Date of Award

9-12-2024

Publication Type

Thesis

Degree Name

M.Sc.

Department

Biological Sciences

Keywords

Circadian Rhythms;Colorectal Cancer;Organoids

Supervisor

Phillip Karpowicz

Abstract

Dysregulation of the circadian clock is correlated with the development of colorectal cancer. APC is a tumour suppressor in >80% colorectal cancers and, previously, our lab used Apcmin/+ mouse models to demonstrate the clock-gene Bmal1 represses tumorigenesis in the intestine. RNA-seq data shows that organoids derived from healthy or cancerous intestinal epithelial tissue exhibit significant differences in gene expression in clock genes, and their target genes such as Hmgcs2. Hmgcs2 is a metabolic enzyme and regulator of intestinal stem cells (ISCs), that has Bmal1-dependent circadian rhythms in its expression. Recent work on murine models has shown that time-restricting feeding (TRF), a regimen that restricts all calorie intake to match daily rhythms of activity, can improve circadian-regulated pathways and upregulate the rhythmicity of Hmgcs2 in most organs. To investigate how TRF affects clock gene and Hmgcs2 expression in the intestinal epithelium, I subjected mouse colon organoids to a TRF regimen of high and low glucose. I first attempted to test TRF using a Luciferase reporter, and tested the growth and differentiation of organoids which underwent TRF. I next collected organoids at four timepoints across 24-hours to test gene expression of Nr1d1, Cry1, and Hmgcs2 using rt-qPCR. Since the control group failed to show circadian rhythms, no conclusions can be drawn on the influence of TRF on circadian rhythms. However, I showed that organoids, which generally require strict parameters for survival, can grow to the same size and proliferating to the same capacity as controls when subjected to TRF. To clarify the role of rhythmic metabolic activity in vivo, I tested the presence of Hmgcs2 in intestinal tumours and healthy epithelial tissue of Apcmin/+ mice lacking a functional clock. My findings show that Hmgcs2 expression is slightly higher in intestinal tumours lacking a functional clock when compared to control tumours, although this difference is not significant.

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