Construction of a miRNA panel as possible biomarker for separation of glioblastoma and brain metastasis patients with adenocarcinoma origin using next-generation sequencing

Next Generation Sequencing technique was performed to compare the miRNA expression pattern of tumor brain tissue sample of 6 Glioblastoma patients, and 6 patients with brain metastases (BM) originated from lung adenocarcinoma (LUAD). In order to analyse the difference on miRNA expresion level between GBM and LUAD-BM cases, we applied cluster analysis on the NGS dataset of 6 samples for each of the two goups with iDEP 1.1 software. Log2FC values were calculated to determine the exact level of up- and downregulation in case of the deregulated miRNAs, using the iDEP 1.1 web tool applying the DESeq2 algorithm. Evaluate the results of analysis 99 known miRNAs were detected with altered expression using a threshold of false discovery rate (FDR) <0.05 and fold-change> 2. Among them, 62 miRNAs were upregulated (log2FC > 2) and 37 miRNAs were downregulated (log2FC < -2) with biological revelance in tissue LUAD-BM samples compared with the GBM samples. To validate our results obtained by NGS, four upregulated miRNAs: hsa-miR-200c-5p, hsa-miR-141-5p, hsa-miR-200a-5p, hsa-miR-375-3p and two downregulated miRNAs: hsa-miR-410-3p, hsa-miR-9-5p were chosen for RT-qPCR analysis. As the result of that hsa-miR-200c-5p, hsa-miR-141-5p, hsa-miR-200a-5p, hsa-miR-375-3p was significantly upregulated, while hsa-miR-410-3p, hsa-miR-9-5p was significantly downregulated in LUAD-BM - GBM comparison. Overall design: Tissue samples were gathered from brain tumor region of six individuals diagnosed with GBM, and from brain tumor region of six patients with LUAD-BM. All samples in both group were confirmed histopathologically. These intraoperative quick-frozen tissue samples were stored at -80°C until further processing. Total RNA was isolated from the tissue samples of GBM and LUAD-BM as well. Global transcriptomic profile was studied by RNA-sequencing applying the Illumina NextSeq500 platform including microRNA sequencing. In the case of all samples 3 replicates were included. In order to identify up-, or down-regulated microRNAs the LUAD-BM samples were compared to GBM samples..