Abstract 3445: ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro
Authors: Jiri Sana, Marek Vecera, Romana Butova, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Kamila Souckova, Leos Kren, Radim Lipina, Martin Smrcka, Ondrej Slaby
Journal: Cancer Research (2017)

Abstract Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain tumor of astrocytic origin characterized by very poor prognosis. Despite conventional therapeutic protocol the prognosis of GBM patients is very poor with median of overall survival ranging between 12 and 15 months from diagnosis. Therefore, many financial charges and lot of effort is spent in research of new therapeutic approaches that could prolong the survival of GBM patients. Long non-coding RNAs (lncRNAs) are a relatively new class of noncoding gene regulators playing critical roles in tumor biology, including GBM. From this perspective, lncRNAs seem to be promising therapeutic targets in GBM patients. Material and Methods: We performed next-generation sequencing analysis of fresh-frozen histopatologically confirmed 45 GBM tissues and 5 non-tumor brain tissues obtained from non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient before the treatment. rRNA depletion and cDNA library preparation were performed with GeneRead rRNA Depletion Kit (Qiagen) and NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), respectively. Sequencing was held using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). Statistical analysis evaluated 24 087 protein-coding and 8 414 non-coding RNAs and their sequential variants with non-zerou RPKM (Reads Per Kilobase of transcript per Million mapped reads) at least in one sample. We used CLC genomic workbench for the alignment and target counts. Targeted regulation of ZFAS1 level have been carried out by the transient transfection of specific siRNA in GBM stable cell lines (A172, T98G, U87MG, U251). Viability and migration were analyzed in vitro using MTT and scratch wound healing assay, respectively. Results: Statistical analysis has revealed 274 (P &amp;lt; 0.01) deregulated lncRNAs in GBMs in comparison with non-tumor brain samples. Moreover, the results have showed also 489 deregulated proten-coding RNAs with P value less than 0.001 and 26 protein-coding RNAs with P value less than 0.000001. For subsequent in vitro functional analyses was chosen one of the most upregulated lncRNAs in GBM samples ZFAS1. Targeted downregulation of this molecule led to the significant reduction of viability in all examined GBM cell lines. Decreasing of proliferation potential was observed only in A172 a U251 cell lines. Conclusion: We have demonstrated a deregulation of many lncRNAs and protein-coding RNAs in GBM tissue in comparison with non-tumor brain tissue. Moreover, ZFAS1, one of the most upregulated lncRNAs in GBM tissue, is involved in regulation of viability and migration of GBM cell lines in vitro. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Note: This abstract was not presented at the meeting. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Kamila Souckova, Leos Kren, Radim Lipina, Martin Smrcka, Ondrej Slaby. ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2017-3445