Regulation of microRNA expression in prostate cancer by hypoxia

Abstract

Background: Hypoxia (a pathologically low oxygen level) is a well-established driver of aggressive behaviour in prostate cancer (PCa), the second most common cancer in men worldwide. MicroRNAs (microRNAs) are short non-coding RNA molecules that are essential for many cell processes by regulating gene expression post-transcriptionally. In prostate cancer, several microRNAs are abnormally expressed but the relationship
between microRNAs and the hypoxic response has not been extensively studied. The primary objective of this thesis was to investigate the expression and functional role of microRNAs in response to hypoxia in prostate cancer.
Methods: Three laboratory models of hypoxia were utilised: (i) in vitro PCa cell line culture at 0.1% oxygen, (ii) 3D cell spheroid culture, and (iii) an in vivo murine tumour xenograft experiment. MicroRNA expression was monitored by RT-qPCR to establish a list of hypoxia-responsive microRNAs. The effect on mRNA targets was determined by RT-qPCR, western blot and luciferase assay following transient transfection to overexpress the microRNAs. For miRNAs that were upregulated by hypoxia in all three models, functional bioassays were used to examine the effect on PCa cell proliferation, migration and invasion. Regulation of miRNA expression by DNA methylation was explored by pyrosequencing. The laboratory work was complemented by in silico and next-generation sequencing (NGS) analyses of data collected from human prostate tumours, including prostate cancer datasets archived in The Cancer Genome Atlas (TCGA) repository. Bioinformatics analyses was also utilised in collaborative work investigating the impact of androgen receptor variants (ARV) on gene expression. Finally, a systematic review and meta-analysis on the role of appetite-regulating hormones in prostate cancer development was performed.
Results: The hypoxic models generated novel data demonstrating that miR-210, miR-21 and miR-196a were upregulated by hypoxia. In the case of miR-210 and miR-21, functional assays showed they could contribute to tumour progression by regulation of an adhesion protein (NCAM) and by increasing clonogenicity and migration, respectively. In silico TCGA analyses revealed that the expression of both microRNAs correlated
significantly with markers of hypoxia and tumour aggressiveness in clinical samples, providing strong evidence for their role in prostate tumour progression. NGS data analyses and methylation analysis generated results to show how bioinformatics can identify key networks of cellular signalling associated with hypoxia and epigenetic regulation of microRNA expression. Collaborative bioinformatics analyses also utilised NGS data to demonstrate how ARV can contribute to prostate cancer progression. The systematic review revealed that obesity related hormones may also be important in prostate cancer development.
Conclusions: This thesis provides evidence that microRNAs play a crucial role in the progression of prostate cancer through hypoxia-related mechanisms. The data presented builds upon previous research of microRNA function in prostate cancer by presenting novel results linking individual microRNAs with
hypoxia-associated tumour progression. Increasing our understanding of the functionality of specific microRNAs in prostate cancer will be crucial in developing this exciting field of research so that it can inform precision medicine and improve patient outcome. This work provides evidence that microRNA profiling has great potential for improving diagnostic, prognostic, and therapeutic approaches in this disease.

Date of Award	Feb 2020
Original language	English
Supervisor	Colum Walsh (Supervisor) & Declan Mc Kenna (Supervisor)