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.

This study was approved by the Institutional Review Board of the Chonnam National University Hwasun Hospital and University of South Carolina. TMA samples composed of breast cancer and matched adjacent normal breast tissues were provided by the Biobank of Chonnam National University Hwasun Hospital, a member of the Korea Biobank Network.
1. Solution Preparation
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	<li>Prepare 1 L of DNase and RNase free water. Make all chemical solutions using diethylpyrocarbonate (DEPC) treated water. Tr...

<ol>
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The goal of this study was to determine the level of miRNA expression in breast cancer tissues using miRNA in situ hybridization. It must be noted that in this experiment, all conditions were optimized for breast cancer tissue. Further optimization might be required for other tissue types. All solutions must be made with DEPC water and all containers to be used should be rinsed with DEPC treated water and subsequently autoclaved. Even slight RNase contamination can interfere with the final outcome of the experim...

Breast cancer is among the most common malignancies affecting the female population across the globe. Over 1.3 million cases of breast cancer are reported every year worldwide1,2. Although tumor cells have been traditionally regarded as biologically homogenous and highly proliferative, it has become evident that breast cancer is genetically and clinically heterogeneous. Resistance to prevalent anticancer drugs is a hallmark of advanced breast cancers, leading to the mortality in the majority of patients through its facilitation of cancer progression and distance metastasis3.
MicroRNAs (miRNAs) are a class of small RNA molecules approximately 22 nucleotides long that regulate gene expression by blocking mRNA translation or mediating mRNA degradation4. More than 2,000 different miRNAs have so far been identified in humans, which are linked to human diseases5. An increasing array of studies have demonstrated that miRNAs can function as oncogenes and tumor suppressors and are often dysregulated in tumors6. miRNAs strongly impact all of the primary tumorigenesis by controlling the major regulators of cell cycle progression, senescence, apoptosis and autophagy, along with those of tumor cell motility, invasion and metastasis7.
Aberrant miRNA expression has been also associated with clinical implications such as stage, differentiation, prognosis and response to adjuvant therapy8. In particular, increased miR-146 expression is correlated with poor prognosis in lung cancer patients9. Another study has demonstrated that lost expression of miRNA-let7b is linked to malignant phenotypes and, consequently, poor survival6. Understanding the types of cells and structures that express miRNAs is an important part of understanding the mechanics through which alterations in miRNA expression influence cell and tissue phenotypes10. Certain miRNAs found to be secreted in stromal cells are taken in by epithelial cells and specifically act upon their targets. In the same vein, miR-212 and miR-132 are secreted by stroma and regulate the epithelial-stromal interactions required for ductal outgrowth during mammary gland development11. The overall objective of this paper is to explain a detailed protocol to detect miRNA expression in breast cancer tissues through miRNA in situ hybridization. We have optimized all conditions to assay the levels of miRNA-489 expression in breast cancer patient samples. To this end, we used a DIG labelled locked nucleic acid (LNA) probe in our experiment. Some of its nucleotides had an LNA modification, that is, a methylene bridge connecting the 2&#39; oxygen atom and 4&#39; carbon atom of the ribose backbone which fixes the nucleotide such that it stays &#34;locked in place&#34; after hybridization. As a result, it is much more difficult for the hybridized complex to denature12,13.

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			<td>Before use spin down it at 16.1 RCF and take supernatant&#160;</td>
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			<td height="21" style="height:21px;width:216px;">Diethyl Pyrocarbonate&#160;</td>
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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.

Human breast cancer tissue was used to determine miRNA-489 expression. Mammary gland duct and epithelial cells were found to express significantly higher miRNA-489 levels than adjacent tumor tissue in two patients (Figure 1A and 1B). This clearly demonstrates that miRNA-489 expression has been lost in tumor tissue, suggesting tumor suppressive activity of miRNA-489. To further confirm these results, tumor tissue and adjacent normal tissue from the same pa...

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.

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 ...

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