Cervical cancer arises when high-risk strains of human papilloma virus (HR-HPV) become integrated into the host genome in cervical epithelial cells and loss of episomal expression of the viral transcriptional regulator gene E2 occurs. However, not all women who acquire HPV infection develop cervical cancer: about 60% of HPV infections are transient and resolve on their own due to innate and adaptive immunity, indicating other risk factors. Among other known factors, dietary methyl donor nutrients that influence the methyl donor cycle may also be important in determining cervical cancer risk. They are required for the maintenance of DNA methylation, modification of which is associated with cancer progression through dysregulation of gene expression. The aim of this study is to identify molecular mechanisms by which alterations in methyl donor nutrients availability affect cervical cancer risk and progression. A bioinformatics analysis was carried out to identify gene clusters susceptible to both methyl donor depletion and HPV integration using the DAVID, ClueGo and GeneMania software. The C4-II cervical cancer cell model of methyl donor depletion was used for this experimental study. Cells were grown for 8 days in complete medium, or medium depleted of folate, methionine or combined folate and methionine. The cell model was validated by measuring the intracellular folate, intracellular methionine and extracellular homocysteine concentrations. The STAT1, RSAD2, OAS1, IFIT1 and ISG15 genes expression were measured using RT-qPCR. A comprehensive DNA methylation profiling was performed using the Infinium Methylation EPIC BeadChip, followed by methylation data analysis with the RnBeads and bioinformatics software. Additionally, the TNF-α and IL-1β cytokine production in THP-1 methyl donor depleted cells was measured using the Quantikine ELISA kit. Gene clusters associated with host defence mechanisms, cell cycle, cell death and cell signalling were highly enriched. Additionally, a cluster of genes involved in antiviral defence induced by the type I interferon (IFN) pathway appeared to be most affected by methyl donor nutrients availability. These genes (RSAD2, OAS1, IFIT1 and ISG15) were reported to be significantly upregulated with methionine or combined folate and methionine depletion; while STAT1 gene expression was downregulated in all depleted conditions. Folate deficiency did not cause significant alteration to the expression of these genes. TNF-α and IL-1β cytokine production was significantly reduced in all methyl donor depleted conditions. A majority of CpG sites were significantly hypomethylated in all conditions where methionine was deficient, while changes in folate depleted medium was not statistically significant. A high proportion of genes associated with host defence mechanisms, immune response as well as mitosis, cell differentiation, cell motility and angiogenesis were found to be hypomethylated. This study highlights the importance of methionine over folate as a methyl donor in the regulation of gene expression, and also the regulatory functions of both folate and methionine on IFN-stimulated gene expression associated with antiviral immunity in a cervical cancer cell line. By identifying changes that occur in methyl donor regulated genes, there is a potential diagnostic or therapeutic approach based on epigenetic changes occurring due to nutrient availability for those who are in the at-risk group.
