Author: Saira Ali
Ali, Saira, 2020 Epigenetic regulation of gene expression in colorectal cancer cells, Flinders University, College of Medicine and Public Health
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Despite improvements in colorectal cancer (CRC) diagnosis and therapy, CRC remains the second leading cause of cancer-related deaths worldwide; hence, there is a need for innovative and novel treatment approaches. CRC development and progression involves several epigenetic alterations including changes in histone modification patterns, DNA methylation and dysregulated non-coding RNA expression (microRNAs (miRNAs) and long non-coding RNAs (lncRNAs)). Dietary molecules, including the gut fermentation product butyrate, can alter CRC cell gene expression and consequently cell behaviour through epigenetic mechanisms. While specific miRNAs have been shown to sensitise CRC cells to butyrate by increasing its anticancer potential (Humphreys et al. 2014b), there have been no reports of an unbiased process to discover butyrate-sensitising ncRNAs. Using functional high-throughput screening and sequencing technologies, the role of miRNAs and other ncRNAs in the butyrate sensitisation of CRC cells was investigated by exploring proliferative and apoptotic cell changes. The roles of downstream interactors were also investigated in the butyrate response of CRC cells.
Unbiased high-throughput screening revealed 13 miRNAs with butyrate-sensitising properties in CRC cells. Several miRNAs increased the anti-proliferative and pro-apoptotic potential of butyrate in CRC cells by a synergistic mechanism. Predicted miRNA target genes were identified as regulators of cell growth and death pathways including WNT signalling, PI3K-AKT signalling and apoptosis. The silencing of a PI3K subunit revealed potent anti-proliferative effects in combination with butyrate in CRC cells.
In addition, a systems biology approach was used to examine butyrate-regulated transcriptomic changes and complex RNA interactions contributing to the butyrate response of CRC cells. Total and small RNA-seq analyses revealed thousands of butyrate-regulated protein-coding and ncRNA species. Differentially expressed (DE) protein-coding transcripts were enriched for genes encoding components of the cell cycle pathway. Network analyses highlighted key miRNA-target pairs, including tumour suppressor miRNAs that were upregulated by butyrate and consequently reduced cell cycle regulators. miRNAs sensitised cells to butyrate through synergistic mechanisms. RNAi in CRC cells resulted in potent anti-proliferative effects in combination with butyrate.
Unbiased high-throughput screening using a lncRNA-targeting siRNA library revealed that silencing of several lncRNAs in combination with butyrate affected CRC cell apoptosis. Network and pathway analyses investigating lncRNA-miRNA-mRNA axes revealed that oncogenic MALAT1 was highly connected in the constructed apoptosis network. MALAT1 was predicted to interact with several miRNAs which potentially targeted apoptotic-related genes. Apoptosis was enhanced when CRC cells were exposed to MALAT1 siRNAs in combination with butyrate. Further investigation is required to confirm regulation of downstream targets.
In summary, these data contribute to the growing knowledge of the role of ncRNAs in the response of CRC cells to butyrate and the cellular pathways that are involved. These results provide the basis for further investigation of a novel therapeutic approach combining RNAi and HDACi treatment in CRC. Further investigation is also warranted in other cancer models due to the broad-acting effects of these epigenetic molecules.
Keywords: colorectal cancer, cancer, epigenetics, microRNAs, miRNAs, long non-coding RNAs, lncRNAs, butyrate
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2020
School: College of Medicine and Public Health
Supervisor: Michael Michael