Epigenetic silencing of Klotho expression correlates with poor prognosis of human hepatocellular carcinoma

Abstract

Klotho is identified as a tumor suppressor in several tumors, but the expression of the Klotho gene (KL) and its regulative mechanism are not reported in hepatocellular carcinoma (HCC). The messenger RNA and protein levels of Klotho were measured in 64 HCC tumor tissues by real-time polymerase chain reaction and immunohistochemistry, respectively. The methylation of KL promoter DNA was examined by bisulfite-based polymerase chain reaction. The correlation of Klotho protein expression and methylation with survival of HCC was analyzed using Kaplan-Meier analysis. The interference of KL gene expression was conducted in HCC cells by DNA demethylating agent and/or histone deacetylase inhibitor. In HCC tissues, a significant loss of Klotho messenger RNA and protein expression was observed, which parallels the increased methylation in KL promoter DNA. Both Klotho expression and methylation correlated with the poor prognosis of HCC. Experiments with HCC cell lines showed that a combination of DNA demethylating agent and histone deacetylase inhibitor fully recovered Klotho expression and subsequently induced cell apoptosis. In conclusion, Klotho is a tumor suppressor in HCC. Both hypermethylation and acetylation are involved in the loss of Klotho expression in HCC cells. Both KL gene expression and its promoter DNA methylation are predictive factors for the poor prognosis of HCC. Our study also suggests that the Klotho gene could be a target for therapy of HCC.