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

Summary

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.

Abstract

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.

Introduction

miRNAs offer an unmatched opportunity for the development of a broad gene therapy approach to many diseases^1,2,3, including cancer^4,5. 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.

Protocol

1. Characterization of Functionally Associated miRNAs in Glioblastoma

1. Analysis of broad differential miRNA expression in glioblastoma vs. brain
   1. First, determine the most significantly deregulated miRNAs in the tumor. This can be achieved using at least three different methods:
      1. Mine the Cancer Genome Atlas, found at https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga, for sequencing data¹¹.
      2. Perform microarray analysis from a fresh operative specimen¹².
      3. 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.
2. Condition-specific miRNA expression analysis
   1. Induction of cell differentiation:
      1. 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 µg/mL. Use 1 mL of solution per 25 cm² surface. Rinse 2x with PBS 6 h after application of the PDL solution.
      2. 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 µM retinoic acid)¹⁴. The protocol for oligodendrocyte differentiation requires addition of IGF-1 (200 ng/mL) after EGF/FGF removal^14,15.
      3. After cell plating, place in incubator for 1 week at 37 °C, 5% CO₂.
   2. RNA extraction:
      1. After 7 days, remove the cells from the incubator and remove the medium.
      2. 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.
      3. Proceed to total RNA isolation following the manufacturer's instructions.
   3. miRNA expression analysis:
      1. 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's protocols for reverse transcription and PCR amplification (Figure 1).
3. Validation of miRNA clusters by stress-specific challenges
   1. 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.
   2. Starting 48 h after plating, add doubling concentrations of DNA alkylating agent temozolomide (TMZ) every 5 days, as follows:
      1. Start with 5 µM 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 µM TMZ and incubate for another 5 days.
      2. Repeat step 1.3.2.1, doubling TMZ concentration each time, until a concentration of at least 100 µM is reached and cells become resistant to the drug¹⁰.
   3. In a parallel experiment, irradiate G34 cells as follows:
      1. 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.
      2. Repeat step 1.3.3.1 4x until a total of 10 Gy of energy is administered.
      3. Return the cells to the incubator and let them recover in fresh medium for at least 5 days before downstream analysis.
   4. After the induction of resistance, lyse cells using lysis reagent and extract total RNA according to the manufacturer's protocol.
   5. Analyze the expression of the specific miRNAs identified in sections 1.1-1.2 as described in step 1.2.1 (Figure 2).
4. Characterization of the functional convergence of clustered miRNAs
   1. 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.
      1. From the front page of Targetscan, select the miRNA of interest from the pre-populated drop-down menu. Click the Submit button.
      2. Download the resulting list of targets as a spreadsheet using the Download table link (Supplementary Figure 1).
      3. 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.
   2. 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.
      1. Paste the list of targets obtained from step 1.4.1 in the "Training gene set" window, using HGNC symbol as the entry type. Click Submit > Start. The program will provide an output table showing the most significant "Go" categories for the entered list of genes (Supplementary Figure 2).
   3. 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.
      1. 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.
      2. 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.

2. Assembly of miRNA Modules into a Polycistronic Transgenic Cluster

1. Preparation of transgenic scaffold based on miR-17-92 cluster locus
   1. Obtain the nucleotide sequence of the miR-17-92 cluster from the Ensembl genome browser²¹. Select the ~800 base pair "core" sequence encompassing all six encoded miRNA hairpins of the locus and at least 200-nucleotide flanking sequences both upstream (5') and downstream (3') of the core sequence.
   2. Paste the sequence above into any word editing program.
   3. 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 "core" sequence. Any sequences between each hairpin represent spacer sequences, which are important for the correct processing of the transgene.
   4. Remove the native hairpin sequences from the "core" sequence, with the exception of 3-5 nucleotides at both the 5' and 3' ends of each hairpin, which will serve as an acceptor for the new hairpins.
2. Building a new transgene encoding multiple miRNAs of choice
   1. 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.
   2. Replace the removed hairpins (step 2.1.4) with the hairpin sequences of the desired miRNAs obtained in step 2.2.1.
   3. 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.
   4. 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.
3. Verifying the two-dimensional structure of the transgene
   1. Copy the full transgenic sequence into the RNA structure prediction software program RNAweb Fold²³.
   2. Using standard program settings, click Proceed.
   3. 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).

3. Obtaining Transgenes by DNA Synthesis

1. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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