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

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

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 worldwide^1,2. 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' oxygen atom and 4' carbon atom of the ribose backbone which fixes the nucleotide such that it stays "locked in place" after hybridization. As a result, it is much more difficult for the hybridized complex to denature^12,13.

Protocol

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

1. 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 µl 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.
2. Prepare 1x PBS from 10x PBS using DEPC treated water.
3. 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.
4. 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.
5. Prepare 50 µg/µl proteinase K in buffer (50 mM Tris-HCl, pH 8.0 in DEPC treated water).
6. Prepare hybridization buffer composed of 500 µl of formamide, 250 µl of 20x SSC, 100 µl of 50x Denhardt's solution, 12.5 µl of t-RNA, 2.5 µl of herring sperm DNA, 30 µl of RVC, 0.02 g of blocking reagent and 50 µl of DEPC water. Prepare fresh.

2. Breast Tissue Sections

1. Cut 5 µm 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.

3. Tissue Preparation

1. 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.
2. Immerse the tissue in DEPC treated water for 5 min. At this point make boundaries around tissue section with hydrophobic barrier pen.
3. Fix the tissue with 4% PFA for 15 min followed by a wash with PBS.
4. Immerse the tissue in 0.3% Triton X-100/PBS for 10 min followed by a wash with PBS.
5. Incubate sample with 50 µg/µl Proteinase K solution for 15 min at 37 °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.

4. Hybridization

1. Wash the tissue thoroughly and incubate the tissue section with hybridization buffer for 2 to 3 hr at room temperature. Typically 120 µl hybridization buffer is enough to cover the tissue section.
2. Dilute 5'-DIG labelled LNA probe to final concentration of 3 pM in 120 µl hybridization solution. As the stock concentration usually is 40 ng/µl, add 3 µl of stock to 120 µl hybridization buffer and incubate mixture at 95 °C for 5 min.
3. Incubate tissue section with probe overnight at 54 °C (Tm) in a humid chamber. Typically 120 µl 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.
4. Note: Tm may be different for different miRNA. 54 °C is mention for miR-489.

5. Stringency Wash

1. Wash the tissue with 5x SSC for 7 min (after the hybridization step, RNase free conditions are no longer required).
2. Wash the tissue section with 1x SSC for 7 min twice at 57 °C.
3. Wash the tissue section with 0.2x SSC for 7 min at 57 °C.
4. Wash the tissue section with 0.2x SSC for 7 min at room temperature.
5. Incubate tissue section in PBS for 10 min.

6. Blocking

1. 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 µl of Tween-20 and 3 µl of BSA in 18 m of PBS. Store blocking buffer at 4 °C).

7. Primary Antibody Incubation and Development

1. Dilute 1:300 anti-DIG-AP antibody in blocking buffer and incubate the tissue section with antibody at 4 °C overnight.
2. Wash the sections with wash buffer 3 times, for 5 min each (buffer provided by development kit).
3. Develop the tissue section with a commercial kit for alkaline phosphatase that produces CY-3 florescent light.
4. Wash the section with wash buffer for 5 min. Stain section with 100 µl of 2.5 ng/µl Hoechst dye for 5 min followed by a 5 min wash with wash buffer.
5. Mount the section with mounting buffer according to manufacturer's protocol. Use 15 µl per tissue section and wipe excessive mounting solution with tissue wipe. Seal the slide with clear nail polish to prevent leaking.
6. Observe the section florescent microscope using with 10X lens so total magnification is 100X.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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