Name: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization (Video) | JoVE Journal
Uploaded: 2016-06-07T15:00:00.000+00:00
Duration: 6 min 1 s
Description: Learn miRNA in situ hybridization for detecting micro RNA localization in breast cancer tissue samples. This method reveals cellular expression patterns to study tumor suppression mechanisms.

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5 minutes

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
<ol>
	<li>Prepare 1 L of DNase and RNase free water. Make all chemical solutions using diethylpyrocarbonate (DEPC) treated water. Treat 1 L of water with 500 &#181;l of DEPC and incubate for 3 hr at room temperature. Autoclave the water to inactivate DEPC. 
	Caution: Do not inhale DEPC since it is known carcinogen.</li>
	<li>Prepare 1x PBS from 10x PBS using DEPC treated water.</li>
	<li>Prepare 20x SSC (dissolve 175.3 g of NaCl and 88.2 g of Sodium Citrate in 800 ml water, adjust the pH to 7.0 with a few drop of 14 N solution of HCl and adjust the volume to 1 L with water), 4% paraformaldehyde, 95% ethanol, 80% ethanol using DEPC treated water.</li>
	<li>Add 8 ml of triethanolamine and 1.05 ml of HCl in 590 ml of DEPC-treated water and add 1.5 ml of acetic anhydride for acetylation.</li>
	<li>Prepare 50 &#181;g/&#181;l proteinase K in buffer (50 mM Tris-HCl, pH 8.0 in DEPC treated water).</li>
	<li>Prepare hybridization buffer composed of 500 &#181;l of formamide, 250 &#181;l of 20x SSC, 100 &#181;l of 50x Denhardt&#39;s solution, 12.5 &#181;l of t-RNA, 2.5 &#181;l of herring sperm DNA, 30 &#181;l of RVC, 0.02 g of blocking reagent and 50 &#181;l of DEPC water. Prepare fresh.</li>
</ol>
2. Breast Tissue Sections
<ol>
	<li>Cut 5 &#181;m formalin-fixed paraffin-embedded sections with microtome and apply the section to charge slide14. 
	Note: The breast tissue used here was prepared previously from the Korea Biobank Network.</li>
</ol>
3. Tissue Preparation
<ol>
	<li>Remove paraffin from the tissue by immersing slides into Coplin jar 2x in fresh xylene for 10 min each and rehydrate the tissue section by 5 min incubations in decreasing concentrations of ethanol (100%, 95% and 80%) in ddH2O. Perform this step in a fume hood.</li>
	<li>Immerse the tissue in DEPC treated water for 5 min. At this point make boundaries around tissue section with hydrophobic barrier pen.</li>
	<li>Fix the tissue with 4% PFA for 15 min followed by a wash with PBS.</li>
	<li>Immerse the tissue in 0.3% Triton X-100/PBS for 10 min followed by a wash with PBS.</li>
	<li>Incubate sample with 50 &#181;g/&#181;l Proteinase K solution for 15 min at 37 &#176;C. Then incubate tissue sections with triethanolamine and acetic anhydride for 5 min at room temperature, followed by a wash with PBS. No PBS wash is required between Proteinase K treatment and triethanolamine/acetic anhydride treatment.</li>
</ol>
4. Hybridization
<ol>
	<li>Wash the tissue thoroughly and incubate the tissue section with hybridization buffer for 2 to 3 hr at room temperature. Typically 120 &#181;l hybridization buffer is enough to cover the tissue section.</li>
	<li>Dilute 5&#39;-DIG labelled LNA probe to final concentration of 3 pM in 120 &#181;l hybridization solution. As the stock concentration usually is 40 ng/&#181;l, add 3 &#181;l of stock to 120 &#181;l hybridization buffer and incubate mixture at 95 &#176;C for 5 min.</li>
	<li>Incubate tissue section with probe overnight at 54 &#176;C (Tm) in a humid chamber. Typically 120 &#181;l is enough to cover one section. Create humidity by using two wet paper towels. The ideal size of chamber is 10 inches by 8 inches.</li>
	<li>Note: Tm may be different for different miRNA. 54 &#176;C is mention for miR-489.</li>
</ol>
5. Stringency Wash
<ol>
	<li>Wash the tissue with 5x SSC for 7 min (after the hybridization step, RNase free conditions are no longer required).</li>
	<li>Wash the tissue section with 1x SSC for 7 min twice at 57 &#176;C.</li>
	<li>Wash the tissue section with 0.2x SSC for 7 min at 57 &#176;C.</li>
	<li>Wash the tissue section with 0.2x SSC for 7 min at room temperature.</li>
	<li>Incubate tissue section in PBS for 10 min.</li>
</ol>
6. Blocking
<ol>
	<li>Incubate the tissue section with blocking buffer at room temperature for 1 hr in a humid chamber (To make blocking buffer add 2 ml FBS, 10 &#181;l of Tween-20 and 3 &#181;l of BSA in 18 m of PBS. Store blocking buffer at 4 &#176;C).</li>
</ol>
7. Primary Antibody Incubation and Development
<ol>
	<li>Dilute 1:300 anti-DIG-AP antibody in blocking buffer and incubate the tissue section with antibody at 4 &#176;C overnight.</li>
	<li>Wash the sections with wash buffer 3 times, for 5 min each (buffer provided by development kit).</li>
	<li>Develop the tissue section with a commercial kit for alkaline phosphatase that produces CY-3 florescent light.</li>
	<li>Wash the section with wash buffer for 5 min. Stain section with 100 &#181;l of 2.5 ng/&#181;l Hoechst dye for 5 min followed by a 5 min wash with wash buffer.</li>
	<li>Mount the section with mounting buffer according to manufacturer&#39;s protocol. Use 15 &#181;l per tissue section and wipe excessive mounting solution with tissue wipe. Seal the slide with clear nail polish to prevent leaking.</li>
	<li>Observe the section florescent microscope using with 10X lens so total magnification is 100X.</li>
</ol>

<ol><li>Shah, N. R., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNAs+in+pathogenesis+of+breast+cancer%3A+Implications+in+diagnosis+and+treatment%5BTitle%5D)+AND+%22World+J+Clin+Oncol%22%5BJournal%5D)">MicroRNAs in pathogenesis of breast cancer: Implications in diagnosis and treatment.</a> World J Clin Oncol. 5, 48-60 (2014).</li>
<li>Tang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-185+suppresses+tumor+proliferation+by+directly+targeting+E2F6+and+DNMT1+and+indirectly+upregulating+BRCA1+in+triple-negative+breast+cancer%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">miR-185 suppresses tumor proliferation by directly targeting E2F6 and DNMT1 and indirectly upregulating BRCA1 in triple-negative breast cancer.</a> Mol Cancer Ther. 13, 3185-3197 (2014).</li>
<li>Ye, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetic+silencing+of+miR-375+induces+trastuzumab+resistance+in+HER2-positive+breast+cancer+by+targeting+IGF1R%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R.</a> BMC Cancer. 14, 134(2014).</li>
<li>Oneyama, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA-mediated+downregulation+of+mTOR%2FFGFR3+controls+tumor+growth+induced+by+Src-related+oncogenic+pathways%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.</a> Oncogene. 30, 3489-3501 (2011).</li>
<li>McGuire, A., Brown, J. A., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metastatic+breast+cancer%3A+the+potential+of+miRNA+for+diagnosis+and+treatment+monitoring%5BTitle%5D)+AND+%22Cancer+Metastasis+Rev%22%5BJournal%5D)">Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring.</a> Cancer Metastasis Rev. 34, 145-155 (2015).</li>
<li>Quesne, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+and+prognostic+associations+of+miR-205+and+let-7b+in+breast+cancer+revealed+by+in+situ+hybridization+analysis+of+micro-RNA+expression+in+arrays+of+archival+tumour+tissue%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue.</a> J Pathol. 227, 306-314 (2012).</li>
<li>Fernandez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-340+inhibits+tumor+cell+proliferation+and+induces+apoptosis+by+targeting+multiple+negative+regulators+of+p27+in+non-small+cell+lung+cancer%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.</a> Oncogene. 34, 3240-3250 (2015).</li>
<li>Yu, L., Zhang, J., Guo, X., Li, Z., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA-224+upregulation+and+AKT+activation+synergistically+predict+poor+prognosis+in+patients+with+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Cancer+Epidemiol%22%5BJournal%5D)">MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma.</a> Cancer Epidemiol. 38, 408-413 (2014).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((microRNA-146+up-regulation+predicts+the+prognosis+of+non-small+cell+lung+cancer+by+miRNA+in+situ+hybridization%5BTitle%5D)+AND+%22Exp+Mol+Pathol%22%5BJournal%5D)">microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization.</a> Exp Mol Pathol. 96, 195-199 (2014).</li>
<li>Kriegel, A. J., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+in+situ+hybridization+for+formalin+fixed+kidney+tissues%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">MicroRNA in situ hybridization for formalin fixed kidney tissues.</a> J Vis Exp. , e50785(2013).</li>
<li>Ucar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-212+and+miR-132+are+required+for+epithelial+stromal+interactions+necessary+for+mouse+mammary+gland+development%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development.</a> Nat Genet. 42, 1101-1108 (2010).</li>
<li>Nuovo, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+detection+of+microRNAs+in+paraffin+embedded%2C+formalin+fixed+tissues+and+the+co-localization+of+their+putative+targets%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets.</a> Methods. 52, 307-315 (2010).</li>
<li>Kierzek, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+influence+of+locked+nucleic+acid+residues+on+the+thermodynamic+properties+of+2%27-O-methyl+RNA%2FRNA+heteroduplexes%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes.</a> Nucleic Acids Res. 33, 5082-5093 (2005).</li>
<li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paraffin+embedding+tissue+samples+for+sectioning%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Paraffin embedding tissue samples for sectioning.</a> CSH Protoc. 2008, (2008).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequential+expression+of+miR-182+and+miR-503+cooperatively+targets+FBXW7%2C+contributing+to+the+malignant+transformation+of+colon+adenoma+to+adenocarcinoma%5BTitle%5D)+AND+%22J+Pathol%22%5BJournal%5D)">Sequential expression of miR-182 and miR-503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma.</a> J Pathol. 234, 488-501 (2014).</li>
</ol>

There is nothing to disclose.

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.

<table><tbody><tr><td>ELF-97</td><td>Life technology&amp;nbsp;</td><td>E6604</td><td></td></tr><tr><td>50x Denhard&#039;t&amp;nbsp;</td><td>Life technology&amp;nbsp;</td><td>750018</td><td></td></tr><tr><td>t-RNA</td><td>Roche</td><td>109541</td><td>Reconstitute in DEPC water</td></tr><tr><td>Herring Sperm DNA</td><td>Promega&amp;nbsp;</td><td>D1815</td><td></td></tr><tr><td>Roche Blocking&amp;nbsp;</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>RVC</td><td>Fisher</td><td>50-812-650</td><td>Before use spin down it at 16.1 RCF and take supernatant&amp;nbsp;</td></tr><tr><td>miR-489 probe&amp;nbsp;</td><td>Exiqon</td><td>38599-01</td><td></td></tr><tr><td>Nuclease free BSA</td><td>Roche&amp;nbsp;</td><td>711454</td><td></td></tr><tr><td>Primary antibody-anti DIG</td><td>Roche&amp;nbsp;</td><td>11093274910</td><td></td></tr><tr><td>Diethyl Pyrocarbonate&amp;nbsp;</td><td>Sigma</td><td>1609478</td><td></td></tr></tbody></table>

clinicopathological analysis of mirna expression in breast cancer tissues by 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 (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...

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

The overall goal of this Micro RNA In Situ Hybridization is to detect the localization of the Micro RNA of interest in breast cancer tissue. So the message can answer the key question in breast cancer field. Such as the was the micro RNA of interest is unclear or tumor suppressant.The major advantage of this technique is that it can detect cellular nuclearization of of interest in breast cancer tissue. Begin tissue preparation by removing paraffin from previously prepared breast tissue sections. Immerse the slides into a Coplin jar in fresh Xyline two times for ten minutes each.Re-hydrate the tissue section with incubations in decreasing concentrations of ethanol in double distilled water for five minutes each. Next, immerse the tissue in diethylpyrocarbonate or DEPC treated water for five minutes. While the tissue is immersed make boundaries around the tissue section with a hydrophobic barrier pen.After the DEPC immersion, fix the tissue with 4%PFA for 15 minutes. Follow the fixation with a PBS wash. Then, immerse the tissue in 0.3%Triton X-100 in PBS for ten minutes.Incubate the sample with 50 micrograms per microliter of Proteinase K solution for 15 minutes at 37 degrees Celsius. Then incubate the tissue sections with triethanolamine and acetic anhydride for five minutes at room temperature. And follow with a PBS wash.Wash the tissue thoroughly. And incubate the tissue section with 120 microliters of hybridization buffer for two hours at room temperature. Next, dilute the five prime DIG labeled NLA probe to a final concentration of 3 picamolar in 120 microliters of hybridization solution.Then add three microliters of 40 nanograms per microliter stock to the 120 microliters of hybridization buffer. And incubate the mixture at 95 degrees Celsius for five minutes. Insert the tissue section into a ten by eight inch container and cover the tissue with 120 microliters of the probe so that it is completely covered.Wet two paper towels and cover the container to create a humidity chamber. Incubate the tissue section overnight at 54 degrees Celsius. After hybridization, wash the tissue with five X saline sodium citrate buffer or SSC for seven minutes at 57 degrees Celsius.Next, wash the tissue section with one X SSC twice. And wash the tissue section with 0.2 X SSC. Then, wash the tissue section with 0.2 X SSC for seven minutes at room temperature.Incubate the tissue section in PBS for ten minutes. Incubate the tissue section with blocking buffer at room temperature for one hour in a humid chamber. Dilute the anti DIG AP antibody one to 300 in blocking buffer.And incubate the tissue section with the antibody at 4 degrees Celsius overnight. The next day, wash the sections with wash buffer three times for five minutes each. Follow the ELF 97 commercial kit instructions to develop the tissue section with alkaline phosphatase that produces size three fluorescent light.Next, wash the section with wash buffer for five minutes. Stain the section with 100 microliters of 2.5 nanograms per microliter Hoeshct dye for five minutes. And follow the stain with a five minute wash with wash buffer.Use 15 microliters of mounting buffer per tissue section. And wipe excessive mounting solution with a tissue wipe. Then, seal the slide with clear nail polish to prevent leaking.Finally observe the section with a fluorescent microscope at a total magnification of 100 X.Human breast cancer tissue from two patients was used to determine Micro RNA 489 expression. Memory gland duct and epithelial cells were found to express significantly higher Micro RNA 489 levels than adjacent tumor tissue. After watching this video, you should have a good understanding of how to perform Micro RNA In Sito hybridization by using breast cancer patient samples.

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Learn miRNA in situ hybridization for detecting micro RNA localization in breast cancer tissue samples. This method reveals cellular expression patterns to study tumor suppression mechanisms.

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

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