Name: mirna expression analyses in prostate cancer clinical tissues
Uploaded: 2015-09-08T14:00:00.000+00:00
Duration: 11 min 29 s
Description: Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

We thank Dr. Roger Erickson for his support and assistance with preparation of the manuscript.
This work was supported by the National Cancer Institute at the National Institutes of Health
(Grant Number RO1CA177984; RO1CA138642), VA program project on prostate cancer (BX001604).

Formalin-fixed, paraffin-embedded (FFPE) or fresh frozen prostate cancer samples were obtained from the SFVAMC. Samples were from prostate cancer patients who underwent radical prostatectomy at SFVAMC. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research. Alternatively, prostate cancer tissues microarrays were procured from commercial sources and used for miRNA analyses by ISH. Clinicopathological and follow up information for analyzed prostate cancer patients was collected.
1. Tissue Samples
<ol>
	<li>Cut prostate cancer tissue samples into 10 &#181;m sections using a microtome and stain with H &#38; E following the manufacturer&#8217;s protocol.</li>
	<li>Review stained slides for the identification of prostate cancer foci as well as adjacent normal glandular epithelium. 
	Note: A board certified pathologist should review the H &#38; E stained slides and mark for tumor and normal areas. Use the marked slides as guides in the subsequent sections for miRNA analyses from tumor vs normal areas.</li>
</ol>
2. miRNA Expression Analyses by Quantitative Real-time PCR
Note: This workflow involves the following steps: Laser Capture microdissection, isolation of total RNA (including miRNA and mRNA), assaying of mature miRNAs using the TaqMan MicroRNA expression assays as detailed in the following sections.
<ol>
	<li>RNA Extraction
	<ol>
		<li>Isolation of miRNA from Prostate Cancer FFPE Tissues
		<ol>
			<li>Laser capture microdissection (LCM) 
			Note: Perform steps 2.1.1.1.1- 2.1.1.1.4 in a chemical fume hood.
			<ol>
				<li>Prepare tissue slides (10 &#181;m sections) for LCM. Deparrafinize tissue sections by soaking in Xylene (2x), for 10 min each, then rehydrate the tissues by incubating the slides for 5 min each in graded ethanol (100%, 95%, 90%, 80%, 70%) followed by distilled water (5 min).</li>
				<li>Following rehydration, stain sections with hematoxylin for 30 sec followed by water.</li>
				<li>Place tissues in graded ethanol (70%, 95% 90%, 80%, 70%) (5 min each) and xylene (5 min).</li>
				<li>Dry slides in a fume hood and then place in the LCM instrument for microdissection.</li>
				<li>Perform microdissections with the LCM System in accordance with the manufacturer&#8217;s instructions24. Use pathologist marked up slides as guides for the identification of prostate cancer foci as well as adjacent normal tissue. Use the laser beam at a 10-20 &#181;m diameter pulses with a power of 70-90 mW.</li>
				<li>Capture areas of interest with infrared laser pulses onto LCM caps. Combine cells from 2-5 caps for miRNA extraction per sample. To ensure integrity of extracted RNA, immediately process captured cells for miRNA extraction.</li>
				<li>Alternatively, lyse cells in the miRNA extraction buffer (according to manufacturer&#8217;s protocol) and store lysates at -80 &#186;C.</li>
			</ol>
			</li>
			<li>miRNA Extraction and Analyses from LCM Microdissected Tissues
			<ol>
				<li>Extract total RNA from microdissected FFPE tissues using a commercial kit following the manufacturer&#8217;s instructions. 
				Note: The yield of RNA from laser capture miscrodissection is typically low. Therefore, RNA is eluted into low volumes (20-30 &#181;l) and used for expression profiling using Taqman microRNA expression assays following manufacturer&#8217;s instructions (also described in section 2.2). Optimization of input RNA for real time PCR detection of mature miRNAs suggests that 5 &#181;l of the eluted RNA is optimal for each miRNA-specific reverse transcription reaction. As an endogenous control, RNU48/RNU24 is used. For control reactions, 2 &#181;l of the eluted RNA is used for the reverse transcription reaction.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Isolation of miRNA from Prostate Cancer Frozen Tissues
		<ol>
			<li>Homogenize frozen resected prostate tissues by grinding the tissues in liquid nitrogen using a mortar and pestle. Transfer homogenized tissue to a microcentrifuge tube using a cooled spatula. Maintain the mortar/pestle and spatula at the same temperature by dipping in liquid nitrogen so homogenized tissue can be easily transferred.</li>
			<li>Add commercial guanidine isothiocyanate-phenol reagent (1 ml/0.1 g of tissue) to the homogenized tissue and incubate at room temperature for 5 min. 
			Note: Homogenized tissues in the commercial guanidine isothiocyanate-phenol reagent can be stored at -80 &#186;C for several months.</li>
			<li>Add chloroform to the homogenate (0.2 ml chloroform/ml) and shake vigorously for 30 sec. Incubate samples at RT for 3-5 min followed by centrifugation at 10,000 x g for 20 min at 4 &#186;C.</li>
			<li>Transfer the upper aqueous phase to a fresh sterile RNase-free 1.5 ml tube.</li>
			<li>Add an equal volume of isopropyl alcohol. Mix and incubate for 10 min at RT followed by centrifugation at 12,000 x g for 20 min at 4 &#186;C to pellet the RNA.</li>
			<li>Aspirate the supernatant and wash the RNA pellet with 1 mL of 70% ethanol. Centrifuge at 12,000 x g for 10 min at 4 &#186;C.</li>
			<li>Carefully aspirate the supernatant. Dry RNA pellets at RT for 5-10 min. Dissolve RNA in 50-100 &#181;l nuclease-free water.</li>
			<li>Perform quantification of RNA using a nanodrop spectrophotometer and determine RNA integrity with a bioanalyzer. Adjust RNA concentrations to 10 ng/&#181;l. Use 10-50 ng RNA for miRNA- specific cDNA reaction followed by real-time PCR analyses (Section 2.2).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Quantitative Real-time PCR for miRNA Expression Analyses 
	Note: Assay mature miRNAs using a two-step RT-PCR protocol as described below.
	<ol>
		<li>Reverse Transcription (RT)
		<ol>
			<li>Reverse transcribe cDNA from RNA using a miRNA reverse transcription kit in accordance with the manufacturer&#39;s instructions. Use 10-50 ng of total RNA with miRNA specific primer from the MicroRNA Assays and RT kit. Use RNU48/RNU24/RNU6B as controls. Dilute cDNA 1:5 to 1:10 (depending on the abundance of the analyzed miRNA) and use in Real-time PCR detection as described in the following step.</li>
		</ol>
		</li>
		<li>Real-time PCR for Detection of Mature miRNA
		<ol>
			<li>Amplify PCR products from cDNA samples using the MicroRNA assays with a Fast Universal master mix in accordance with manufacturer&#8217;s instructions. Normalize samples to RNU48/RNU24/RNU6B control. Use the comparative Ct (threshold cycle) method to calculate the relative changes in gene expression on the Fast Real Time PCR System. Analyze each sample in triplicate.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. miRNA Expression Analyses by In Situ Hybridization (ISH)
<ol>
	<li>Pre-Treatment of Tissues
	<ol>
		<li>Cut 5 &#181;m sections from FFPE prostate cancer tissue blocks using a microtome.</li>
		<li>Fix tissue sections by incubating the slides at 56 &#176;C for 1 hr. 
		Note: Slides can be stored at RT for several weeks for miRNA analyses.</li>
		<li>Deparrafinize the tissues by soaking the slides with xylene (2x), for 15 min each.</li>
		<li>Rehydrate the tissues by incubating the slides for 5 min each in graded ethanol (100% (2x), 95%, 90%, 80%, 70%) followed by distilled water (5 min).</li>
		<li>Fix the slides with 4% paraformaldehyde in PBS at room temperature for 20 min.</li>
		<li>Wash the slides with PBS (2x) at room temperature for 5 min each.</li>
		<li>Treat the slides with 10 &#181;g/ml Proteinase K at 37 &#186;C for 10 min in pre-warmed proteinase K buffer (5 mM Tris-HCL pH 7.4, 1 mM EDTA, 1 mM NaCl).</li>
		<li>Following Proteinase-K treatment, rinse the slides with 0.2% glycine in PBS for 30 sec.</li>
		<li>Wash slides in PBS (1x) at room temperature for 5 min.</li>
		<li>Fix the slides with 4% Paraformaldehyde in PBS for 15 min.</li>
		<li>Rinse slides with PBS for 5 min.</li>
	</ol>
	</li>
	<li>Hybridization
	<ol>
		<li>Pre-hybridize the slides with pre-hybridization solution for 3-4 hr at 55 &#186;C in a humidified chamber. Use tissue lab wipes soaked in 50% formamide/50% 5x SSC to keep the chamber humidified.</li>
		<li>Use 5&#8217; digoxigenin labeled probes at a concentration of 20-50 nM in hybridization buffer. Dilute miRNA-specific probe (20-50 nM) and small nuclear RNA U6 control probe (20 nM), heat at 90 &#186;C for 4 min, place it on ice, and add to ice cold hybridization buffer (2-4 ng/&#181;l).</li>
		<li>Remove pre-hybridization solution, add hybridization solution (probe + hybridization buffer, 100 &#181;l per slide), incubate for 12-16 hr at probe-specific hybridization temperature (Hybridization temperature =Tm probe-21 &#186;C). Perform hybridizations in a humidified chamber (50% formamide/50% 5x SSC).</li>
	</ol>
	</li>
	<li>Washing Steps
	<ol>
		<li>Wash the slides with 2x SSC for 10 min at 45 &#186;C.</li>
		<li>Wash the slides with 1.5x SSC for 10 min at 45 &#186;C.</li>
		<li>Wash the slides with 0.2x SSC (2x) at 37 &#186;C for 20 min each.</li>
		<li>Incubate slides with 1x blocking solution for 1-2 hr at room temperature</li>
	</ol>
	</li>
	<li>Detection
	<ol>
		<li>Incubate the slides with 1:100 PBS diluted AP-conjugated anti-digoxigenin antibody for 1-4 hr or overnight at 4 &#186;C.</li>
		<li>Wash the slides with PBS (3x) at room temp for 10 min each.</li>
		<li>Wash the slides (2x) with Alkaline Phosphatase buffer (100 mM Tris pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20) at RT for 5 min each.</li>
		<li>Incubate the slides in BM Purple AP substrate in the dark at RT for 1-20 hr.</li>
		<li>Rinse the slides with PBS containing 0.1% Tween-20 and wash (2x) in water.</li>
		<li>Mount the slides using an aqueous mounting media and examine under a microscope.</li>
	</ol>
	</li>
</ol>

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The authors have no financial disclosures.

In this article, we describe a simplified workflow for miRNA expression profiling in archived FFPE or fresh frozen prostate cancer clinical tissues. In prostate cancer, several studies suggest an important role of microRNAs in prostate cancer initiation, progression and metastasis. However, conflicting results are often obtained on a specific miRNA22 since the miRNA extraction and analyses methods differ widely. In view of the emerging evidence supporting the potential application of miRNAs as alternative pros...

Cancer of the prostate gland is a commonly diagnosed male malignancy that is one of the leading causes of cancer related mortality among men. In US, an estimated 220,800 new cases and 27,540 deaths will be reported in 20151.
Prostate cancer is a heterogeneous disease with highly variable disease course- tumors can be indolent or very aggressive. A critical challenge in prostate cancer clinical management is posed by the inadequacy of currently used methods/biomarkers for disease screening, diagnosis, prognosis and treatment2. Current screening methods include prostate specific antigen (PSA) testing and a digital rectal examination (DRE) followed by prostate biopsies3. Prostate specific antigen (PSA) is the most widely used prostate cancer biomarker that has significantly revolutionized clinical management and improved survival rates4. However, due to inherent limitations of PSA including lack of specificity, PSA-based screening has led to over diagnosis and over treatment of the disease. In view of this, intensive efforts are being directed towards a search for alternate prostate cancer biomarkers, particularly those which can predict the aggressiveness of the disease and drive better treatment decisions4,5. Over the last few years, microRNAs (miRNAs) have emerged as promising alternate prostate cancer biomarkers.
MicroRNAs (miRNAs) constitute an evolutionarily conserved class of small non-coding RNAs that suppress gene expression post-transcriptionally via sequence-specific interactions with the 3&#8217;- untranslated regions (UTRs) of cognate mRNA targets. It is estimated that &#62;60% of mRNAs are conserved targets of miRNAs6. miRNA genes are located in intergenic regions or within introns or exons of protein/non-protein coding genes7. These genes are preferentially transcribed by RNA Polymerase II into primary miRNAs (pri-miRNAs, several kilobases long) that form hairpin shaped stem loop secondary structures. These pri-miRNAs are processed into precursor miRNAs (pre-miRNAs, 60-75 nucleotide long) that are exported to the cytoplasm and further processed into mature miRNAs (18-25 nucleotide long)8-10. miRNAs regulate key cellular processes including proliferation, development, differentiation and apoptosis11. Studies suggest a widespread dysregulation of miRNA expression profiles in various human malignancies including prostate cancer12-15. miRNA expression profiles have been reported to be widely dysregulated in primary and metastatic prostate cancer. Altered miRNA expression have been linked with prostate cancer progression, aggressiveness and recurrence highlighting the prognostic potential of miRNAs12,14,16-19. A growing body of evidence indicates that miRNAs play important mechanistic roles in prostate cancer initiation, development, progression and metastasis. Overall, miRNAs are emerging as promising alternate biomarkers for prostate cancer diagnosis and prognosis that can distinguish between normal and cancer tissues and aid in stratification of prostate tumors12. Also, miRNAs are important targets for development of effective therapeutics against prostate cancer20.
Owing to their small size and resistance to endogenous RNase activity, miRNAs are stable biomarkers that can be readily detected in formalin-fixed tissues21 and in prostate biopsies22. Moreover, the expression profiles of miRNAs have been compared in frozen and formalin fixed tissues and have been found to be strongly correlated21. However, miRNA expression profiling in prostate cancer clinical tissues is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. Here we describe a simplified workflow used in our lab for miRNA expression profiling in archived FFPE or fresh frozen prostate cancer clinical specimens. We employ a combination of quantitative real-time PCR and in situ hybridization for miRNA analyses of clinical specimens, with the former yielding more quantitative information and the latter for visualizing the differential expression of potential miRNA biomarkers in an array of tissues. Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues, expression analyses by Taqman-probe based miRNA RT-PCR and miRNA in situ hybridization technique using locked nucleic acid (LNA)-based probes23. LNA-based probes offer increased sensitivity and specificity compared to DNA- or RNA- based probes and enables robust detection of all miRNA sequences, regardless of their GC content and also allow discrimination of miRNA families. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA), with the latter offering the potential to accelerate miRNA biomarker discovery.

<table><tbody><tr><td>Microtome</td><td>Leica Biosystems&amp;nbsp;</td><td>RM2255</td><td></td></tr><tr><td>Arcturus Autopix for LCM</td><td>Arcturus/ Life Technologies</td><td>LCM1621/LCM1110</td><td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&amp;nbsp;</td></tr><tr><td>CapSure Macro LCM Caps</td><td>Life Technologies</td><td>LCM0211</td><td></td></tr><tr><td>miRNeasy FFPE Kit&amp;nbsp;</td><td>Qiagen</td><td>217504</td><td></td></tr><tr><td>7500 Fast Real-time PCR System&amp;nbsp;</td><td>Applied Biosystems/ Life Technologies</td><td>4351106</td><td></td></tr><tr><td>Taqman MicroRNA Reverse Transcription kit&amp;nbsp;</td><td>Applied Biosystems/ Life Technologies</td><td>4366596</td><td></td></tr><tr><td>Taqman Fast Universal PCR master mix&amp;nbsp;</td><td>Applied Biosystems/ Life Technologies</td><td>4352042</td><td></td></tr><tr><td>DIG labeled LNA probe for U6</td><td>Exiqon</td><td>99002-01</td><td></td></tr><tr><td>BM Purple AP substrate</td><td>Roche</td><td>11442074001</td><td></td></tr><tr><td>Pre-hybridization solution&amp;nbsp;</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-1&lt;/a&gt;</td><td></td></tr><tr><td>Hybridization solution&amp;nbsp;</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-2&lt;/a&gt;</td><td></td></tr><tr><td>Blocking solution&amp;nbsp;</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-8&lt;/a&gt;</td><td></td></tr><tr><td>AP-conjugated anti-digoxigenin antibody</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-7&lt;/a&gt;</td><td></td></tr><tr><td>Aqueous mounting media&amp;nbsp;</td><td>Vector Laboratories&amp;nbsp;</td><td>H-5501&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Trizol (guanidine isothiocyanate-phenol reagent)&amp;nbsp;</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/15596018&quot;&gt;15596-018&lt;/a&gt;</td><td></td></tr><tr><td>Harris hematoxylin</td><td>Statlab</td><td>SL200</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Statlab</td><td>SL201&amp;nbsp;</td><td></td></tr></tbody></table>

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).

Expression profiling of miR-203 in LCM primary prostate cancer clinical specimens by RT-PCR analyses (Figure 1)
RT-PCR analyses of relative miR-203 expression in LCM primary prostate cancer tissues and the matched adjacent normal regions was carried out as described in Saini et al.15 RNU48 was used as a control. The Table below summarizes the relative miR-203 expression in prostate cancer tumor tissues relative to adjacent normal tissues.
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Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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, ...

mirna-expression-analyses-in-prostate-cancer-clinical-tissues

Research

JoVE Journal

Medicine

10.8K Views.  University of California San Francisco. 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).

Video: miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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 ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Cancer Research

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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 ...

TMEM200A: A Novel Oncogene Regulating EMT and PI3K/AKT in Gastric Cancer

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in common cancers by multiomics analysis and used in vitro cell cultures of gastric cells to verify the results. The expression of TMEM200A in several human cancer types was assessed using the RNA-seq data from the UCSC Xena database. Bioinformatic analysis revealed a potential role of TMEM200A as a diagnostic and prognostic biomarker. 
Cultures of normal gastric and cancer cell lines were grown and TMEM200A was knocked down. The expression levels of TMEM200A were measured by using quantitative real-time polymerase chain reaction and western blotting. In vitro loss-of-function studies were then used to determine the roles of TMEM200A in the malignant behavior and tumor formation of gastric cancer (GC) cells. Western blots were used to assess the effect of the knockdown on epithelial-mesenchymal transition (EMT) and PI3K/AKT signaling pathway in GC. Bioinformatic analysis showed that TMEM200A was expressed at high levels in GC. 
The proliferation of GC cells was inhibited by TMEM200A knockdown, which also decreased vimentin, N-cadherin, and Snai proteins, and inhibited AKT phosphorylation. The PI3K/AKT signaling pathway also appeared to be involved in TMEM200A-mediated regulation of GC development. The results presented here suggest that TMEM200A regulates the tumor microenvironment by affecting the EMT. TMEM200A may also affect EMT through PI3K/AKT signaling, thus influencing the tumor microenvironment. Therefore, in pan-cancers, especially GC, TMEM200A may be a potential biomarker and oncogene.

Author Spotlight: Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies 

Here, a protocol is presented in which multiple bioinformatic tools are combined to study the biological functions of TMEM200A in cancer. In addition, we also experimentally validate the bioinformatics predictions.

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

Bioengineering

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

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 ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Summary

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Chapters in this video

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

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

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

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging