Abstract
m6A modification has been shown to play a role in regulating HBV RNA expression. However, it remains unclear whether the m6A levels of HBV pregenomic RNA (pgRNA) are related to HBV replication and liver function. Serum HBV pgRNA-m6A levels were determined by T3 DNA ligase assay. HBV DNA copy numbers and gene expression were detected by TaqMan assay and RT-qPCR, respectively. Liver function was evaluated using clinical laboratory indicators. The TET-off stable HBV-producing cell line HepAD38 and targeted pgRNA demethylation by SunTag system (TRADES) were used for the site-specific m6A editing. Flow cytometry was used to measure the cytokine levels in the supernatant of cell culture. Serum HBV pgRNA-m6A levels were positively correlated with HBV DNA copy number, pgRNA expression and liver function indicators (all p 6A levels and pgRNA expression were significantly increased in patients with high virus load (HBV DNA > 1.0 × 107 IU/mL). Site-specific demethylation of pgRNA1907-m6A significantly reduced HBV DNA, hepatitis B e antigen (HBeAg) and HBV pgRNA, while induced levels of interferon alfa 2 (IFN-α2) and apolipoprotein B mRNA editing enzyme catalytic subunit 3A (APOBEC3A). pgRNA-m6A modification of HBV associates with viral replication and liver function indicators. Site-specific m6A demethylation of pgRNA provides novel insight into anti-HBV treatment.