Investigation of the association and carcinogenesis of Fusobacterium nucleatum, DNMT3a, and the hypermethylation of SEPT9 in colorectal cancer (CRC)

Pang, Siew Wai * (2024) Investigation of the association and carcinogenesis of Fusobacterium nucleatum, DNMT3a, and the hypermethylation of SEPT9 in colorectal cancer (CRC). Doctoral thesis, Sunway University.

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Abstract

The FDA has recently approved the screening of colorectal cancer (CRC) using methylated SEPT9 (mSEPT9) as a highly sensitive biomarker. Meanwhile, Fusobacterium nucleatum (FN) has been implicated in CRC progression. However, the association between FN and SEPT9 in CRC remains unexplored, as does the role of DNMT3a, a DNA methyltransferase known to methylate SEPT9 in liver cells but with an unknown function towards SEPT9 in CRC. To address these gaps, this study primarily aims to demonstrate the clinical significance of FN and SEPT9 methylation in CRC patients, investigate the involvement of DNMT3a in regulating SEPT9 methylation in CRC, and define the relationship between FN, DNMT3a expression, and SEPT9 methylation in CRC. Quantitative polymerase chain reaction (qPCR) analysis of the DNA from FFPE tissue collected from CRC patients reveals that FN is prevalent in CRC patients (87.95 %) and more abundant in cancer tissue than in normal tissue (p < 0.05). Although no significant associations were found between FN and clinicopathological features of CRC patients, a high bacterial load of FN was significantly associated with larger tumour size and the presence of KRAS mutations (p < 0.05). Moreover, SEPT9 is more prevalent in CRC patients (90.36 %). Elevated levels of mSEPT9 were detected in CRC tissue, particularly in patients aged 50 years and above, and were associated with larger tumour size (p < 0.05). Importantly, high levels of mSEPT9 were correlated with elevated levels of FN in CRC tissue, regardless of the cancer status of the tissue. For the in vitro study, we see a reduction in SEPT9 hypermethylation due to the silencing of DNMT3a in HCT116 colon cancer cells. However, the resulting downregulation of SEPT9 protein was unexpected. This finding revealed the complex and multifaceted roles of DNMT3a in the regulation of SEPT9 at the methylation, gene expression, and protein expression levels. On the other hand, the FN infection assay demonstrated that FN induces greater changes in CRC cell line (HCT116) compared to normal colon cell line (CCD-112). Interestingly, we see a downregulation of DNMT3a and SEPT9 protein expression in response to FN infection despite an upregulation of the mRNA expression of DNMT3a and SEPT9 in HCT116 and CCD-112 cells. No observable trends in SEPT9 hypermethylation were observed in the FN infected cells. In summary, this study provides valuable insights into the association between FN and SEPT9 methylation in CRC. It sheds light on the complex involvement of DNMT3a in regulating SEPT9 methylation in CRC cells. These findings contribute to a better understanding of CRC and may pave the way for the development of novel diagnostic and therapeutic approaches targeting FN, SEPT9, and DNMT3a in the management of CRC.

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