Hepatitis B virus promotes hepatocellular carcinoma progression by disrupting NSUN2/YBX1-mediated m5C modification of PTEN mRNA.

Hepatitis B virus (HBV) has been implicated in hepatocellular carcinoma (HCC) progression, partly through regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Although transcriptional regulation of PTEN by HBV is well characterized, its post-transcriptional regulation remains poorly understood. Because RNA 5-methylcytosine (m5C) modification influences post-transcriptional gene control and cancer development, we investigated whether HBV modulates PTEN through m5C. Methylated RNA immunoprecipitation (MeRIP)-quantitative polymerase chain reaction showed a marked reduction in m5C on PTEN mRNA in HBV-producing cells. MeRIP sequencing further identified decreased m5C within the PTEN coding sequence region (chr10:89717747-89717771) in HBV-producing HepAD38/tetracycline-off cells, with chr10:89717756 emerging as a critical site where HBV suppresses m5C enrichment and PTEN expression. Mechanistically, the m5C "writer" NOP2/Sun RNA methyltransferase 2 (NSUN2) and the "reader" Y-box binding protein 1 (YBX1) stabilized PTEN mRNA in an m5C-dependent manner. HBV disrupted this pathway, decreasing PTEN mRNA stability via NSUN2- and YBX1-mediated m5C. Overexpression of NSUN2 or YBX1 attenuated HBV-driven proliferation, migration, and invasion, and these effects were partially reversed by the PTEN inhibitor VO-Ohpic. The small hepatitis B surface antigen and hepatitis B X protein downregulated NSUN2 and YBX1, linking viral proteins to PTEN suppression. Further, HBV is associated with reduced NSUN2 expression in HBV transgenic (HBV-Tg) mice, HBV-infected primary human hepatocytes as well as HBV-positive clinical HCC specimens, supporting the physiological and clinical relevance of this finding. Together, these findings identify the NSUN2/YBX1/PTEN axis as a potential therapeutic target in HBV-associated HCC.