Abstract 908: Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients
Authors: Sana Jiri , Siegl Frantisek , Vecera Marek , Fadrus Pavel , Juracek Jaroslav , Pokorna Petra , Trachtova Karolina , Kazda Tomas , Slaby Ondrej 
Published: June 2022

Abstract
Introduction: Glioblastoma (GBM) is the most common malignancy affecting the CNS. Despite radical therapy, recurrence of GBM is a frequent and relatively early event in the course of the disease. One of the putative mechanisms of early recurrence of GBM is the existence of glioblastoma stem cells (GSCs) that can resist therapy and subsequently establish new GBM foci. PIWI-interacting RNAs (piRNAs), responsible for maintaining genome stability, may play a key role not only in the biology of germ and stem cells but also GSCs since their dysregulated expression has been described in several cancers, including GBM. Thus, the identification of specific piRNAs in GSCs could allow the distinction of these cells from other GBM cells and their eventual targeting. Some piRNAs could also be promising biomarkers useful for prognosis and prediction of disease progression.
Material and Methods: Native GBM tissues dissociated using the Papain Dissociation System were used for culturing of paired cell primary cultures. Cells were divided into two aliquots with different culture conditions. GSCs were cultured in DMEM/F12 medium supplemented with bFGF and EGF growth factors. DMEM medium containing 10% fetal bovine serum (FBS) was used to culture non-stem GBM cells. Subsequently, the presence of neural stem cell markers CD133 and SOX2, the ability of GSCs to establish tumors in immunodeficient mice, and the ability to differentiate into more mature cell types were analyzed. RNA isolated from the paired primary cultures was used for small-RNA sequencing (RNA-seq). QIAseq miRNA Library Kit was used for the preparation of cDNA libraries. Sequencing analysis was performed using the NextSeq 500/550 High Output v2 Kit (75 cycles) and the NextSeq 500 sequencer.
Results: Twelve paired primary cultures were prepared to global piRNA expression profiling. Small RNA-seq bioinformatics analysis identified between 3.45 and 5.51% of unique mapped piRNA reads in all samples; GSCs expressing higher levels of piRNA molecules in comparison with paired GBM cells. Subsequently, we identified a set of significantly differentially expressed piRNAs in GSCs. Expression of selected piRNAs was then artificially regulated in primary GSCs in vitro, and the effect of these molecules on neurosphere formation, viability, and cell genome stability was analyzed. Finally, we also observed an association of piR-23231 with overall survival of GBM patients. All results will be presented at the AACR Annual Meeting 2022.
Conclusion: Based on the results so far, piRNAs seem to play an important role in GSCs as well as GBM biology and their targeting may be a promising tool for therapy of GBM patients. The study was supported by the programme project of the Ministry of Health of the Czech Republic with reg. no. NV19-03-00501 and NV19-03-00559.
Citation Format: Jiri Sana, Frantisek Siegl, Marek Vecera, Pavel Fadrus, Jaroslav Juracek, Petra Pokorna, Karolina Trachtova, Tomas Kazda, Ondrej Slaby. Small RNA-seq analysis of PIWI-interacting RNAs in glioblastoma stem cells: Identification of new therapeutic targets in glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 908.