Chromatin immunoprecipitation (ChIP) assay optimization and Runx3 targets analysis in glioblastoma cells
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2017-12-01 |
eISSN 1648-9144.
Glioblastomas are the most aggressive malignant brain tumours with significant heterogeneity in molecular profile and patient outcome. Tumour heterogeneity is also determined by genes behaving as bifunctional modulators. Runt-related transcription factor 3 (Runx3) was described as both tumour suppressor and activator in vast of cancers as well as in gliomas. Bi-functioning of Runx3 may be explained by transcription factor ability to initiate or repress target genes expression. The aim of this study was to optimize ChIP experimental conditions and to analyze expression of selected Runx3 targets in human glioblastoma cells. Chromatin immuneprecipitation – ChIP-qPCR assay was applied to analyze transcription factor Runx3 and its targets interaction in human glioblastoma U87 cell line. U87 cells were cross-linked using 1% formaldehyde at 70% confluence. The following steps included cell lysis, DNA ultrasonication, immunoprecipitation, immunocoplex purification with magnetic beads, DNA extraction and qPCR. Optimization part showed that initial cell number, time of crosslinking and quenching as well as sonication and immunoprecipitation setting are critical ChIP factors. Runx3 showed high transcriptional interaction rates with AKT1, CLDN1, AKT1, ABCC1 genes, 78%, 54%, 51% and 36%, respectively, as compared to input control. ChIP assay is commonly used for research of protein-DNA interactions that are present in living cells. The optimization of ChIP assay is one of the most complicated steps, which is the key to success of the entire research. Runx3 role in glioblastoma is undeniable, nevertheless the exact functioning is still unclear and Runx3 is often described as a “tumour modifier”. ChIP data revealed the presence of Runx3-target complexes in U87 cells demonstrating Runx3 role for its targets regulation. Nevertheless, further analysis is necessary to show whether the increase or decrease in Runx3-target interact[...].