Correlation of Sox9 expression and its promoter site methylation status in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the third most fatal cancer in Hong Kong. Molecular hepatocarcinogenesis involves the accumulation of genetic and epigenetic alterations leading to oncogene activation or tumor suppressor gene inactivation. Sry (sex determining region Y)-box9 (Sox9) is a crucial gene that regulates embryonic development of various organs such as the cartilage, testes, heart etc. Sox9 is also implicated in many human cancers and associated with Cancer stem cell (CSC) features in some. Our preliminary result demonstrated that Sox9 was overexpressed in HCC and conferred stemness properties. To date, the epigenetic regulation of Sox9 in HCC has not been addressed.

Hypothesis and aims: We hypothesized that Sox9 expression in HCC is modulated by methylation status of its promoter site. Overexpression of Sox9 in HCC tissues is mediated through promoter hypomethylation. In this study, we aimed at investigating the methylation status of Sox9 in HCC and to correlate it with the expression level.

Methodology: This study involved three main parts. First, Sox9 expression of SMMC-7721and LO2with and without hypomethylating agent, 5-Aza-2’-deoxycytidine(5-Aza)treatment was analyzed by quantitative polymerase chain reaction (qPCR)and compared. Next, Sox9 promoter region methylation status of SMMC-7721, LO2,and Huh7was analyzed by bisulfite genomic sequencing(BGS). Lastly, methylation specific-polymerase chain reaction(MS-PCR) of 20 clinical primary HCC samples was performed toassess the methylation status of Sox9 promoter region in tumor (compared with non-tumorous tissues).

Results: By treatment of HCC cell lineSMMC-7721and the immortalized liver cell line LO2, which both show a low endogenous Sox9 expression, with 5-Aza,Sox9 mRNA expression was increased in a dose-dependent manner. Based on computational prediction, 3 CpG Islands in Sox9 promoter were selected. These 3 regions of Sox9 promoter were analyzed by BGS in three cell lines (SMMC-7721, LO2,and Huh7), and MS-PCR in 20 primary HCC cases. The BGS result showed that Region 3 included most abundant CpG dinucleotide and also highest chance of methylation. MS-PCR result showed 53% (10/19) hypomethylation, 26% (5/19) hypermethylation, and no alteration 21% (4/19) in the HCC cases (with one case produced invalid result). The concordance of MS-PCR and qPCR results was 47%. The correlation was further analyzed by dividing 19 cases into groups according to the MS-PCR or qPCR result (preliminary result). For the hypomethylated Sox9 promoter group, 5/10 cases were with Sox9 upregulated expression. Meanwhile, among the 7 cases with upregulated Sox9 by qPCR, 5 cases showed hypomethylation at Sox9 promoter site. Conclusion: Methylation status of Sox9 promoter is possibly one of the regulatory mechanisms forSox9 expression in HCC. Promoter hypomethylation is a non-negligible mechanism leading to Sox9 overexpression in HCC.