Name: clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization
Uploaded: 2016-06-07T15:00:00
Description: Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

Este estudo foi aprovado pelo Institutional Review Board do Hospital Chonnam National University Hwasun e University of South Carolina. amostras TMA compostas de câncer de mama e tecidos mamários normais adjacentes combinados foram fornecidos pelo Biobank de Chonnam National Hwasun Hospital Universitário, um membro da Coreia do Biobank rede. 1. Preparação de Soluções <ol><li> Prepare 1 litro de DNase e RNase água livre. Faça todas as soluções químicas usando dietilpirocarbonato...

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O objetivo deste estudo foi determinar o nível de expressão miRNA em tecidos de câncer de mama utilizando miRNA hibridização in situ. Deve ser notado que nesta experiência, todas as condições foram optimizadas para o tecido do cancro da mama. Optimização adicional pode ser necessário para outros tipos de tecidos. Todas as soluções deve ser feita com água com DEPC e todos os recipientes a ser usado deve ser lavado com água tratada com DEPC e subsequentemente autoclavadas. Mesmo fraca contaminaçã...

O cancro da mama é um dos tumores malignos mais comuns que afetam a população feminina em todo o mundo. Mais de 1,3 milhões de casos de câncer de mama são notificados a cada ano no mundo 1,2. Embora as células tumorais têm sido tradicionalmente considerados como biologicamente homogênea e altamente proliferativa, tornou-se evidente que o câncer de mama é geneticamente e clinicamente heterogênea. A resistência aos fármacos anticancerígenos prevalentes é uma característica de cancros da mama avançado, conduzindo à mortalidade na maioria dos pacientes através da sua promoção da progressão do cancro e metástases à distância 3. MicroRNAs (miRNAs) são uma classe de pequenas moléculas de RNA de aproximadamente 22 nucleótidos de comprimento que regulam a expressão do gene através do bloqueio da tradução do mRNA ou mediar a degradação de ARNm 4. Mais de 2.000 miRNAs diferentes foram até agora identificados em humanos, que estão ligadas a doenças humanas 5. Uma crescente variedade de estudos have demonstraram que miARN pode funcionar como oncogenes e supressores tumorais e são frequentemente desreguladas em tumores 6. miARNs impacto fortemente toda a tumorigénese principal, controlando as principais reguladores da progressão do ciclo celular, senescência, apoptose e autofagia, juntamente com os da motilidade de células tumorais, invasão e metástase 7. Expressão aberrante miRNA foi também associada a implicações clínicas, tais como estágio, diferenciação, prognóstico e resposta ao adjuvante terapia 8. Em particular, o aumento da expressão de miR-146 está correlacionada com mau prognóstico em pacientes com câncer de pulmão 9. Outro estudo demonstrou que a expressão perdida de miRNA-let7b está ligada a fenótipos malignas e, consequentemente, pior sobrevida 6. Compreender os tipos de células e estruturas que expressam miRNAs é uma parte importante de compreender os mecanismos através dos quais alterações nas células de expressão influência miRNA etecido fenótipos 10. Certos miARNs encontrado para ser segregado em células do estroma são tomadas por células epiteliais e, especificamente, agir sobre os seus objectivos. Na mesma linha, o miR-212 e miR-132 que são segregadas por estroma e regular as interacções epiteliais-estromal requeridas para crescimento ductal durante 11 desenvolvimento da glândula mamária. O objetivo geral deste trabalho é explicar um protocolo detalhado para detectar a expressão de miRNA em tecidos de cancro da mama através de miRNA hibridização in situ. Temos todas as condições optimizadas para ensaiar os níveis de expressão de miARN-489 em amostras de pacientes de cancro da mama. Para este fim, foi utilizado um DIG marcado bloqueado ácido sonda (LNA) nucleico em nosso experimento. Alguns dos seus nucleótidos tinha uma modificação LNA, que é, uma ponte metileno que liga &#39;átomo de oxigénio e 4&#39; a 2 átomos de carbono do esqueleto ribose que fixa as sequências de nucleótidos de tal modo que ele fica &quot;bloqueado no lugar&quot; após a hibridização. Como resultado, é muito mais difícilpara o complexo hibridado para desnaturar 12,13.

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clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

tecido do cancro da mama humano foi utilizado para determinar a expressão de miARN-489. Ducto da glândula mamaria e células epiteliais foram encontrados para expressar significativamente mais elevados de miARN-489 níveis que o tecido adjacente do tumor em dois pacientes (Figura 1A e 1B). Isto demonstra claramente que a expressão de miARN-489 foi perdido no tecido tumoral, sugerindo que a actividade supressora do tumor de miARN-489. Para confirmar ai...

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Análise clínico-patológico de miRNA Expressão em cancro da mama tecidos usando miRNA<em&gt; In Situ</em&gt; Hibridização

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

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Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

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