Stefan Reguli
University Hospital Ostrava Poruba, Czech Republic
Title: Long non-coding RNA analysis in glioblastoma
Biography
Biography: Stefan Reguli
Abstract
Glioblastoma Multiforme (GBM) is the most frequent primary brain tumor of astrocytic origin. The prognosis of GBM patients is very poor with median of overall survival ranging between 12 and 15 months from diagnosis despite conventional therapeutic protocol consisting of maximal surgical resection followed by concomitant chemo-radiotherapy with Temozolomide and adjuvant Temozolomide in monotherapy. Therefore, a lot of financial resources and a great deal of effort are spent in the 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. We performed NGS analysis of fresh-frozen histopathologically confirmed GBM tissues and non-tumor brain tissues obtained from non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy with no signs of dysplastic changes. 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 done using NextSeq 500 high output kit and NextSeq 500 instrument (both Illumina). Statistical analysis evaluated protein-coding and non-coding RNAs and their sequential variants with non-zero 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 has been carried out by the transient transfection of specific siRNA in GBM stable cell lines (A172, U87MG and T98G). The success of transfection and viability were analyzed in vitro using qRT-PCR and MTT assay, respectively. We have demonstrated a dysregulation of many lncRNAs and protein-coding RNAs in GBM tissue in comparison with non-tumor brain tissue. However, the forced down regulation of ZFAS1, one of the most up-regulated lncRNAs in GBM tissue, was not found to have an impact on the viability of GBM cell lines in vitro.