Mapping the MicroRNA Landscape in Central Nervous System Malignancies: Functional Role and Clinical Relevance

Glioblastoma (GB) is the most aggressive and lethal brain tumour found in adults and is characterised by poor prognosis and limited treatment options. Identifying putative biomarkers for early diagnosis and developing new therapeutic strategies are necessary to improve patient outcomes. Liquid biopsy-based microRNAs (miRNAs) have emerged as promising diagnostic biomarkers due to their stability in body fluids and their ability to reflect tumour-specific changes. This study aimed to identify miRNA diagnostic biomarkers for GB and evaluate alternative therapeutic approaches. The primary focus was to assess the dysregulation of miRNAs and their target genes, explore their potential as diagnostic tools, and evaluate new therapies, including in-house synthesised anticancer compounds. Initially, we performed in silico analyses to identify the most dysregulated miRNAs in GB and their associated target genes. Selected miRNAs and their target genes were further validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in GB cell lines. Western blotting, immunofluorescence (IF) and immunohistochemistry (IHC) were used to further confirm the expression profiles of selected proteins in GB cells and patient samples, respectively. To expand the clinical relevance of the study, the expression of selected miRNAs and their target genes was also assessed in highly malignant paediatric brain tumours, such as medulloblastomas (MBs). To evaluate liquid biopsy-based diagnostics, exosomes from GB patients were extracted and analysed using small RNA-sequencing (sRNA-seq) and proteomics to screen the miRNome and proteome in these patients. Transient transfections and functional assays were conducted to understand the mechanisms underlying selected miRNAs and their roles as alternative therapeutics for GB and MB tumours. Additionally, two newly synthesised anticancer compounds were evaluated as potential therapeutics against GB cell line models. In vitro functional assays, further bioinformatics analyses, and sRNA-seq were utilised to assess the cytotoxic effects and the impact of these compounds on the miRNome of GB cells. Data from these analyses were integrated to determine the therapeutic potential of both natural and synthetic compounds, particularly focusing on their ability to modulate miRNAs involved in GB progression. Our in silico analyses revealed that miR-34a and miR-128a were downregulated in GB and targeted the potential oncogenes CORO1C and SV2B, demonstrating their tumour-suppressive properties. However, the affinity of miR-34a and miR-128a towards CORO1C and SV2B was relatively weak, leading to the selection of miR-206 and miR-383 for further analysis. MiR- 206 and miR-383 demonstrated stronger targeting affinity for CORO1C and SV2B, respectively, as shown by our in silico analyses, making them more suitable candidates for potential miRNA-mimic therapies aimed at suppressing these oncogenes in GB. Notably, CORO1C was particularly elevated in paediatric patients with high synaptic plasticity, highlighting its diagnostic potential for aggressive brain tumours. Elevated SV2B expression was associated with shorter survival, emphasising its role in tumour prognosis. Western blotting and IF analyses confirmed these findings by showing the upregulation and cytoplasmic cellular appearance of CORO1C and SV2B in GB and MB cell lines, suggesting their potential roles in tumour progression. Immunohistochemistry results further supported our in vitro findings, showing elevated protein expression of CORO1C and SV2B in both GB and MB patient tissues. Additionally, sRNA-seq of paediatric MB patient samples showed the downregulation of miR-206 and miR-383, whilst transcriptomic analysis revealed upregulation of CORO1C and SV2B. However, exosomal GB sRNA-seq and proteomics analysis did not show these miRNAs or proteins as dysregulated, suggesting that CORO1C and SV2B may have specialised roles in intracellular processes rather than in exosome-mediated communication. Functional assays demonstrated that miR-206 and miR-383 effectively targeted and downregulated CORO1C and SV2B, leading to reduced cell viability and impaired colony formation in GB and MB cells. In addition, two synthesised compounds, SHG-8 and SHG-44 were evaluated for their therapeutic potential in GB. Both compounds significantly inhibited cell viability, migration, and proliferation in GB cells. SHG-8 downregulated CORO1C and inhibited the Wnt signalling pathway, while SHG-44 modulated GB-associated oncomiRs and tumour suppressor miRNAs. This study identified miR-206/CORO1C and miR-383/SV2B axes as highly promising therapeutic targets due to their observed tumour-suppressive effects in vitro. MiR-206 and miR- 383 were shown to effectively downregulate their respective targets, CORO1C and SV2B, resulting in significant reduction in cell viability and tumour progression, emphasising their potential use in targeted miRNA-mimic therapies for brain malignancies. Furthermore, the synthesised compounds SHG-8 and SHG-44 exhibited strong inhibitory effects on GB cells, demonstrating their potential as alternative treatments by modulating key miRNAs and oncogenic pathways, including the suppression of CORO1C and the Wnt signalling pathway. These findings enhance our understanding of miRNA-based diagnostics and therapeutics in brain cancer, highlighting the miR-206/CORO1C and miR-383/SV2B axes as candidates in the development of personalised treatment strategies and improving patient outcomes in neurooncology.