The expression and promoter methylation of SLIT2 gene in human primary hepatocellular carcinomas

Purpose To study the SLIT2 expression and mechanism of its silencing in hepatocellular carcinoma (HCC). Methods Genomic DNA and total RNA were extracted from 3 normal liver tissue samples, 32 HCC samples and 8 liver cancer cell lines, respectively. The expression of SLIT2 was evaluated by semi-quantitative RT-PCR. The methylation specific PCR (MSP) and bisulfite DNA sequencing were performed to examine the methylation status of SLIT2 gene promoter. Results Promoter methylation of SLIT2 gene was found in 27 of 32 liver tumor tissues (84.3%) and 18 of 32 non-cancerous liver tissues (56.3%) from 32 HCC patients, and 6 of 8 liver cancer cell lines (75%), whereas SLIT2 methylation was not detected in normal liver tissues from healthy controls. There was no correlation between promoter hypermethylaion and pathological data for primary HCC patients, including tumor size, lymph metastasis, and clinical stage. However, the frequency of methylation in elder patients (≥60 years old) was significantly higher than that in younger patients (60 years old) (P0.05). Interestingly, the expression of SLIT2 could be induced by the demethylating agent 5-aza-2-deoxycytidine in HCC cell lines with the promoter hypermethylation of SLIT2 gene. Conclusions Promoter hypermethylation of SLIT2 gene may occur as an early event in the development of HCC. Methylation is possibly a major mechanism for inactivation of SLIT2 gene in HCC.