Abstract: Objective To assess genome-wide DNA methylation level of non-alcoholic fatty liver disease concurrent with chronic hepatitis B (NAFLD + CHB) and investigate the effect of betaine intervention on genome-wide DNA methylation in the cellular model of NAFLD+CHB. Methods In this study, a total of five groups were included. First, a free fatty acid mixture with a concentration of 0.75 mmol/L was used to intervene in human liver cancer cells (HepG2) and HepG2 cells transfected with hepatitis B virus (HepG2.2.15) to establish the non-alcoholic fatty liver disease (NAFLD) group and the NAFLD+CHB group cell models. Subsequently, 2, 4, and 8 mmol/L of betaine were used to intervene in the NAFLD+CHB cells to establish three betaine intervention groups. The cell viability of liver cells, the levels of genome-wide DNA methylation, and the mRNA expression levels of DNA methylation-related enzymes (DNMTs, MBDs, TETs) were measured. Results Compared with the NAFLD group, the genome-wide DNA methylation level of the NAFLD+CHB group exhibited a significant decrease (1.070%±0.166% vs. 1.848%±0.238%, t=4.64, P<0.05). Additionally, there was a down-regulation in mRNA expressions of DNMT3B and MBD1, while an up-regulation was observed in mRNA expressions of DNMT3A, MBD4-6, and TET2. Treatment with betaine at concentrations of 2 mmol/L, 4 mmol/L, and 8 mmol/L in the NAFLD+CHB cell resulted in an increase of genome-wide DNA methylation level (1.895%±0.234%, 1.581%±0.089%, and 1.892%±0.066% vs. 1.070%±0.166%, F=19.14, P<0.01), along with a down-regulation in mRNA expressions of MBD1-3, MBD5-6, and TET1-3. Conclusions The genome-wide DNA methylation levels are found to be lower in NAFLD+CHB cells compared with NAFLD cells. Furthermore, betaine intervention demonstrates potential for increasing the genome-wide DNA methylation level in NAFLD+CHB cells.

Key words: Non-alcoholic fatty liver disease, Chronic hepatitis B, Genome-wide DNA methylation, Betaine