Author: He Lin
Lin, He, 2023 The circular RNAs, circPPP1R13B(2-4) and circNFASC(26,27), are upregulated in Glioblastoma and reduce cellular tumorigenicity, Flinders University, College of Medicine and Public Health
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Patients with glioblastoma (GBM), the most common malignant brain tumour, have a median survival of 12-15 months after diagnosis, with less than 5% of patients surviving 5 years. While the average survival rate of most cancers has improved by ~20% in the past 30 years, survival from GBM has not improved. Therefore, a deeper fundamental understanding of GBM is required for identifying the disease at an earlier stage and for identifying targets for more effective therapies. Circular RNAs (circRNAs) are the most contemporary family of largely non-coding RNA molecules which are particularly abundant in brain tissue. CircRNAs arise from alternative splicing of pre-mRNA transcripts and have been shown to be functional molecules offering potential as both cancer biomarkers and therapeutic targets.
Our laboratory has previously profiled circRNAs by high-throughput RNA-seq on five healthy brain tissue samples and five GBM tumours. From over 72,000 high confidence circRNAs in this dataset, 5,366 circRNAs showed > 2-fold differential expressions between the tissue with 3,647 circular RNAs downregulated in GBM. The hypothesis of this project is that overexpression of circRNAs downregulated in GBM can affect the oncogenic characteristics of GBM cell lines. The aims of the project were to (1) Identify candidate circRNAs which are downregulated in GBM tumours compared with control healthy brain tissue, (2) Generate GBM cell lines stably overexpressing candidate circRNAs and (3) Assess phenotypes in circRNA overexpressing GBM cell lines
By transgenically overexpressing three downregulated circRNAs, circPPP1R13B(2-4), circKCNN2(8) and circNFASC(26,27) in the two most widely used GBM cell lines (U87 and U251) their effects on molecular and cellular biology was investigated. Despite showing no effect on cell proliferation, overexpressing circPPP1R13B(2-4) in U251 cells and overexpressing circNFASC(26,27) in U87 cells was shown to reduce the tumorigenicity in vitro using a colony formation assay by 36% and 29%, respectively. High-content fluorescence microscopy was performed to study cellular and nuclear morphology which are known to be distorted in a number of cancers. Cell morphology assay shows circKCNN2(8) increased cell size by 37% in U87 cells, and overexpressing circNFASC(26,27) in U87 cells increased both nucleus and cell areas by 27% and 64%, respectively.
The results of this project indicate that circRNAs can change the phenotypes of GBM cells. Exploring more circRNAs is worthwhile because they have the potential to reduce the tumorigenicity of GBM cells, thus leading us into a new era in the treatment of GBM.
Keywords: Circular RNAs, Glioblastoma, cellular tumorigenicity, circPPP1R13B(2-4), circNFASC(26-27)
Subject: Biotechnology thesis
Thesis type: Masters
Completed: 2023
School: College of Medicine and Public Health
Supervisor: Professor Simon Conn