LINE-1 Methylation Analysis in Mesenchymal Stem Cells Treated with Osteosarcoma-Derived Extracellular Vesicles

Summary

Described here is the use of a methylation-specific probe amplification method to analyze methylation levels of LINE-1 elements in mesenchymal stem cells treated with osteosarcoma-derived extracellular vesicles. Ultracentrifugation, a popular procedure for separating extracellular vesicles from fetal bovine serum, is also demonstrated.

Abstract

Methylation-specific probe amplification (MSPA) is a simple and robust technique that can be used to detect relative differences in methylation levels of DNA samples. It is resourceful, requires small amounts of DNA, and takes around 4–5 h of hands-on work. In the presented technique, DNA samples are first denatured then hybridized to probes that target DNA at either methylated or reference sites as a control. Hybridized DNA is separated into parallel reactions, one undergoing only ligation and the other undergoing ligation followed by HhaI-mediated digestion at unmethylated GCGC sequences. The resultant DNA fragments are amplified by PCR and separated by capillary electrophoresis. Methylated GCGC sites are not digested by HhaI and produce peak signals, while unmethylated GCGC sites are digested and no peak signals are generated. Comparing the control-normalized peaks of digested and undigested versions of each sample provides the methylation dosage ratio of a DNA sample. Here, MSPA is used to detect the effects of osteosarcoma-derived extracellular vesicles (EVs) on the methylation status of long interspersed nuclear element-1 (LINE-1) in mesenchymal stem cells. LINE-1s are repetitive DNA elements that typically undergo hypomethylation in cancer and, in this capacity, may serve as a biomarker. Ultracentrifugation is also used as a cost-effective method to separate extracellular vesicles from biological fluids (i.e., when preparing EV-depleted fetal bovine serum [FBS] and isolating EVs from osteosarcoma conditioned media [differential centrifugation]). For methylation analysis, custom LINE-1 probes are designed to target three methylation sites in the LINE-1 promoter sequence and seven control sites. This protocol demonstrates the use of MSPA for LINE-1 methylation analysis and describes the preparation of EV-depleted FBS by ultracentrifugation.

Introduction

DNA methylation is a major epigenetic modification occurring in human cells. DNA methylation refers to the linkage of methyl groups to cytosine residues in CpG dinucleotides. Such dinucleotides are usually found in clusters (CpG islands) at the 5' region of genes¹. In normal cells, most of these dinucleotides exist in an unmethylated state, which allows DNA transcription. Incidentally, many cancers are associated with hypermethylated CpG islands and transcriptomic silencing², especially in tumor suppressor genes, which in turn contribute to various hallmarks of cancer³.

Protocol

This study was approved by the Ethics Committee of Helsinki and Uusimaa Hospital District (ethical approval D. No. 217/13/03/02/2015).

1. Preparation of EV-depleted FBS by ultracentrifugation

1. Take FBS in (ultra)centrifuge tubes and place them in ultracentrifuge buckets. To ensure that the ultracentrifugation runs smoothly and safely, balance the buckets within 10 mg of each other.
2. Load the buckets on a swinging rotor (type SW28, k-factor 246). Place the rotor in the ultracentrifuge and run at 100,000 x g for 19 h at 4 °C.
3. Carefully collect the light-colored upper layer of the supernatant (approx....

Results

The main goal of this study was to evaluate the epigenetic effects of OS-EVs on MSCs. OS-EVs were isolated from HOS-143B cells using the standard differential centrifugation method. Expression of the typical EV markers CD63, Hsp70, and TSG101 by western blotting confirmed the presence of OS-EVs. (Figure 2A). Absence of calnexin signal indicated purity of the OS-EV isolate. Additional indication of purity was observed with TEM, with intact vesicles of various sizes being pres.......

Discussion

This study illustrates how MSPA can be used to detect and quantify the methylation status of a specific genetic element. LINE-1 was the focus here, but the probes can be designed to target a range of genes and sequences. Moreover, there is a growing list of probe mixes available for different applications. MSPA is a simple and robust technique for DNA methylation analysis that does not require bisulfite conversion¹⁰. The complete procedure from sample preparation to data analysis takes around 2 da.......

Materials

Name	Company	Catalog Number	Comments
1 mL syringe	Terumo	SS+01T1	for NTA
24-well plate	Corning	3524	MSC cell culture
3730xl DNA Analyzer	Applied Biosystems, ThermoFisher Scientific	3730XL
50 mL centrifuge tube	Corning	430829
Beckman Optima LE-80K Ultracentrifuge	Beckman
BlueStar Prestained Protein Marker	Nippon Genetics	MWP03	WB: protein marker
Calnexin (clone C5C9)	Cell Signaling Technology	2679	WB, dilution 1:800
CD63 (clone H5C6)	BD Biosciences	556019	WB, dilution 1:1000
Centrifuge 5702 R	Eppendorf	5703000010	For conditioned media and cells
Centrifuge 5810	Eppendorf	5810000010	For spinning down 96-well plate
Centrifuge tube (polyallomer, 14x95 mm)	Beckman	331374	Ultracentrifugation
DMEM/F-12 + GlutaMAX medium	Gibco, Life Technologies	31331-028	For AT-MSC culture
Fetal bovine serum	Gibco, Life Technologies	10270-106
GeneScan 500 LIZ size standard	Applied Biosystems, Life Technologies	4322682	for capillary electrophoresis
GenomePlex Complete Whole Genome Amplification (WGA) Kit	Sigma	WGA2-10RXN	for MSPA negative control
Hi-Di formamide	Applied Biosystems, Life Technologies	4311320	for capillary electrophoresis
HOS-143B cell line	ATCC	CRL-8303
Hsp70 (clone 5G10)	BD Biosciences	554243	WB, dilution 1:1000
IRDye 800CW Goat anti-mouse	Li-Cor	926-32210	WB: secondary
IRDye 800CW Goat anti-rabbit	Li-Cor	926-32211	WB: secondary
LINE-1 probe-mix primers	IDT		Sequences in Table 1
MicroAmp Optical 96-well reaction plate with barcode	Applied Biosystems, Life Technologies	4306737	also requires sealing film
Micro BCA Protein Assay kit	ThermoFisher Scientific	23235	measure protein concentration
MiniProtean TGX 10% gels	Bio-Rad	456-1034	WB: gel electrophoresis
NanoSight LM14C	Malvern Instruments		for NTA
Nitrocellulose membrane 0.2 µm	Bio-Rad	1620112	WB: protein transfer
NucleoSpin Tissue XS	Macherey-Nagel	740901.50	for DNA extraction
Odyssey Blocking Buffer	Li-Cor	927-40000	WB: blocking, antibodies
PBS, 1X	Corning	21-040-CVR
Penicillin-streptomycin	Gibco, Life Technologies	DE17-602E	Antibiotics for culture media
Protein LoBind tube, 0.5 mL	Eppendorf	22431064	For storing Evs
REVERT Total Protein Stain and Wash Solution Kit	Li-Cor	926-11015	WB: total protein staining
RKO cell line	ATCC	CRL-2577	for MSPA positive control
RPMI medium 1640 + GlutaMAX	Gibco, Life Technologies	61870-010	For HOS-143B cell culture
SALSA MLPA HhaI enzyme	MRC-Holland	SMR50
SALSA MLPA reagent kit	MRC-Holland	EK1-FAM
SALSA MLPA P300 probe-mix	MRC-Holland	P300-100R
Swinging rotor SW-28	Beckman Coulter	342207	Ultracentrifugation
Syringe filter, 0.22 µm	Jet Biofil	FPE-204-030	sterile filtering FBS
Tecnai 12	FEI Company		equipped with Gatan Orius SC 1000B CCD-camera (Gatan Inc., USA); for TEM
TBS, 1X tablets	Medicago	09-7500-100	WB: buffer
Trans-Blot Turbo	Bio-Rad		WB: transfer
Thermal cycler	ThermoFisher Scientific	TCA0096
TrypLE Express	Gibco	12604-021	for trypsinization of cells
TSG101 (clone 4A10)	Sigma	SAB2702167	WB, dilution 1:500