This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A3B03035662); and Hallym University Research Fund, 2017 (HRF-201703-003).

<ol>
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The authors have nothing to disclose.

Strategies for the determination of bona fide miRNA targets with the functions of a miRNA of interest are indispensable for the understanding of multiple roles of miRNAs. Identification of miRNA target genes can be a guideline for interpreting the cell signaling events modulated by miRNAs in a cell. An unveiling of functionally important target genes of miRNAs can provide the fundamental knowledge to develop a miRNA-based therapy in cancer.
Several methods such as microarrays, small RNA librar...

MicroRNAs (miRNAs) are the small regulatory RNAs that mainly modulate the process of translation and degradation of messenger RNAs (mRNAs) by reacting to the 3&#8217; untranslated regions (3&#8217; UTR) in bona fide target genes1. Expression of miRNAs can be regulated by transcriptional and post-transcriptional mechanisms. The imbalance of such regulatory mechanisms brings uncontrolled and distinctive miRNAs expression levels in numerous diseases including cancers2. A single miRNA can have multiple interactions with diverse mRNAs. Correspondingly, an individual mRNA can be controlled by various miRNAs. Therefore, intracellular signaling networks are intricately influenced by distinctively expressed miRNAs by which physiological disorders and diseases can be initiated and deteriorated2,3,4,5,6. Although the altered expression of miRNAs has been observed in various types of cancers, the molecular mechanisms that modulate the manners of cancer cells in conjunction with miRNAs are still largely unknown.
Accumulating evidence has been showing that the oncogenic or tumor-suppressive roles of miRNAs depend on the types of cancers. For example, by targeting forkhead box o3 (FOXO3), miR-155 promotes the cell proliferation, metastasis, and chemoresistance of colorectal cancer7,8. In contrast, the restriction of glioma cell invasion is induced by miR-107 via the regulation of neurogenic locus notch homolog protein 2 (NOTCH2) expression9. The assessment of miRNA-target interactions in connection with miRNA functions is an indispensable part to better understand how miRNAs regulate various biological processes in both healthy and diseased states10. In addition, the discovery of bona fide target(s) of miRNAs can further provide a fine-tuned strategy for a miRNA-based therapy with various anti-cancer drugs. However, the main challenge in the field of miRNAs is the identification of direct targets of miRNAs. Here, detailed methods are presented as reproducible experimental approaches for the miRNA target gene determination. Successful experimental design for the miRNA target identification involves various steps and considerations (Figure 1). Comparison of mature miRNA levels in tumor cells and normal cells can be one of the common procedures to select a miRNA of interest (Figure 1A). The functional study of a selected miRNA to detect the effects of a miRNA on cell proliferation is important to narrow down the list of best potential candidate targets of a miRNA of interest (Figure 1B). Based on the experimentally validated functions of miRNAs, a systematic review of literature and database in company with a miRNA target prediction program is required to search the most relevant information on gene functions (Figure 1C). The identification of real target genes of a miRNA of interest can be achieved by implementing experiments such as the luciferase assay along with the cloning of 3&#8217; UTR of a gene, real-time PCR, and western blotting (Figure 1D). The goal of the current protocol is to provide comprehensive methods of key experiments, the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene. The current protocol can be useful for a better understanding of the functions of individual miRNAs and the implication of a miRNA in cancer therapy.

<table><tbody><tr><td>15 mL conical tube</td><td>SPL Life Sciences</td><td>50015</td><td></td></tr><tr><td>24-well plate</td><td>Thermo Scientific</td><td>142475</td><td></td></tr><tr><td>50 mL conical tube</td><td>SPL Life Sciences</td><td>50050</td><td></td></tr><tr><td>6-well plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>6x DNA loading dye</td><td>Real Biotech Corporation</td><td>RD006</td><td>1 mL</td></tr><tr><td>8-cap strip</td><td>Applied Biosystems</td><td>N8010535</td><td>For cDNA synthesis</td></tr><tr><td>8-tube strip</td><td>Applied Biosystems</td><td>N8010580</td><td>For cDNA synthesis</td></tr><tr><td>96-well plate</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>A6283-1L</td><td>1 L</td></tr><tr><td>Agarose A</td><td>Bio Basic</td><td>D0012</td><td>500 g</td></tr><tr><td>Alkaline phosphatase</td><td>New England Biolabs</td><td>M0290S</td><td>10,000 U/mL</td></tr><tr><td>Ampicillin</td><td>Bio basic Canada Inc</td><td>AB0028</td><td>25 g</td></tr><tr><td>AriaMx 96 tube strips</td><td>Agilent Technologies</td><td>401493</td><td>For real time PCR</td></tr><tr><td>AriaMx real-time PCR system</td><td>Agilent Technologies</td><td>G8830A</td><td>qPCR amplification, detection, and data analysis</td></tr><tr><td>AsiSI</td><td>New England Biolabs</td><td>R0630</td><td>10,000 units/mL</td></tr><tr><td>CAPAN-1 cells</td><td>ATCC</td><td>HTB-79</td><td></td></tr><tr><td>Cell culture hood</td><td>Labtech</td><td></td><td>Model: LCB-1203B-A2</td></tr><tr><td>Counting chambers with V-slash</td><td>Paul Marienfeld</td><td>650010</td><td>Cells counter</td></tr><tr><td>CutSmart buffer</td><td>New England Biolabs</td><td>B7204S</td><td>10X concentration</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td>500 mL</td></tr><tr><td>DNA gel extraction kit</td><td>Bionics</td><td>DN30200</td><td>200 prep</td></tr><tr><td>DNA ladder</td><td>NIPPON Genetics EUROPE</td><td>MWD1</td><td>1 Kb ladder</td></tr><tr><td>DNase I</td><td>Invitrogen</td><td>18068015</td><td>100 units</td></tr><tr><td>Dual-luciferase reporter assay system</td><td>Promega</td><td>E1910</td><td>100 assays</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>26140-079</td><td>500 mL</td></tr><tr><td>HIT competent cells</td><td>Real Biotech Corporation(RBC)</td><td>RH617</td><td>Competent cells</td></tr><tr><td>HPNE cells</td><td>ATCC</td><td>CRL-4023</td><td></td></tr><tr><td>LB agar broth</td><td>Bio Basic</td><td>SD7003</td><td>250 g</td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668-027</td><td>0.75 mL</td></tr><tr><td>Lipofectamine RNAiMax</td><td>Invitrogen</td><td>13778-075</td><td>0.75 mL</td></tr><tr><td>Luminometer</td><td>Promega</td><td></td><td>Model: E5311</td></tr><tr><td>Microcentrifuge tube</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>Microplate reader</td><td>TECAN</td><td></td><td>Infinite F50</td></tr><tr><td>miRNA control mimic</td><td>Ambion</td><td>4464058</td><td>5 nmole</td></tr><tr><td>miRNA-107 mimic</td><td>Ambion</td><td>4464066</td><td>5 nmole</td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td>50 prep</td></tr><tr><td>Mupid-2plus (electrophoresis system)</td><td>TaKaRa</td><td></td><td>Model: AD110</td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R3189</td><td>20,000 units/mL</td></tr><tr><td>Oligo explorer program</td><td>GeneLink</td><td></td><td>For primer design</td></tr><tr><td>Optical tube strip caps (8x Strip)</td><td>Agilent Technologies</td><td>401425</td><td>For real time PCR</td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>31985-070</td><td>500 Ml</td></tr><tr><td>PANC-1 cells</td><td>ATCC</td><td>CRL-1469</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140-122</td><td>100 mL</td></tr><tr><td>Phosphate buffer saline</td><td>Gibco</td><td>14040117</td><td>1000 mL</td></tr><tr><td>Plasmid DNA miniprep S&amp;amp; V kit</td><td>Bionics</td><td>DN10200</td><td>200 prep</td></tr><tr><td>PrimeSTAR GXL DNA polymerase</td><td>TaKaRa</td><td>R050A</td><td>250 units</td></tr><tr><td>Shaker</td><td>TECAN</td><td></td><td>Shaking platform</td></tr><tr><td>Shaking incubator</td><td>Labtech</td><td></td><td>Model: LSI-3016A</td></tr><tr><td>Sigmaplot 14 software</td><td>Systat Software Inc</td><td></td><td>For dose-response curve generation</td></tr><tr><td>Sulforhodamine B powder</td><td>Sigma</td><td>S1402-5G</td><td>5 g</td></tr><tr><td>SYBR green master mix</td><td>Smobio</td><td>TQ12001805401-3</td><td>Binding fluorescent dye for dsDNA</td></tr><tr><td>T4 DNA ligase</td><td>TaKaRa</td><td>2011A</td><td>25,000 U</td></tr><tr><td>TaqMan master mix</td><td>Applied Biosystems</td><td>4324018</td><td>200 reactions, no AmpErase UNG</td></tr><tr><td>TaqMan microRNA assay (hsa-miR-107)</td><td>Applied Biosystems</td><td>4427975</td><td>Assay ID: 000443 (50RT, 150 PCR rxns)</td></tr><tr><td>TaqMan microRNA assay (hsa-miR-301)</td><td>Applied Biosystems</td><td>4427975</td><td>Assay ID: 000528 (50RT, 150 PCR rxns)</td></tr><tr><td>TaqMan miR RT kit</td><td>Applied Biosystems</td><td>4366597</td><td>1,000 reactions</td></tr><tr><td>Thermo CO&lt;sub&gt;2&lt;/sub&gt; incubator (BB15)</td><td>ThermoFisher Scientific</td><td></td><td>37 &amp;deg;C and 5% CO&lt;sub&gt;2&lt;/sub&gt; incubation</td></tr><tr><td>Trichloroacetic acid</td><td>Sigma</td><td>91228-100G</td><td>100 g</td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T4661-100G</td><td>100 g</td></tr><tr><td>Ultrapure water</td><td>Invitrogen</td><td>10977-015</td><td>500 mL</td></tr><tr><td>Veriti 96 well thermal cycler</td><td>Applied Biosystems</td><td></td><td>For amplification of DNA (or cDNA)</td></tr><tr><td>XhoI</td><td>New England Biolabs</td><td>R0146</td><td>20,000 units/mL</td></tr></tbody></table>

an in vitro protocol for evaluating microrna levels, functions, and associated target genes in tumor cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

Successful and accurate confirmation of miRNA levels is important for the interpretation of data by which the classification of miRNAs is possible based on the anticipated roles of miRNAs in the development and progression of a disease. The levels of miRNA-107 and miRNA-301 were measured in three pancreas cell lines using the probe-based quantitative PCR. The synthesis of cDNAs of both a specific miRNA and a reference gene in the same reaction can increase the reproducibility of data. PANC-1 and CAPAN-1 are human pancrea...

Watch this Scientific Journal Video about An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells at JoVE.com

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

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Research

JoVE Journal

Genetics

8.2K Views.  Hallym University. This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3’ untranslated regions (3’ UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

Video: An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Cancer Research

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Medicine

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

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.

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.

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

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

Summary

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Chapters in this video

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

In Vitro Assay to Study Tumor-macrophage Interaction

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library