Analysis of microrna expression in brain metastases using next-generation sequencing

Abstract BACKGROUND Brain metastases (BMs) are intracranial tumors that frequently occur in adult cancer patients. Because the prognosis of patients with BM individually varies, it would be useful to improve prognostic scoring tools by including new high-performance biomarkers. MicroRNAs (miRNAs) appear to be promising in this regard as they are highly stable and, thus, suitable for both next-generation sequencing (RNA-Seq) and retrospective analyses in formalin-fixed and paraffin-embedded (FFPE) tissues.Material and METHODS Total RNA enriched for miRNAs was isolated from 71 freshly frozen histopathologically confirmed BMs with origin in 5 tumor types (ca lung - 37%, melanoma - 23%, ca breast - 18%, RCC - 15%, CRC - 7%) using mirVana miRNA Isolation Kit (Thermo Fisher Scientific). Sequencing libraries were prepared from RNA using the QIAseq miRNA Library Kit (Qiagen) and sequenced using the NextSeq 500 platform (Illumina). The miRNA molecules were subsequently transcribed from total RNA samples isolated from a retrospective set of 119 FFPE tissues using the TaqMan Advanced miRNA cDNA Synthesis Kit, and the expression of selected miRNAs was validated in a pilot experiment by qPCR using TaqMan Fast Advanced Master Mix and appropriate TaqMan MicroRNA Assays (all from Thermo Fisher Scientific). RESULTS The differential analysis identified 373 miRNAs with significantly different expression among the 5 BMs groups (p< 0.001). A molecular classifier based on the expression of 32 miRNAs was able to classify all samples correctly. Out of these, seven, including miR-122-5p, miR-141-3p, miR-146a-5p, miR-194-5p, miR-200c-3p, miR-211-3p, and miR-215-5p, were chosen for subsequent validation and their significantly different expression in 5 BMs groups was validated. CONCLUSIONS Presented results confirm the importance of studying dysregulated miRNA expression in BM and the diagnostic potential of validated miRNAs. The study was prepared with the grant support of the Ministry of Health of the Czech Republic - grant No. NV18-03-00398.