Quantificational Methylation Analysis of APC and AXIN2 in HBV-related Hepatocellular Carcinoma

Background: Aberrant activation of the Wnt signaling is one of the most common events in hepatocarcinogenesis. The aim of this study is to investigate the role of methylation modulated APC, AXIN2, APC2 and NKD2 genes expression silencing in hepatitis B virus (HBV) related heptocellular carcinoma (HCC). Methods: Eighty paired tumor and adjacent non-tumor tissues were collected from HCC patients with chronic HBV infection and underlying cirrhosis. The promoter CpG islands (CGI) methylation status of the above genes in paired tissues and 7 human hepatoma cell lines were quantitatively analyzed. Realtime RT-PCR was performed to determine the mRNA levels of these genes. Results: Compared to the adjacent non-tumor tissues, tumor tissues exhibited significantly increased APC hypermethylation both in frequency (56.2% vs. 78.7%, P = 0.0024) and in intensity (15.0% vs. 34.4%, P < 0.0001). Accordantly, APC expression in HCC tissues was significantly lower than that in non-tumor tissues (P = 0.0031). AXIN2 CGI hypermethylation in both non-tumor and tumor tissues were found 91.4% and 85.7% in frequency, and 31.6% and 32.5% in intensity, both were significantly higher than that in disease-free liver tissues (P = 0.0015 and 0.0008, respectively). Consistent with the methylation pattern, no statistical difference of the AXIN2 mRNA level between the paired tissues was observed. Intriguingly, stratified analysis revealed AXIN2 mRNA expression level in tumor tissues was significantly down-regulated in under 55-year-old male group (P = 0.0138) and lower AXIN2 mRNA expression level in tumor tissues implicated earlier onset age. In contrary, in over 55-year-old group, tumor tissues' AXIN2 mRNA level increased in parallel with the increase of patients age (P = 0.019). No methylation-mediated inactivation of APC2 and NKD2 gene was observed in hepatoma cell lines. Conclusion: Our findings suggested that methylation caused APC and AXIN2 expression silencing might be involved in hepatocarcinogenesis arisen from cirrhotic liver.