Identification of Circulating MicroRNAs as Biomarkers in Glioblastoma

Abstract

MicroRNAs (miRNAs) are small RNA sequences 22-25 nucleotides in length which play a role in post-transcriptional gene regulation by binding to target mRNA sequences preventing translation. Changes in miRNA expression can contribute to disease pathogenesis, including gliomagenesis. The release of glioblastoma specific exosomes containing miRNA into the circulation of patients provides a source of biomarkers which could be utilised in a relatively non-invasive diagnostic test. The primary aim of this thesis was to identify secreted biomarkers in biofluids of glioblastoma patients for the diagnosis and prognosis of glioblastoma mulitforme (GBM).
This is the first report of miRNA expression based on the age and sex of GBM patients. GBM and non-cancerous control patients were grouped into age categories (20-39, 40-59 and 60+ years old) and gender. Initial analysis was performed using miScript Brain Cancer Array (n=3 per category) and a total of 28 dysregulated miRNAs were identified in GBM patient serum as candidate biomarkers for further study.
Using a new patient cohort (n=3 per category), further analysis of the 28 miRNAs by qPCR identified five miRNAs with altered expression in GBM serum: miR-34a-5p, miR-92a-3p, miR-20a-5p, miR-30c-5p, and miR-150-5p. Further validation following power analysis identified four of the five miRNA biomarkers: miR-34a-5p, miR-92a-3p, miR-20a-5p and miR-30c-5p to be significantly dysregulated in the serum of GBM patients. Grouping of patients by age and gender identified miR-34a-5p as significantly increased in aged 60+ patients (p < 0.05); miR-92a-3p expression was significantly higher in male GBM patients compared to female GBM patients (p < 0.05) and miR-20a-5p was significantly higher in a sub group of GBM patients (p < 0.01). Moreover, increased expression of miR-20a-5p in the serum of GBM patients was associated with a better median survival compared to those with no change in miR-20a-5p expression.
Investigation into the origin of the serum miRNA biomarkers using qPCR, in situ hybridisation and GBM tissue data from The Cancer Genome Atlas (TCGA) identified potential differences in origin of the four miRNA biomarkers; miR-20a-5p in the serum of GBM patients was likely to originate from the GBM. MiR-34a-5p showed increased expression in the GBM tissue analysed by TCGA. In contrast, analysis of matched patient serum and tissue lysate samples using qPCR demonstrated a higher expression of miR-34a-5p in the serum of GBM patients compared to tissue expression, possibly due to increased miRNA secretion by neighbouring non-cancerous cells, or from leukocytes as part of an immune response. Further work utilising larger patient samples could confirm the origin of miR-34a-5p.
Overall four miRNAs were identified in this thesis with altered expression in GBM patients. Further studies could evaluate their use as diagnostic and prognostic serum biomarkers for glioblastoma which could provide a relatively non-invasive alternative to current diagnostic methods requiring surgery.