunohistochem Mol Morphol ; 23(3): 188-95, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25265435

RESUMO

OncomiRs miR-21 and miR-155 have been linked to lymphomagenesis, but information on their implication in diffuse large B-cell lymphoma (DLBCL) is limited. Here, we used locked nucleic acid-based in situ hybridization (ISH) detection techniques on formalin-fixed paraffin-embedded DLBCL tissue samples to identify miR-155 and miR-21 at the cellular level in 56 patients diagnosed with DLBCL, and compared them to miR array data. miR-155 was observed in tumor cells in 19/56 (33.9%) of the samples evaluated by ISH. miR-21 was localized to the stromal compartment in 41/56 (73.2%). A subset of these, 16/56 (28.6%), also showed labeling in tumor cells. When comparing ISH-scores and miR array data, miR-155 in tumor cells, identified by ISH, was associated with miR-155 expression in miR array data (P=0.030). Equally, miR-21 expression by miR array data were highly associated with miR-21 ISH-scores in the stromal cells (P=0.002), whereas no association between miR array data and ISH of miR-21 in tumor cells was observed (P=0.673). We found no association of miR-155 and miR-21 with overall survival or germinal center B-cell-like (GCB) versus non-GCB-like subtypes of DLBCL. In conclusion, miR-ISH added to the biological interpretation of miR expression in DLBCL compared with miR array data, but miR-155 and miR-21 ISH did not add prognostic information in this series.


Assuntos
Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/metabolismo , MicroRNAs/biossíntese , RNA Neoplásico/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Células Estromais/metabolismo , Células Estromais/patologia
12.
PLoS One ; 9(10): e109430, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25329796

RESUMO

PURPOSE: We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs. RESULTS: In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts. CONCLUSIONS: We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.


Assuntos
Neoplasias Colorretais/genética , Desoxicitidina/análogos & derivados , Fluoruracila/análogos & derivados , MicroRNAs/biossíntese , Compostos Organoplatínicos/administração & dosagem , Análise Serial de Tecidos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Humanizados/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bevacizumab , Capecitabina , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Desoxicitidina/administração & dosagem , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Oxaliplatina , Prognóstico , Resultado do Tratamento
13.
J Mol Diagn ; 16(1): 106-15, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24211363

RESUMO

Carcinomas of unknown primary origin constitute 3% to 5% of all newly diagnosed metastatic cancers, with the primary source difficult to classify with current histological methods. Effective cancer treatment depends on early and accurate identification of the tumor; patients with metastases of unknown origin have poor prognosis and short survival. Because miRNA expression is highly tissue specific, the miRNA profile of a metastasis may be used to identify its origin. We therefore evaluated the potential of miRNA profiling to identify the primary tumor of known metastases. Two hundred eight formalin-fixed, paraffin-embedded samples, representing 15 different histologies, were profiled on a locked nucleic acid-enhanced microarray platform, which allows for highly sensitive and specific detection of miRNA. On the basis of these data, we developed and cross-validated a novel classification algorithm, least absolute shrinkage and selection operator, which had an overall accuracy of 85% (CI, 79%-89%). When the classifier was applied on an independent test set of 48 metastases, the primary site was correctly identified in 42 cases (88% accuracy; CI, 75%-94%). Our findings suggest that miRNA expression profiling on paraffin tissue can efficiently predict the primary origin of a tumor and may provide pathologists with a molecular diagnostic tool that can improve their capability to correctly identify the origin of hitherto unidentifiable metastatic tumors and, eventually, enable tailored therapy.


Assuntos
MicroRNAs/genética , Técnicas de Diagnóstico Molecular/métodos , Neoplasias Primárias Desconhecidas/classificação , Neoplasias Primárias Desconhecidas/genética , Análise de Sequência de RNA/métodos , Algoritmos , Sequência de Bases , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Primárias Desconhecidas/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Especificidade de Órgãos/genética , Inclusão em Parafina
14.
APMIS ; 121(11): 1020-4, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24033365

RESUMO

Recently, miR-155 has been implicated in cutaneous T-cell lymphoma (CTCL). Thus, elevated levels of miR-155 were observed in skin lesions from CTCL patients as judged from qPCR and micro-array analysis and aberrant, high miR-155 expression was associated with severe disease. Moreover, miR-155 promoted proliferation of malignant T cells in vitro. Little is, however, known about which cell types express miR-155 in vivo in CTCL skin lesions. Here, we study miR-155 expression using in situ hybridization (ISH) with a miR-155 probe, a negative control (scrambled), and a miR-126 probe as a positive control in nine patients with mycosis fungoides, the most frequent subtype of CTCL. We provide evidence that both malignant and non-malignant T cells stain weakly to moderately positive with the miR-155 probe, but generally negative with the miR-126 and negative control probes. Reversely, endothelial cells stain positive for miR-126 and negative for miR-155 and the control probe. Solitary T cells with a malignant morphology display brighter staining with the miR-155 probe. Taken together, our findings suggest that both malignant and non-malignant T cells express miR-155 in situ in CTCL. Moreover, they indicate heterogeneity in miR-155 expression among malignant T cells.


Assuntos
Linfoma Cutâneo de Células T/genética , MicroRNAs/fisiologia , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Hibridização In Situ , Masculino , MicroRNAs/análise , Pessoa de Meia-Idade
15.
Dev Biol ; 358(1): 56-67, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21802414

RESUMO

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.


Assuntos
Metaloproteinase 9 da Matriz/deficiência , Gravidez/fisiologia , Fenômenos Fisiológicos da Pele , Ativador de Plasminogênio Tipo Uroquinase/deficiência , Cicatrização/fisiologia , Animais , Western Blotting , Pesos e Medidas Corporais , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Técnicas Histológicas , Hibridização In Situ , Camundongos , Cicatrização/genética
16.
PLoS One ; 5(9): e12746, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20856796

RESUMO

BACKGROUND: Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficient mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficient mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Fibrinolisina/metabolismo , Pele/fisiopatologia , Ativador de Plasminogênio Tecidual/deficiência , Ativador de Plasminogênio Tipo Uroquinase/imunologia , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/tratamento farmacológico , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Testes de Neutralização , Pele/efeitos dos fármacos , Pele/lesões , Ativador de Plasminogênio Tecidual/genética , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/fisiopatologia
17.
Front Biosci (Landmark Ed) ; 14(6): 2103-14, 2009 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-19273187

RESUMO

The uPAR-associated protein (uPARAP/Endo180), a type-1 membrane protein belonging to the mannose receptor family, is an endocytic receptor for collagen. Through this endocytic function, the protein takes part in a previously unrecognized mechanism of collagen turnover. uPARAP/Endo180 can bind and internalize both intact and partially degraded collagens. In some turnover pathways, the function of the receptor probably involves an interplay with certain matrix-degrading proteases whereas, in other physiological processes, redundant mechanisms involving both endocytic and pericellular collagenolysis seem to operate in parallel. On certain cell types, uPARAP/Endo180 occurs in a complex with the urokinase plasminogen activator receptor (uPAR) where it seems to fulfill other functions in addition to collagenolysis. uPARAP/Endo180 is expressed on various mesenchymal cells, including subpopulations of fibroblasts, osteoblasts and chondrocytes, generally in conjunction with matrix turnover and collagenolysis. A striking expression is found in developing bone where the collagenolytic function of uPARAP/Endo180 is one of the rate-limiting steps in growth. In murine breast tumors, the endocytic function of the receptor in collagen breakdown seems to be involved in invasive tumor growth.


Assuntos
Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Mitogênicos/metabolismo , Humanos , Lectinas de Ligação a Manose/química , Glicoproteínas de Membrana/química , Conformação Proteica , Receptores de Superfície Celular/química , Receptores Mitogênicos/química
18.
Am J Gastroenterol ; 103(9): 2350-8, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18844621

RESUMO

OBJECTIVES: Chronic inflammation of the intestinal wall is the common characteristic of Crohn's disease and ulcerative colitis; disorders, which in some cases can be difficult to distinguish. The inflammation also affects the local neuronal plexuses of the enteric nervous system. It is known that plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) are upregulated in neurons after experimental peripheral nerve injury and have been linked to nerve regeneration. METHODS: The expression of PAI-1 and uPAR in neuronal cells in lesions of the gastrointestinal tract was analyzed by immunohistochemical techniques. RESULTS: PAI-1 was found in a subset of neurons primarily located in the submucosal plexus of the small and large intestine in 24 of 28 cases (86%) with Crohn's disease, but in none of 17 cases with chronic ulcerative colitis and other severe inflammatory conditions in the intestinal wall. The PAI-1 was seen in the perikarya of the neurons and a few proximal axons, whereas nerves were negative. uPAR was seen in nerves in all types of lesion varying from 21% to 88% of the cases, most frequent in colon adenocarcinomas. No uPAR-positive nerves were detected in normal colon. CONCLUSIONS: PAI-1-positive neurons in inflammatory bowel disease are linked to chronic inflammation in Crohn's disease, implying PAI-1 as a potential parameter for the differential diagnosis between Crohn's disease and ulcerative colitis. The findings also suggest that PAI-1 in neurons is related to pain and that both PAI-1 and uPAR are involved in neuronal repair in the inflamed tissue.


Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Neurônios/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Colite Ulcerativa/terapia , Doença de Crohn/terapia , Feminino , Humanos , Técnicas Imunoenzimáticas , Mucosa Intestinal/metabolismo , Intestinos/inervação , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Pessoa de Meia-Idade , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo
19.
PLoS One ; 3(8): e2959, 2008 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-18698413

RESUMO

Matrix metalloproteinase (MMP) 13 (collagenase 3) is an extracellular matrix remodeling enzyme that is induced in myofibroblasts during the earliest invasive stages of human breast carcinoma, suggesting that it is involved in tumor progression. During progression of mammary carcinomas in the polyoma virus middle T oncogene mouse model (MMTV-PyMT), Mmp13 mRNA was strongly upregulated concurrently with the transition to invasive and metastatic carcinomas. As in human tumors, Mmp13 mRNA was found in myofibroblasts of invasive grade II and III carcinomas, but not in benign grade I and II mammary intraepithelial neoplasias. To determine if MMP13 plays a role in tumor progression, we crossed MMTV-PyMT mice with Mmp13 deficient mice. The absence of MMP13 did not influence tumor growth, vascularization, progression to more advanced tumor stages, or metastasis to the lungs, and the absence of MMP13 was not compensated for by expression of other MMPs or tissue inhibitor of metalloproteinases. However, an increased fraction of thin collagen fibrils was identified in MMTV-PyMT;Mmp13(-/-) compared to MMTV-PyMT;Mmp13(+/+) tumors, showing that collagen metabolism was altered in the absence of MMP13. We conclude that the expression pattern of Mmp13 mRNA in myofibroblasts of invasive carcinomas in the MMTV-PyMT breast cancer model recapitulates the expression pattern observed in human breast cancer. Our results suggest that MMP13 is a marker of carcinoma-associated myofibroblasts of invasive carcinoma, even though it does not make a major contribution to tumor progression in the MMTV-PyMT breast cancer model.


Assuntos
Antígenos Transformantes de Poliomavirus/farmacologia , Fibroblastos/enzimologia , Fibroblastos/virologia , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Metaloproteinase 13 da Matriz/biossíntese , Animais , Primers do DNA , Progressão da Doença , Feminino , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/irrigação sanguínea , Metaloproteinase 13 da Matriz/deficiência , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Knockout , Metástase Neoplásica , Estadiamento de Neoplasias , Neovascularização Patológica
20.
Int J Cancer ; 122(4): 734-41, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17849475

RESUMO

C4.4A is a glycolipid-anchored membrane protein with structural homology to the urokinase-type plasminogen activator receptor (uPAR). Although C4.4A was identified as a metastasis-associated protein little is known about its actual expression and possible function in malignant disease. In the present study, we have therefore analyzed the expression of C4.4A in 14 esophageal squamous cell carcinomas (ESCC). Normal squamous esophageal epithelium shows a strong cell surface associated C4.4A expression in the suprabasal layers, whereas basal cells are negative. Upon transition to dysplasia and carcinoma in situ the expression of C4.4A is abruptly and coordinately weakened. Double immunofluorescence staining of normal and dysplastic tissue showed that C4.4A colocalizes with the epithelial cell surface marker E-cadherin in the suprabasal cells and has a complementary expression pattern compared to the proliferation marker Ki-67. A prominent, but frequently intracellular, C4.4A expression reappeared in tumor cells located at the invasive front and local lymph node metastases. Because C4.4A was reported previously to be a putative laminin-5 (LN5) ligand, and both proteins are expressed by invasive tumor cells, we analyzed the possible coexpression of C4.4A and the gamma 2-chain of LN5 (LN5-gamma 2). Although these proteins are indeed expressed by either neighboring cancer cells or in a few cases even coexpressed by the same cells in the tumor front and metastases, we found no evidence for a general colocalization in the extracellular compartment by confocal microscopy. In conclusion, C4.4A is expressed during invasion and metastasis of human ESCC and may thus provide a new histological marker in this disease.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Moléculas de Adesão Celular/metabolismo , Neoplasias Esofágicas/metabolismo , Adulto , Idoso , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Epitélio/metabolismo , Epitélio/patologia , Neoplasias Esofágicas/patologia , Feminino , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Laminina/metabolismo , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia
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