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Title: microRNA expression in colorectal tumours : identification of novel mechanisms of gene regulation
Author: Amankwatia, Edward Bobie
ISNI:       0000 0004 5347 7435
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2014
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Colorectal cancer is a major cause of cancer-related death in the UK, in part as a consequence of a failure to detect the disease at its early stages and also to treatment failure due to the development of drug and radiation resistance. MicroRNAs (miRNAs), small non-coding RNAs, regulate the expression of tumour suppressor genes and oncogenes including KRAS and may influence cancer development and drug and radiation response in colorectal cancer. MiRNAs therefore have potential as biomarkers of disease progression and treatment response. Quantitative real time PCR analysis, in the form of Taqman Low Density Array (TLDA) miRNA cards, was firstly used to prolife colorectal adenomas, cancers and matched normal mucosae and isogenic KRAS mutant and wild-type colorectal cancer cell lines to identify potential candidate miRNAs that regulate KRAS signalling and are involved in colorectal cancer progression. The over-expression of miR-224 was identified to be an early and persistent event in colorectal cancer as it was increased in colorectal adenomas and cancers compared to patient-matched normal tissue. MiR-224 expression was also increased in KRAS WT cells compared to mutant cells and in KRAS/BRAF WT colorectal cancers compared to BRAF mutant cancers. MiR-224 knockdown in KRAS WT cells increased the amount of GTP-bound activated KRAS, increased ERK 1/2 phosphorylation and also increased cellular 5-FU sensitivity thus mimicking a KRAS mutant phenotype. MiR-224 knockdown also reduced cell invasion in vitro and miR-224 expression was additionally increased in liver metastases compared to patient-matched primary colorectal cancers. To identify novel mechanisms of drug resistance, two colorectal cancer cell lines HCT116 and DLD-1 were made resistant to 5-FU or oxaliplatin, following continuous incremental drug selection, and chemosensitivity to the colorectal cancer drugs 5-FU, oxaliplatin and irinotecan were compared using MTT cytotoxicity assays. MiRNA and messenger RNA (mRNA) expression differences in paired drug sensitive and resistant cells were identified using TLDA miRNA cards and Illumina HT-12 BeadChip mRNA expression arrays. To identify miRNAs involved in KRAS-mediated radiation resistance, isogenic KRAS WT and mutant colorectal cancer cell lines were treated with 5 Gy of ionising radiation and profiled using TLDA miRNA cards. MiRNA target prediction databases (mirDB, miRANDA, miRBase, TargetMiner and TargetScan) identified common candidate target genes for each differentially expressed miRNA, and Metacore analysis predicted key processes and pathways involved in drug and radiation resistance. In the paired drug sensitive and resistant cell lines, miRNA and mRNA profiling and bioinformatics analysis predicted cancer-related pathways and processes involved with cell invasion, cell cycle regulation and glycolysis as drug resistance mechanisms, which were then experimentally validated. I identified candidate drug resistance genes (ACTB, TUBB, ANGPTL4, MCM4, ALDOA, PGAM1, and AKR1C3) involved in the aforementioned cancer-related pathways and processes as well as candidate drug resistance miRNAs predicted to regulate the expression of my candidate drug resistance genes. In radiation treated KRAS WT and mutant cells, the pathways and processes involved in radiation response were similar to those predicted in acquired drug resistance. I also identified a number of miRNAs, including miR-224, that were differentially expressed in irradiated KRAS WT and mutant cells and that may modulate KRAS-mediated radiation resistance. My data suggests that miR-224 could be a useful disease progression biomarker, in conjunction with other markers, to aid in determining patient prognosis. Furthermore, this study has identified novel candidate drug and radiation resistance signatures that could aid as additional markers of treatment response.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available