利用miRNA的miRNA表达的临床病理分析乳腺癌组织<em>原位</em>杂交
Summary
Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.
Abstract
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.
Introduction
乳腺癌是影响世界各地的女性人口中最常见的恶性肿瘤之一。超过130万例乳腺癌，每年全世界1,2报告。虽然肿瘤细胞已被传统上被视为生物学均匀和高度增殖，它已成为明显的是，乳腺癌是遗传和临床异质性。耐流行抗癌药物是先进乳腺癌的标志，导致通过它的癌症进展和远处转移3的便利在大多数患者的死亡率。
微RNA（miRNAs）是一类通过阻断mRNA翻译或介导的mRNA降解4调节基因表达的小RNA分子约22个核苷酸长的。超过2000种不同的miRNA迄今已在人类中，它们与人类疾病5识别。越来越的研究阵列甲肝Ë证实的miRNA可以作为癌基因和肿瘤抑制基因的功能，并且经常在肿瘤中6失调。通过控制细胞周期进程，衰老，凋亡和自噬的主要调节的miRNA强烈影响所有初级肿瘤发生的，与这些肿瘤细胞运动性，侵入和转移7的沿。
miRNA表达异常，已与临床意义，如分期，分化程度，预后和应对辅助治疗8也被相关。特别是，增加的miR-146表达与肺癌患者9预后差相关。另一项研究已经证明了miRNA-let7b失去表达是与恶性表型，因此，生存差6。理解表达的miRNA在细胞类型和结构是理解机制的重要组成部分，通过它miRNA表达影响细胞改变和组织表型10。发现在基质细胞被分泌某些miRNA的取在由上皮细胞并在它们的靶标特异性作用。本着同样的精神中，miR-212和miR-132是由间质分泌和调节乳腺发育11期间导管生长所需的上皮-间质相互作用。本文的总体目标是解释一个详细的协议，并通过miRNA的原位杂交技术检测乳腺癌组织中的miRNA表达。我们优化所有条件以测定乳腺癌患者样品中的miRNA-489的表达水平。为此，我们使用标有DIG在我们的实验锁核酸（LNA）探针。它的一些核苷酸的有一个LNA修饰，即，亚甲基桥连接的2'氧原子和4'核糖主链固定，使得它保持“锁定就位”杂交后的碱基的碳原子上。其结果，这是更为困难用于杂交复合变性12,13。
Protocol
Representative Results
Discussion
本研究的目的是确定在原位杂交使用的miRNA乳腺癌组织的miRNA的表达水平。必须指出，在该实验中，所有条件为乳腺癌组织优化。进一步的优化可能需要的其它类型的组织。所有解决方案必须用DEPC水进行，也可以使用所有的容器应用DEPC处理过的水冲洗，随后高压灭菌。即使是轻微的RNase污染可以用实验的最终结果产生干扰。作为miRNA的是一个非常短的序列，其有必要使用的LNA探针，以便于与…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| ELF-97 | Life technology | E6604 | |
| 50X Denhard't | Life technology | 750018 | |
| t-RNA | Roche | 109541 | Reconstitute in DEPC water |
| Herring Sperm DNA | Promega | D1815 | |
| Roche Blocking | Roche | 11096176001 | |
| RVC | Fisher | 50-812-650 | Before use spin down it at 16.1 RCF and take supernatant |
| miR-489 probe | Exiqon | 38599-01 | |
| Nuclease free BSA | Roche | 711454 | |
| Primary antibody-anti DIG | Roche | 11093274910 | |
| Diethyl Pyrocarbonate | Sigma | 1609478 |
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