Name: Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy (Video) | JoVE Journal
Uploaded: 2019-10-04T14:30:00.000+00:00
Duration: 9 min 40 s
Description: Discover artificial miRNA cluster engineering for glioblastoma gene therapy using bioinformatics and DNA assembly. This computational method enables simultaneous modulation of multiple microRNAs in cancer cells.

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5 minutes

The authors wish to thank the members of the Harvey Cushing Neuro Oncology Laboratory for support and constructive criticism. This work was supported by NINDS grants K12NS80223 and K08NS101091 to P. P.

1. Characterization of Functionally Associated miRNAs in Glioblastoma
<ol>
	<li>Analysis of broad differential miRNA expression in glioblastoma vs. brain
	<ol>
		<li>First, determine the most significantly deregulated miRNAs in the tumor. This can be achieved using at least three different methods:
		<ol>
			<li>Mine the Cancer Genome Atlas, found at https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga, for sequencing data11.</li>
			<li>Perform microarray analysis from a fresh operative specimen12.</li>
			<li>Use previously published datasets13. 
			NOTE: Whichever method is chosen, the output provides a bulk set of miRNAs whose expression is statistically correlated (either directly or inversely) with tumor biology. This initial group constitutes the pool of miRNAs that are further analyzed for the identification of a subset of miRNAs displaying the most stringent functional association by performing a more dynamic analysis of expression changes, as discussed below.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Condition-specific miRNA expression analysis
	<ol>
		<li>Induction of cell differentiation:
		<ol>
			<li>Use pre-coated poly-D-lysine (PDL) plastic dishes to favor the formation of a monolayer culture. For dish coating, dissolve PDL in water at a concentration of 100 &#181;g/mL. Use 1 mL of solution per 25 cm2 surface. Rinse 2x with PBS 6 h after application of the PDL solution.</li>
			<li>Plate human neural stem cells in equal amounts (100,000 cells/5 mL) either in stem cell medium (neurobasal medium + B27 supplement + 20 ng/mL EGF/FGF), astrocytic differentiation medium (DMEM + 10% FBS), or neuronal differentiation medium (neurobasal medium + B27 supplement, + 2 &#181;M retinoic acid)14. The protocol for oligodendrocyte differentiation requires addition of IGF-1 (200 ng/mL) after EGF/FGF removal14,15.</li>
			<li>After cell plating, place in incubator for 1 week at 37 &#176;C, 5% CO2.</li>
		</ol>
		</li>
		<li>RNA extraction:
		<ol>
			<li>After 7 days, remove the cells from the incubator and remove the medium.</li>
			<li>Wash the cells with PBS (5 mL). Then, add 1 mL of lysis reagent (see Table of Materials) to lyse the cells and scrape the cells into 1.5 mL microfuge tube using a cell scraper. Keep the tubes containing the lysed cells at room temperature (RT) for 5 min.</li>
			<li>Proceed to total RNA isolation following the manufacturer&#39;s instructions.</li>
		</ol>
		</li>
		<li>miRNA expression analysis:
		<ol>
			<li>Quantify the differential expression of specific miRNAs identified in step 1.1 among the three differentiation patterns by real-time PCR, using TaqMan probes and following the manufacturer&#39;s protocols for reverse transcription and PCR amplification (Figure 1).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Validation of miRNA clusters by stress-specific challenges
	<ol>
		<li>Culture G34 glioblastoma stem-like cells in neurobasal medium + B27 supplement + 20 ng/mL EGF/FGF. Start with 1 x 106 cells in 5 mL in a 25 cm2 low attachment flask.</li>
		<li>Starting 48 h after plating, add doubling concentrations of DNA alkylating agent temozolomide (TMZ) every 5 days, as follows:
		<ol>
			<li>Start with 5 &#181;M for 5 days. After 5 days, remove the medium and replace with fresh medium without temozolomide, to allow the surviving cells to recover. After 48 h, add 10 &#181;M TMZ and incubate for another 5 days.</li>
			<li>Repeat step 1.3.2.1, doubling TMZ concentration each time, until a concentration of at least 100 &#181;M is reached and cells become resistant to the drug10.</li>
		</ol>
		</li>
		<li>In a parallel experiment, irradiate G34 cells as follows:
		<ol>
			<li>Starting 48 h after plating 1 x 106 cells in 5 mL in a 25 cm2 low attachment flask, irradiate cells with 2 Gy of energy (any irradiator providing photon emission is acceptable). Return the cells to the incubator and do not disturb. After 48 h, irradiate the cells again with additional 2 Gy of energy.</li>
			<li>Repeat step 1.3.3.1 4x until a total of 10 Gy of energy is administered.</li>
			<li>Return the cells to the incubator and let them recover in fresh medium for at least 5 days before downstream analysis.</li>
		</ol>
		</li>
		<li>After the induction of resistance, lyse cells using lysis reagent and extract total RNA according to the manufacturer&#39;s protocol.</li>
		<li>Analyze the expression of the specific miRNAs identified in sections 1.1-1.2 as described in step 1.2.1 (Figure 2).</li>
	</ol>
	</li>
	<li>Characterization of the functional convergence of clustered miRNAs
	<ol>
		<li>Obtain the predicted targetome of each miRNAs defined in sections 1.2-1.3 obtained using miRNA targeting prediction tools. 
		NOTE: We generally resort to TargetScan, as its algorithm is periodically updated16. Additional prediction tools are miRanda17, miRDB18, and DIANA-miRPath19. All these programs are free, web-based applications.
		<ol>
			<li>From the front page of Targetscan, select the miRNA of interest from the pre-populated drop-down menu. Click the Submit button.</li>
			<li>Download the resulting list of targets as a spreadsheet using the Download table link (Supplementary Figure 1).</li>
			<li>To decrease the chances of false positive predictions, include only targets within the conserved sites column in downstream analyses. Also, further stringency can be obtained by using additional miRNA prediction programs (see above) and only including targets that are common to all algorithms.</li>
		</ol>
		</li>
		<li>Classify the resulting targetomes according to Gene Ontology categories using ToppGene Suite20 to evaluate for the enrichment of pathways that are common to each miRNA.
		<ol>
			<li>Paste the list of targets obtained from step 1.4.1 in the &#34;Training gene set&#34; window, using HGNC symbol as the entry type. Click Submit &#62; Start. The program will provide an output table showing the most significant &#34;Go&#34; categories for the entered list of genes (Supplementary Figure 2).</li>
		</ol>
		</li>
		<li>To finally establish the contribution of each miRNA to the regulation of a common pathway or cellular process (in this case, chromatin regulation), check each targetome obtained in step 1.4.1 against the full list of genes involved in the specific cellular process (i.e., chromatin regulation), using the Venn diagram function provided in the Bioinformatics and Evolutionary Genomics website http://bioinformatics.psb.ugent.be/webtools/Venn. 
		NOTE: This program allows the intersection of multiple groups at the same time (Figure 3). The specific unique targets provided by each miRNA are then annotated and selected for downstream functional experiments.
		<ol>
			<li>In the front page of the program, upload or copy/paste the list if target mRNAs for each miRNA or interest in the respective windows. Name each list with a unique identifier.</li>
			<li>Click Submit. The program will provide a visual output of a Venn diagram with numbers of genes in each sector, as well as a full list of mRNAs for each subset and intersection combinations.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Assembly of miRNA Modules into a Polycistronic Transgenic Cluster
<ol>
	<li>Preparation of transgenic scaffold based on miR-17-92 cluster locus
	<ol>
		<li>Obtain the nucleotide sequence of the miR-17-92 cluster from the Ensembl genome browser21. Select the ~800 base pair &#34;core&#34; sequence encompassing all six encoded miRNA hairpins of the locus and at least 200-nucleotide flanking sequences both upstream (5&#39;) and downstream (3&#39;) of the core sequence.</li>
		<li>Paste the sequence above into any word editing program.</li>
		<li>Define the sequence of each one of the six native hairpins by retrieving them in miRBase22. Mark each one of these sequences within the span of the previously identified &#34;core&#34; sequence. Any sequences between each hairpin represent spacer sequences, which are important for the correct processing of the transgene.</li>
		<li>Remove the native hairpin sequences from the &#34;core&#34; sequence, with the exception of 3-5 nucleotides at both the 5&#39; and 3&#39; ends of each hairpin, which will serve as an acceptor for the new hairpins.</li>
	</ol>
	</li>
	<li>Building a new transgene encoding multiple miRNAs of choice
	<ol>
		<li>Obtain hairpin sequences of miRNAs intended to be overexpressed in the transgene from miRBase. Carefully note the specific nucleotides that are the sites of microprocessor cleavage.</li>
		<li>Replace the removed hairpins (step 2.1.4) with the hairpin sequences of the desired miRNAs obtained in step 2.2.1.</li>
		<li>Add desired restriction sequences at both flanking regions of the transgene to facilitate subcloning into delivery vectors of choice. Verify that the chosen restriction sequences are not present within the sequence itself.</li>
		<li>For negative controls, use 20-nucleotide scrambled sequences to replace the natural 20-nucleotide sequence of each mature miRNA. Generate these scrambled sequences using an online tool such as https://genscript.com/tool/create-scrambled-sequence.</li>
	</ol>
	</li>
	<li>Verifying the two-dimensional structure of the transgene
	<ol>
		<li>Copy the full transgenic sequence into the RNA structure prediction software program RNAweb Fold23.</li>
		<li>Using standard program settings, click Proceed.</li>
		<li>Analyze the graphical output, particularly for the presence of well-defined hairpins and presence of double-stranded stem structures at least 11 nucleotides proximal to the microprocessor cleavage site. Also, look for the absence of branching points within the hairpin sequences (Figure 4).</li>
	</ol>
	</li>
</ol>
3. Obtaining Transgenes by DNA Synthesis
<ol>
	<li>After the design and in silico validation of the chimeric miRNA cluster, obtain the working sequence using DNA synthesis from commercial vendors24 and proceed with downstream cloning into vectors and transgene delivery. We have used lentiviral vectors for in vitro delivery10 and adeno-associated viruses (AAVs) for in vivo intracranial delivery25.</li>
</ol>

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</ol>

The authors report no conflicts of interest.

This protocol is based on the notion that rather than functioning in isolation, miRNAs are biologically relevant by working in groups, and these groups are transcriptionally determined by specific cellular contexts26. To justify this approach from a translational perspective, a follow-up protocol that allows recreation of this multi-miRNA pattern in cells/tissues is introduced. This is possible by taking advantage of the relatively simple biogenesis of miRNAs, whereby the recognition of the charac...

miRNAs offer an unmatched opportunity for the development of a broad gene therapy approach to many diseases1,2,3, including cancer4,5. This is based on several unique features of these biological molecules, including their small size6, simple biogenesis7, and natural tendency to function in association8. Many diseases are characterized by specific miRNA expression patterns, which often converge on the regulation of complex biological functions9. The purpose of this method is first to define a strategy to identify groups of miRNAs that are synergistically relevant for specific cellular functions. Consequently, it provides a strategy for the re-establishment of such miRNA combinations in downstream studies and applications.
This method allows for functional analysis of multiple miRNAs at once, leveraging on their simultaneous targeting of a large number of mRNAs, thus recapitulating the complex landscapes of diseases. This approach has been recently employed to define a group of three miRNAs that 1) are simultaneously downregulated in brain cancer and 2) show a strong co-expression pattern during neural differentiation as well as in response to genotoxic stress by radiation or a DNA alkylating agent. The combinatorial re-expression of this module of three miRNAs by the clustering method described below results in profound interference with the biology of cancer cells and can be easily used as a gene therapy strategy for preclinical studies10. This protocol may be of particular interest to those involved in miRNA research and its translational applications.

<table><tbody><tr><td>0.4% low melting temperature agarose&amp;nbsp;</td><td>IBI Scientific</td><td>IB70058</td><td></td></tr><tr><td>0.45 &amp;micro;M sterile filter unit</td><td>Merck Millipore</td><td>SLH033RS</td><td></td></tr><tr><td>1.5 mL Microcentrifuge tube</td><td>Eppendorf</td><td>22431081</td><td></td></tr><tr><td>6-Well plates&amp;nbsp;</td><td>Greiner Bio-One</td><td>657160</td><td></td></tr><tr><td>Athymic mice (FoxN1 nu/nu)</td><td>Envigo</td><td>069(nu)/070(nu/+)</td><td></td></tr><tr><td>B-27 Supplement&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>12587010</td><td></td></tr><tr><td>Cell culture flask</td><td>Greiner Bio-One</td><td>660175</td><td></td></tr><tr><td>Cell Scraper, 16cm</td><td>Sarstedt</td><td>83.1832</td><td></td></tr><tr><td>Cesium 137 irradiator&amp;nbsp;</td><td>JL Sheperd and Associates</td><td>Core Facility (Harvard Medical School)</td><td></td></tr><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>439142-4L</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11995040</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate-buffered saline&amp;nbsp;</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Eosin Y solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E4009</td><td></td></tr><tr><td>Fetal Bovine Serum&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F9665</td><td></td></tr><tr><td>Formalin solution</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>GlutaMAX Supplement&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>HEK-293</td><td>American Type Culture Collecti</td><td>ATCC CRL-1573</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>1051750500</td><td></td></tr><tr><td>Human primary glioma stem-like cells (GBM62)</td><td></td><td></td><td>Provided by Dr. E. A. Chiocca (Brigham and Women&amp;rsquo;s Hospital, Boston, MA)</td></tr><tr><td>Human primary glioma stem-like cells (MGG4)</td><td></td><td></td><td>Provided by Dr. Hiroaki Wakimoto (Massachusetts General Hospital, Boston, MA)</td></tr><tr><td>Lentiviral vector pCDH-CMV-MCS-EF1-copGFP</td><td>System Biosciences</td><td>CD511B-1</td><td></td></tr><tr><td>Lipofectamine 2000&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>Microcentrifuge refrigerated</td><td>Eppendorf</td><td>model no. 5424 R, cat. no.5404000138</td><td></td></tr><tr><td>Mounting medium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>4112APG</td><td></td></tr><tr><td>Nalgene High-Speed Polycarbonate Round Bottom Centrifuge Tubes&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>3117-0380PK</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Fisher Scientific</td><td>2000c</td><td></td></tr><tr><td>Neural Progenitor cells (NPC)</td><td></td><td></td><td>Provided by Dr. Jakub Godlewski (Brigham and Women&amp;rsquo;s Hospital, Boston, MA)</td></tr><tr><td>Neurobasal Medium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>21103049</td><td></td></tr><tr><td>Nikon eclipse Ti motorized fluorescent microscope system</td><td>Nikon, Japan</td><td>14314</td><td></td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher Scientific</td><td>31985088</td><td></td></tr><tr><td>PCR tubes&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CLS6571-960EA</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Petri-Dishes 94/16&amp;nbsp;</td><td>Greiner Bio-One</td><td>632180</td><td></td></tr><tr><td>Poly-D-Lysine&amp;nbsp;</td><td>Sigma- Aldrich</td><td>P4707</td><td></td></tr><tr><td>Recombinant Human EGF&amp;nbsp;</td><td>PeproTech&amp;nbsp;</td><td>AF-100-15</td><td></td></tr><tr><td>Recombinant Human FGF-basic&amp;nbsp;</td><td>PeproTech&amp;nbsp;</td><td>AF-100-18B</td><td></td></tr><tr><td>Retinoic acid</td><td>Gibco</td><td>12587-010&amp;nbsp;</td><td></td></tr><tr><td>RNA Miniprep Kit</td><td>Direct-zol</td><td>R2050</td><td></td></tr><tr><td>S1000 Thermal Cycler&amp;nbsp;</td><td>Bio-Rad</td><td>1852196</td><td></td></tr><tr><td>Small Animal Image-Guided Micro Irradiator&amp;nbsp;</td><td>Xstrahal Life Sciences, UK</td><td>Core facility (Dana-Farber Cancer Institute, Boston, MA)</td><td></td></tr><tr><td>Sorvall WX+ Ultracentrifuge&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>75000100</td><td></td></tr><tr><td>StemPro Accutase&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A1110501</td><td></td></tr><tr><td>StepOne Real-Time PCR System</td><td>Applied Biosystems&amp;nbsp;</td><td>4376357</td><td></td></tr><tr><td>SterilGARD biosafety cabinet&amp;nbsp;</td><td>The Baker Company</td><td>SG403A-HE</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>T98-G</td><td>American Type Culture Collecti</td><td>ATCC CRL-1690</td><td></td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>4366596</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Thermo Fisher Scientific</td><td>4324018</td><td></td></tr><tr><td>Temozolomide</td><td>Tocris Bioscience</td><td>2706</td><td></td></tr><tr><td>Tissue-Tek optimum cutting temperature&amp;nbsp;</td><td>Fisher Scientific</td><td>NC9636948</td><td></td></tr><tr><td>TRIzol Reagent&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15596026</td><td>Lysis reagent</td></tr><tr><td>U251-MG</td><td>American Type Culture Collecti</td><td>ATCC HTB-17</td><td></td></tr><tr><td>U87-MG&amp;nbsp;</td><td>American Type Culture Collecti</td><td>ATCC HTB-14</td><td></td></tr><tr><td>ViraPower Lentivector Expression system&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>K4970-00</td><td></td></tr><tr><td>Water, HPLC grade</td><td>Fisher</td><td>W54</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Sigma-Aldrich</td><td>534056</td><td></td></tr></tbody></table>

characterization of functionally associated mirnas in glioblastoma and their engineering into artificial clusters for gene therapy

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated miRNAs rather than the action of a single miRNA. The characterization of these specific miRNAs modules is a fundamental step in maximizing their use in therapy. This is extremely relevant because their combinatorial attributes can be practically exploited. Described here is a method to define a specific miRNA signature relevant to the control of oncogenic chromatin repressors in glioblastoma. The approach first defines a general group of miRNAs that are deregulated in tumors in comparison to normal tissue. The analysis is further refined by differential culture conditions, underscoring a subgroup of miRNAs that are co-expressed simultaneously during specific cellular states. Finally, the miRNAs that satisfy these filters are combined into an artificial polycistronic transgenes, which is based on a scaffold of naturally existing miRNA clusters genes, then used for overexpression of these miRNA modules into receiving cells.

This method allowed characterization of a module of three miRNAs that are consistently downregulated in brain tumors, which are co-expressed specifically during neuronal differentiation (Figure 1) and involved in the tumor survival response after therapy (Figure 2). This is accomplished by regulating a complex oncogenic chromatin repressive pathway. This co-expression pattern suggested a strong synergistic activity among these th...

Described here is a protocol for characterizing modules of biologically synergistic miRNAs and their assembly into short transgenes, which allows simultaneous overexpression for gene therapy applications.

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

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Research

JoVE Journal

Genetics

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

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.

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

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.

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

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Proteomic dysregulation plays an important role in the spread of diffusely infiltrating gliomas, but several relevant proteins remain unidentified. Digital spatial processing (DSP) offers an efficient, high-throughput approach for characterizing the differential expression of candidate proteins that may contribute to the invasion and migration of infiltrative gliomas.

Digital Spatial Profiling for Characterization of the Microenvironment in Adult-Type Diffusely Infiltrating Glioma

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

The protocol describes a tandem immunofluorescence hybridization assay with formalin-fixed paraffin-embedded tissue to detect and quantify microRNA and multiplexed proteins simultaneously.

MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

Discover artificial miRNA cluster engineering for glioblastoma gene therapy using bioinformatics and DNA assembly. This computational method enables simultaneous modulation of multiple microRNAs in cancer cells.

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy (Video) | JoVE Journal

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

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