HBx-induced DNMT3A mediated UNC13D methylation aggravates liver fibrosis by regulating the liver microenvironment

Chronic hepatitis B virus (HBV) infection is the main etiology of liver fibrosis (LF), leading to cirrhosis and liver tumors. HBV-infected hepatocytes activate hepatic stellate cells (HSCs) through microenvironment, but the mechanism remains unclear. In this study, we discovered that DNMT3A expression increased in HBV-infected liver tissue. After constructing 7 HBV-coding protein plasmids, it was discovered that the HBV X protein (HBx) mostly increased the expression of DNMT3A. The reduced representation bisulfite sequencing (RRBS) and RNA-sequencing (RNA-Seq) data demonstrated an increase in genome-wide methylation. And UNC13D expression was decreased as a result of methylation at the promoter CpG site. The lower expression of UNC13D makes HepG2 cells more sensitive to activating the interferon response cGAMP interactor 1 (STING) signaling pathway, which enhances TBK-1 phosphorylation. Furthermore, HepG2 and Hep3B cells overexpressing DNMT3A were constructed using lentivirus, and the culture supernatant was collected to provide conditioned medium for further culture with LX-2 cells. The results showed that LX-2 cells were significantly activated. Decitabine, which targets DNMT3A, can effectively reverse the activation of LX-2 cells. In summary, this study revealed that HBV-infected hepatocytes alter the liver microenvironment through the DNMT3A/UNC13D/STING axis, promoting HSCs activation and leading to liver fibrosis. Targeting DNMT3A could be a promising therapeutic strategy in reversing LF.