Hyper-methylation of RIZ1 tumor suppressor gene is involved in the early tumorigenesis of hepatocellular carcinoma

The retinoblastoma protein-interacting zinc finger gene RIZ1 is a putative tumor suppressor gene, and the inactivation of the RIZ1 is frequently found in tumors through a loss of mRNA expression. In order to understand the role of RIZ1 inactivation in the tumorigenesis of hepatocellular carcinoma (HCC), we detected the RIZ1 promoter methylation status in 39 HCCs using a methylation specific PCR (MSP) method, and carried out LOH study with marker P704. We also assessed the associations between the methylation status and clinicopathological parameters, tumor size, tumor differentiation, and fractional allelic loss (FAL). The results showed that the RIZ1 promoter methylated both in advanced tumors (> 3 cm), (18/ 31, 58.0%) and in early tumors (< 3 cm), ( 4/ 8, 50.0%). There were 54.6% ( 12/ 22) tumors with hyper-methylation in the low FAL group and 45.5% (10/ 22) in the high FAL group. Moreover, the DNA methylation of the RIZ1 promoter was found not only in the poorly differentiated tumors (12/ 22, 54.6%), but also in the well differentiated tumors (10/ 22, 45.5%). Among the 22 HCCs (22/ 39, 56.4%) that showed hypermethylation at the RIZ1 promoter region, 3 cases showed biallelic methylation. Interestingly, one case showed hyper-methylation on one allele and a loss of heterozygosity (LOH) on the other allele. In other words, 4 HCCs showed the biallelic inactivation of the RIZ1. These results suggest that the inactivation of the RIZ1 by DNA methylation at its promoter region is involved in the tumorigenesis of HCC, particularly in the early stage of disease.