MicroRNA‑20 induces methylation of hepatitis B virus covalently closed circular DNA in human hepatoma cells

Abstract

Methylation was suggested to suppress the transcriptional activity of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in hepatocytes. This may be associated with its low replicative activity during the inactive stage of chronic HBV infection; however, the exact mechanisms of methylation in HBV infection remain unknown. We have previously shown that short hairpin RNAs induced the methylation of the HBV genome in hepatoma cell lines. We also reported that the microRNA (miR) 17–92 cluster negatively regulates HBV replication in human hepatoma cells. In addition, miR-20a, a member of the miR 17–92 cluster, has sequence homology with the short hairpin RNA that induces HBV methylation. In the present study, we investigated whether miR-20a can function as an endogenous effector of HBV DNA methylation. The results indicated that overexpression of miR-20a could suppress the replicative activity of HBV and increased the degree of methylation of HBV cccDNA in the HepAD38 hepatoma cell line. Argonaute (AGO)1 and AGO2, effectors of the RNA-induced silencing complex, were detected in the nucleus of HepAD38 cells; however, only AGO2 was bound to HBV cccDNA. In addition, intranuclear AGO2 was determined to be bound with miR-20a. In conclusion, miR-20a may be loaded onto AGO2, prior to its translocation into the nucleus, inducing the methylation of HBV DNA in human hepatoma cells, leading to the suppression of HBV replication.

Methylation profile of hepatitis B virus is not influenced by interferon α in human liver cancer cells

“…The present study hypothesized that cccDNA methylation may also contribute to the IFN-mediated transcriptional suppression of HBV. To test this hypothesis, our previous cell-based model was used to generate a condition for cccDNA methylation, with some modifications ( 39 ). First, direct infection with wild-type HBV virions ( 52 , 53 ) was used instead of plasmid-mediated in vitro HBV models ( 30 ), because CpG-containing plasmids may trigger inflammatory cytokine responses and become inadvertently methylated ( 54 ).…”

“…Since HepG2 cells were maintained in the presence of fetal bovine serum (FBS) throughout the experiments, the possibility that various cytokines in FBS may affect the results cannot be excluded. Serum-free culture condition is difficult to maintain in our cell model because our previous data showed that ≥5 days were needed before methylation of HBV cccDNA was induced ( 39 ). Other related studies also used similar study design with serum in culture media ( 16 , 30 ) and it is hypothesized that possible effect of cytokines may have been adjusted by comparison with the control with same culture condition (except for IFN).…”

“…Southern and northern blots for HBV rcDNA and RNA were performed using a digoxigenin-labeled RNA probe as previously reported ( 39 , 40 ). Briefly, 15 µg of HBV rcDNA was electrophoresed on a 1% agarose/Tris-Borate EDTA gel, which was depurinated with HCl (0.25 M) for 15 min and denatured in NaOH (0.5 M) for 15 min.…”

“…PCR was performed to measure the relative amount of methylation using the same primer pairs for methylation and normalized using the unmethylated primer pair. Bisulfite sequencing was performed to detect the methylation of CpG island II ( 39 ). The cloned sequences were analyzed using a BiQ Analyzer (Max Planck Institut Informatik; ) ( 44 ).…”

Silencing hepatitis B virus covalently closed circular DNA: The potential of an epigenetic therapy approach

“…A recent study by Moon et al [ 175 ] has suggested that miR-20a can act in an epigenetic manner and promote methylation of cccDNA. They found that AGO2-miR-20a binding to the cccDNA may recruit DNMT3 to silence gene expression; however, further studies are needed to confirm the exact mechanism.…”