PO-347 Analysis of global methylation status under the microenvironment in glioblastoma cells

Abstract

Introduction Glioblastoma multiforme is the most aggressive brain tumour and it has reported that cellular subpopulation of tumours display several characteristics of stem cells highly expressing stem cell specific markers such as SOX2. Glioblastoma stem cells (GSCs) are at the root of tumour recurrence and are regarded as a potential target for anticancer therapy. Microenvironment within the tumour might be important to regulate and/or maintain its properties via epigenetic modifications including DNA methylation. In this study, we explored whether different culture conditions lead to alter the status of methylation in glioblastoma cells, U87-MG. Material and methods Cells were maintained in normal culture condition and spheres were cultivated in serum-free on low attachment plate. Genomic DNA was converted using bisulfite conversion kit (Zymo EZ DNA methylation kit). Converted genomic DNA was used for infinium methylation assay. Images were captured by Illumina’s iScan Control software. After correction and filtering raw data, differentially expressed methylation list was determined using |delta_mean|≥0.2 (the difference of methylation signal, avg beta of Case – avg beta of Control) and p-value Results and discussions We observed that expression of SOX2 at the mRNA level consistently increased in cells cultured without serum on the low-attachment plate compared with physiological culture condition. Genome-wide methylation analysis shows that ninety eight genes were significantly hypo-methylated (forty three genes, 43.88%) or hyper-methylated (fifty five genes, 56.12%) under the condition without serum on the low-attachment plate compared with physiological culture condition. Furthermore, these results might give a macroscopic insight into differential methylation status in structure of gene such as 5’ untranslated region (UTR), transcription start site (TSS), and 3’UTR under the low-attachment condition without serum. In addition, methylation status of three miRNAs under the low-attachment condition without serum was altered and their functional roles require further exploration. Conclusion Global methylation status was distinctly changed in glioblastoma spheres under the serum-free and low-attachment condition (Fund no: NRF-2017R1A1A1A05000839).