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 (m 5 C) modification influences post-transcriptional gene control and cancer development, we investigated whether HBV modulates PTEN through m 5 C. Methylated RNA immunoprecipitation (MeRIP)–quantitative polymerase chain reaction showed a marked reduction in m 5 C on PTEN mRNA in HBV-producing cells. MeRIP sequencing further identified decreased m 5 C 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 m 5 C enrichment and PTEN expression. Mechanistically, the m 5 C “writer” NOP2/Sun RNA methyltransferase 2 (NSUN2) and the “reader” Y-box binding protein 1 (YBX1) stabilized PTEN mRNA in an m 5 C-dependent manner. HBV disrupted this pathway, decreasing PTEN mRNA stability via NSUN2- and YBX1-mediated m 5 C. 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. • HBV reduces PTEN mRNA stability by disrupting site-specific m 5 C modification. • NSUN2 and YBX1 are critical for maintaining PTEN mRNA stability via m 5 C modification. • HBV viral proteins SHBs and HBx suppress NSUN2 and YBX1 expression. • Overexpression of NSUN2 and YBX1 inhibit HBV-driven cell proliferation and invasion. • The NSUN2/YBX1/PTEN axis is a potential therapeutic target in HBV-related HCC.