Correlation of TSPY expression level and its promoter methylation status in liver cancer

Abstract

Background: Hepatocellular carcinoma (HCC) is the 6th most common cancer and the 2nd leading cause of cancer deaths globally. TSPY (Testis-specific protein Y-encoded) is normally expressed in testis. Recent studies demonstrated the presence of TSPY transcript in some tumors including HCC, suggesting its tumorigenic role in hepatocarcinogenesis. Methylation status in other cancers was investigated previously, showing TSPY expression might be regulated by DNA methylation. This prompts us to interrogate the potential epigenetic regulation of TSPY expression in HCC. In this study, we determined the methylation status of TSPY promoter site in clinical samples and attempted to correlate it with mRNA expression. Methods: Expression of TSPY was examined using quantitative real-time PCR (qPCR). HCC Cell line Huh7 and immortalized normal liver cell line MIHA, which showed low endogenous TSPY expression, were treated with DNA methyltransferase inhibitor 5-Aza-2'-Deoxycytidine (5-aza). qPCR was performed to compare TSPY expression levels before and after treatment. Huh7 was selected to analyze the methylation status in promoter site of TSPY using Bisulfite genomic sequencing (BGS). Methylation-specific PCR (MS-PCR) of the selected CpG island region was performed in 12 HCC clinical samples to evaluate the methylation status in tumor as compared with the non-tumoral counterpart. Methylation status was then correlated with expression level of TPSY in the clinical samples. Results: By qPCR, upregulation, downregulation and no significant difference of TSPY mRNA expression (tumor compared with non-tumorous tissues) was found in 40% (10 of 25) cases, 36% (9 of 25) cases and 24% (6 of 25) cases respectively. By Western blotting, low endogenous level of TSPY protein expression was found in Huh7 and MIHA cell lines. Subsequent qPCR of these two 5-aza-treated cell lines showed restoration of TSPY mRNA expression. Four potential CpG islands were identified through online programme MethPrimer and one was chosen in this study. BGS results showed a 48.3% probability of methylation in 3 clones of Huh7 cells treated with 2μM 5-aza for 3 days. For MS-PCR result, “hypermethylation”, “hypomethylation” and “no significant differences” status were found in 41.7% (5 of 12), 41.7% (5 of 12) and 16.7% (2 of 12) cases respectively. Correlation between TSPY mRNA expression and promoter site methylation status showed a “concordance” in 33.3% in the clinical samples (4 of 12 cases). Conclusions: Our findings suggested that promoter site methylation status might not be a major regulatory mechanism on the expression of TSPY in HCC. Further studies are recommended to consolidate the findings.