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 functions

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.<....

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