Transcriptional regulation of tumor suppressor gene RASSF1A by HBx.

[INTRODUCTION] The occurrence of liver cancer in China is primarily attributed to chronic hepatitis B virus (HBV) infection. HBV X protein (HBx) has emerged as a significant carcinogenic driver in HBV-related liver cancer. However, the underlying mechanism by which HBx contributes to liver cancer development is not fully understood.

[METHODS] This study investigated HBx's role in regulating the tumor-suppressor gene RASSF1A. Firstly, the RASSF1A plasmid was constructed using a luciferase reporter system. The dual luciferase assay system detected HBx's effect on RASSF1A promoter activity. Western blotting and quantitative PCR methods measured HBx's impact on RASSF1A protein and mRNA expression. Chip was used to test the binding of HBx and SP1. CCK8, transwell, flow cytometry were used to detect the effect of RASSF1A on HCC proliferation. Methylation-specific PCR analyzed HBx's effect on RASSF1A methylation.

[RESULTS] Our results show that HBx significantly enhances RASSF1A promoter activity in an SP1 binding site-dependent manner. When only one SP1 binding site remained, HBx's effect was abolished. RASSF1A can inhibit HCC proliferation. Both mRNA and protein expression levels of RASSF1A were lower in HBx-expressing THLE-2 cells than in control cells, correlating with higher RASSF1A promoter methylation.

[CONCLUSION] These findings suggest HBx enhances RASSF1A promoter activity and upregulates transcription via SP1, potentially preceding RASSF1A promoter methylation. This study provides new insights into HBx's regulation of the tumor suppressor gene RASSF1A in HBV-related liver cancer.